Statistical methodologies for the analysis and normalization of RIP-Chip data

Tese de doutoramento, Estatística e Investigação Operacional (Bioestatística e Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2011Pre-mRNA splicing is an essential step in the post-transcriptional gene expression control involving protein-splicing factors like PTB and U2AF65; the last one is exported to the cytoplasm and involved in some other cellular functions. The identification of PTB- and U2AF65-associated mRNAs under native conditions was performed by immunoprecipitation and hybridization on Chip (RIP-Chip) technology using the Affymetrix GeneChip R Human Genome U133 Plus 2.0. The aim of this thesis is to develop statistical methodologies for low level analysis and enriched gene selection in RIP-Chip experiments. When the most common methodologies for quality assessment, low level analysis (background adjustment, normalization and summarization) and detection of diferentially expressed genes (DEG), are applied to RIP-Chip data the obtained results difer. This probably happens because usually more than 20% of the mRNAs are enriched, while methods for normalization and identification of DEG are developed supposing that only a small proportion of genes (1% or 5%, say) express diferently. Also, methods for detecting diferentially expressed genes may not be the most adequate for gene enrichment selection. In this thesis is implemented a background correction method inspired in a non-specific hybridization method used for pre-processing ChIP-Chip data. Linear regression models are used in each array to model the non-specific hybridization. Probe intensities on the array are standardized using their predicted intensity and the variance of similar predicted intensities. The standardized probe intensities showed no need for further normalization, so the scores could be directly compared. It is proposed a probe set score, a probe set enrichment value and its p-value for enriched gene selection. The genes selected using this new method are practically the same as the ones found experimentally. Additionally, a new methodology based on ranks is presented for enriched gene selection, being applied to the probe set scores proposed. Both methodologies had high accuracy when applied to Spike-In U133 dataset, which is used to benchmark methodologies for analysing Affymetrix microarrays.Nos ultimos anos foram desenvolvidas técnicas de alto rendimento na investiga cão em biologia. Essas técnicas evoluíram fornecendo à comunidade científica instrumentos como: sequenciadores de alta capacidade, que permitem obter milhões de fragmentos de DNA ao mesmo tempo; espectómetros de massa em tandem que permitem a identificação de proteínas ou proteomas completos; ou hibridaçãode microarrays, usados para determinar a expressão dos genes através da identificação mRNAs presentes na célula num momento específico. Os microarrays constituem uma técnica usada para quantificar a expressão de genes e analisar fragmentos de genes, proteínas ou metabolitos. Também têm sido utilizados para clarificar elementos específicos do Dogma Central da Biologia Molecular, envolvidos no controle da transcrição; na busca de dados que expliquem como a expressão do gene começa a partir do DNA; ou como o mRNA em associação o com os ribossomas e traduzido em proteínas nas no citoplasma da célula. Dado o enquadramento biológico descrito acima, o Capítulo 1 introduz os aspectos da biologia relacionados com os dados RIP-Chip utilizados nesta tese, dados esses obtidos por Gama-Carvalho et al. [2006], em que se pretende identifi car os mRNAs associados a PTB e U2AF65 em condições nativas. Estas duas proteínas de ligação a RNA fazem parte do controle pós-transcripcional da expresscão genética em células eucariótas. Este capítulo começa por introduzir conceitos de biologia molecular da célula tal como o dogma central da biologia molecular, onde os processos de transcri cão e tradução são essenciais para manter a vida da célula e onde o controle de expressão genética é um aparelho fundamental na regulação da célula. Como parte do controle da expressão dos genes, o Capítulo 1 apresenta uma visão geral do controle pré- e pós-transcripcional da expressão dos genes. O splicing de pr e-mRNAs e um passo essencial no controle da expressão pós-transcripcional dos genes e envolve factores de splicing tais como as proteínas PTB e U2AF65, sendo U2AF65 exportada para o citoplasma e envolvida em outras funções celulares. O Capítulo 1 mostra como foram obtidos os dados RIP-Chip das proteínas PTB e U2AF65 e apresenta uma breve descri cão da metodologia utilizada por Gama-Carvalho et al. [2006] na sua experiência RIP-Chip. Mostra como a investigação de mRNAs associados a PTB e U2AF65, em condições nativas, foi realizada por imunoprecipitação (IP) após a adição de um anticorpo monoclonal específico (Bb7 anti-PTB mAb ou anti-U2AF65 MC3), seguido de extração de RNA, poliadenilacão, transcricão reversa, etiquetagem final e amplificação por PCR. Os cDNAs gerados foram hibridados com o GeneChip A ymetrix Human Genome U133 Plus 2.0 [Gama-Carvalho et al., 2006]. Este capítulo apresenta uma descricão da tecnologia de microarrays, em particular as características dos microarrays da Affymetrix utilizados na experiência RIP-Chip executada por Gama-Carvalho et al. [2006]. De seguida, o Capítulo 2 apresenta alguns dos métodos mais comuns de análise de dados de microarrays e os resultados de seu desempenho nos dados de Gama-Carvalho et al. [2006]. Para a correcão de background foi utilizado o modelo linear robusto (RMA) de Irizarry et al. [2003a] e uma modificação do mesmo (GCRMA) proposta por Wu et al. [2004], apenas sobre PM (Perfect Match). A normalização foi realizada através da normalização quartílica e a sumariacão das sondas foi feita usando a mediana polish [Irizarry et al., 2003a]. Alternativamente, os dados foram pré-processados usando o programa dChip: apenas para PM; usando o método de normalização invariant set [Li and Wong, 2001]; e o método baseado em modelos de Li and Wong [2001] para calcular os níveis de expressão. Para efeitos de comparacão foram utilizados os dados obtidos após a correcão de background com RMA, a normalização quartílica e sumariacão com a mediana polish. Com base nestes dados, foi feita a seleção de genes enriquecidos usando as seguintes bibliotecas do BioConductor: limma (ajusta um modelo linear para cada gene); eBayes (calcula a estatísticas T moderada, F e B - logaritmo das chances a posteriori); decideTests com um valor-p < 0:05 (baseia-se em testes múltiplos para determinar se cada estatística numa matriz de estatísticas T deve ser considerada significativamente diferente de zero [Smyth, 2004]); RankProd com FDR <0:05 (teste não-paramétrico que deteta itens que são consistentemente classificados como estando no topo da lista [Breitling et al., 2004]). Estes resultados foram comparados com os resultados obtidos com o programa dChip considerando uma taxa de falsas descobertas (FDR) <0:05 e um valor-p <0:05 [Li and Wong, 2003]. Os resultados apresentados no Capítulo 2 mostram como diferentes metodologias aplicadas aos dados de Gama-Carvalho et al. [2006] produziram resultados Diferentes. Parte das diferenças devem-se sobretudo ao facto de mais de 20% dos mRNAs serem enriquecidos e os métodos de normalização comuns terem por base pequenas diferenças entre eles. Como esta tese teve como principal objetivo o desenvolvimento de metodologias estatísticas para análise de baixo nível e selecão de genes enriquecidos em experiências RIP-Chip, o Capítulo 3 é dedicado a apresentar a implementa çao de um novo método de correção de background inspirado num método de hibridação não específica utilizado para pré-processamento de dados ChIp-Chip [Johnson et al., 2006]. Modelos de regressão linear foram usados para modelar em cada microarray a hibridação não específica, representando intera ções entre cada três nucleótidos consecutivos na sequência da sonda. As intensidades das sondas foram padronizadas usando sua intensidade prevista e a variância das sondas de intensidades previstas semelhantes. A nova abordagem aqui proposta utiliza a informação de cada microarray de forma independente, e os valores de intensidade padronizados não revelaram necessidade de normalização adicional. Assim, os microarrays podem ser directamente comparados [Barreto-Hernandez et al., 2011]. O Capítulo 3 apresenta também um score para a sonda; a definicão de um valor de enriquecimento da sonda (ENRval) e respectivos valores-p para a selecão de genes enriquecidos [Barreto-Hernandez et al., 2011]. Os genes enriquecidos obtidos usando esta metodologia, tanto para os dados RIP-Chip de PTB como de U2AF65, estão de acordo com os genes identi ficados experimentalmente por Gama-Carvalho et al. [2006]. Finalmente, o Capítulo 3 apresenta ao desenvolvimento da uma nova metodologia Não-paramétrica baseada em postos (ranks), implementada para selecão de genes enriquecidos e aplicada aos scores propostos en este Capítulo. Esta metodologia tem em conta a variabilidade da intensidade padronizada em cada sonda, em vez de usar o valor de sumariacão de cada sonda (ENRval). Ainda neste capítulo, as metodologias desenvolvidas nesta tese para a selecão de genes enriquecidos são aplicadas aos dados da experiência Spike-In. Esta base de dados foi construída há alguns anos e é usada no desenvolvimento e comparação de métodos de análise de expressão diferencial de genes [Irizarry et al., 2003b]. A experiência Spike-In U133 engloba 42 transcritos adicionados a um complexo transcriptoma humano em concentrações que variam de 0.125pM a 512pM, correspondendo a 14 hibridações separadas com três repetições técnicas. Os transcritos foram incluídos na experiência sob a forma de um quadrado latino clássico [Irizarry et al., 2003b]. Para a análise comparativa, três diferentes hibridações Spike-In foram selecionadas (hibridações 1, 8 e 14) e usadas para simular diferenças de enriquecimento em experiências RIP-Chip através do seguinte procedimento: 1 como Controle e 8 como IP; 1 como IP e 14 como Controle. As duas metodologias desenvolvidas nesta tese para sele cão de genes enriquecidos, apresentam elevada exatidão quando aplicadas aos dados Spike-In U133.Fundação para a Ciência e a Tecnologia (FCT por projectos PEst-OE/MAT/UI0006/2011 e PTDC/MAT/64353/2006

Diseño de un modelo bioinformática para la detección, identificación y clasificación de genes codificantes de b-lactamasas, como herramienta para el cruce de datos moleculares con datos clínicos

El seguimiento epidemiológico y farmacológico de la resistencia a antibióticos b-lactámicos mediada por la presencia de b-lactamasas requiere identificación precisa, lo cual es posible mediante la secuencia del gen, que es identificado con herramientas bioinformáticas que se incorporan a sistemas de información. Para implementar un prototipo de sistema de información que permite clasificar secuencias de genes de b-lactamasas producidas por microorganismos resistentes aislados en hospitales y realizar su cruce con los datos clínicos, se obtuvieron las secuencias de b-lactamasas reportadas a escala mundial en la base de datos Uniprot utilizando el sistema de recuperación de secuencias (SRS) del Instituto Europeo de Bioinformática (EBI), se diseñó un sistema de datos moleculares y clínicos usando “Unified Modeling Languageâ€� (UML), y se generó un modelo implementado en un servidor con sistema operativo UNIX, gestor de bases de datos MySQL para realizar la creación de la base de datos y lenguajes de programación Perl y PHP para implementar programas y cruzar datos. El sistema de información BLA_ID_CLINIC permite hacer el cruce de datos moleculares y clínicos, de tal forma que se puedan identificar las b-lactamasas de microorganismos resistentes en hospitales y hacer un seguimiento del manejo de los antibióticos y el comportamiento epidemiológico, modelo bioinformático permanentemente actualizado, disponible a través de Internet en http://bioinf.ibun.unal.edu.oc/BLA_ID_CLINIC.The epidemiology and pharmacology of the b-lactamic antibiotics resistance at hospital level mediate by the presence of b-lactamasas requires of their precise identification, possible through the gene sequence that can be identified using bioinformatics tools and incorporated into the information systems. Prototype of information system BAL_ID_CLINIC was implemented for allowing genes sequences classification of b-lactamases produced by isolated resistant microorganisms in hospitals and their crossing with the clinical data. The information system was development using Unified Modeling Language UML and the prototype was implemented in an Unix server, using MySQL management system for data base building and Perl and PHP as a programming languages for writing scripts that obtain cross relationship between the different kind of data inside the system and the result report presentation. BLA_ID_CLINIC allows crossing of molecular and clinical data. It can identify b-lactamasas produced by resistant microorganisms isolated in hospital patients and can give information related with the antibiotics management and the epidemiologic behavior. The bioinformatics prototype BLA_ID_CLINIC is permanently updated and available through Internet in http://bioinf.ibun.unal.edu.oc/BLA_ID_CLINIC

Sistema de infromación para consulta y selección de colorantes de uso en medicamentos y cosméticos: sinco

El diseño y desarrollo de formulaciones farmacéuticas está soportado en la selección adecuada y crítica tanto de excipientes como de procesos que le permitan al químico farmacéutico obtener un producto estable, seguro y eficaz. Los auxiliares optativos como los colorantes pueden desempeñar un papel importante en la estabilidad y en la presentación de un producto farmacéutico, pero en ocasiones la información disponible se encuentra dispersa y, en general, no está actualizada, lo que aumenta el tiempo de búsqueda para llevar a cabo una selección adecuada.En el presente trabajo se desarrolló un sistema de información que no sólo recopila información de las propiedades fisicoquímicas de los colorantes aprobados en Colombia, sino que también permite al usuario hacer una selección crítica de los colorantes más apropiados para una formulación farmacéutica, teniendo en cuenta los criterios químicos y físicos que son considerados para obtener un medicamento o cosmético con las propiedades esperadaDesign and development of pharmaceutical formulations is supported by a critical selection of excipients and standard processes that allows Pharmaceutical Chemists to obtain an efficient and safe product. Excipients like colorants are very important in the stability and the final presentation of drugs, but the available information about the colorants used in pharmaceutical preparations is not always updated and not available to the public use, and in general it increases the time for searching and selection. The Information system SINCO was developed for compiling physical and chemical properties data of colorants approved in Colombia that allows the user to make a critical selection of the most compatible colorants for the pharmaceutical formulations

Impact of rice straw management strategies on rice rhizosphere microbiomes

Rice is the third most important crop worldwide. Unfortunately, in most rice-producing countries, crop residues are burned, increasing emissions of greenhouse gases and toxic compounds. Incorporation of rice straw (RS) into the soil, either or not accompanied by a microbial inoculum, may offer a viable alternative. However, the effects of such treatments on soil, including the microbial community structure and function in the next crop cycle, still remain largely unknown. Here, we studied the effect of four different RS management strategies (leaving RS as mulch with or without a microbial inoculum, incorporation of RS into the soil with microbial inoculum, and RS burning) on rice growth and flowering-stage rhizosphere microbiomes. The relevant microbiomes were examined by amplicon sequencing based on the 16S rRNA and ITS1 gene regions. In comparison to the zero situation, all four treatments tended to increase the soil organic carbon content, albeit without significant differences. Furthermore, none of the treatments had major effects on either (rice) crop yield or phytopathogen incidence in the next cycle. However, leaving RS as a mulch incited a decrease in soil pH, and showed a trend of reducing yield by up to 1 ton·ha−1. Moreover, the different RS treatments affected the structures and predicted functions of the bacteriomes and fungomes in the rice rhizosphere. The mulching treatment was associated with an enhanced abundance of Acidobacteria, particularly Bryobacter spp. In contrast, the non-mulch treatments incited raised abundances of GammaProteobacteria, Bacteroidia and Campylobacteria. The rice rhizosphere fungomes, consisting mostly of Ascomycota, were less affected by the treatments, although the microbial inoculum was shown to drive the respective fungome structures

Survey and analysis of microsatellites from transcript sequences in Phytophthora species: frequency, distribution, and potential as markers for the genus

BACKGROUND: Members of the genus Phytophthora are notorious pathogens with world-wide distribution. The most devastating species include P. infestans, P. ramorum and P. sojae. In order to develop molecular methods for routinely characterizing their populations and to gain a better insight into the organization and evolution of their genomes, we used an in silico approach to survey and compare simple sequence repeats (SSRs) in transcript sequences from these three species. We compared the occurrence, relative abundance, relative density and cross-species transferability of the SSRs in these oomycetes. RESULTS: The number of SSRs in oomycetes transcribed sequences is low and long SSRs are rare. The in silico transferability of SSRs among the Phytophthora species was analyzed for all sets generated, and primers were selected on the basis of similarity as possible candidates for transferability to other Phytophthora species. Sequences encoding putative pathogenicity factors from all three Phytophthora species were also surveyed for presence of SSRs. However, no correlation between gene function and SSR abundance was observed. The SSR survey results, and the primer pairs designed for all SSRs from the three species, were deposited in a public database. CONCLUSION: In all cases the most common SSRs were trinucleotide repeat units with low repeat numbers. A proportion (7.5%) of primers could be transferred with 90% similarity between at least two species of Phytophthora. This information represents a valuable source of molecular markers for use in population genetics, genetic mapping and strain fingerprinting studies of oomycetes, and illustrates how genomic databases can be exploited to generate data-mining filters for SSRs before experimental validation

Polyphenol profile by UHPLC-MS/MS, anti-glycation, antioxidant and cytotoxic activities of several samples of propolis from the northeastern semi-arid region of Brazil

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORPropolis has promising biological activities. Propolis samples from the Northeast of Bahia, Brazil – sample A from Ribeira do Pombal and B, from Tucano – were investigated, with new information regarding their biological activities. Objective: This paper55118841893CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIOR407963/2013-8458114/2014-6sem informaçã

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis

Background: In the SOD1G93A mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS. Methods: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1G93A rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset. Results: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1G93A spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1G93A rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %. Conclusions: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.Agencia Nacional de Investigación e Innovació

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS

Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1(G93A) mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1(G93A) rats, numerous cells displayed nuclear p16(INK4a) as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16(INK4a)-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16(INK4a)-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1(G93A) rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1(G93A) microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-beta-Gal) activity as well as positive labeling for p16(INK4a), p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16(INK4a) staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1(G93A) rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS

Gold nanoparticles inhibit steroid-insensitive asthma in mice preserving histone deacetylase 2 and NRF2 pathways

Background: Gold nanoparticles (AuNPs) can inhibit pivotal pathological changes in experimental asthma, but their effect on steroid-insensitive asthma is unclear. The current study assessed the effectiveness of nebulized AuNPs in a murine model of glucocorticoid (GC)-resistant asthma. Methods: A/J mice were sensitized and subjected to intranasal instillations of ovalbumin (OVA) once a week for nine weeks. Two weeks after starting allergen stimulations, mice were subjected to Budesonide or AuNP nebulization 1 h before stimuli. Analyses were carried out 24 h after the last provocation. Results: We found that mice challenged with OVA had airway hyperreactivity, eosinophil, and neutrophil infiltrates in the lung, concomitantly with peribronchiolar fibrosis, mucus production, and pro-inflammatory cytokine generation compared to sham-challenged mice. These changes were inhibited in mice treated with AuNPs, but not Budesonide. In the GCresistant asthmatic mice, oxidative stress was established, marked by a reduction in nuclear factor erythroid 2-related factor 2 (NRF2) levels and catalase activity, accompanied by elevated values of thiobarbituric acid reactive substances (TBARS), phosphoinositide 3-kinases δ (PI3Kδ) expression, as well as a reduction in the nuclear expression of histone deacetylase 2 (HDAC2) in the lung tissue, all of which sensitive to AuNPs but not Budesonide treatment. Conclusion: These findings suggest that AuNPs can improve GC-insensitive asthma by preserving HDAC2 and NRF2