Caracterização molecular de estirpes autóctones de Saccharomyces cerevisiae

Mestrado em MicrobiologiaA relação entre genótipo e fenótipo é altamente complexa. A acumulação de mutações e a adaptação dos microorganismos a diferentes condições ambientais resultam em importante diversidade genética que pode ser detectada como single nucleotide polymorphisms (SNPs), indels (delecções ou inserções), ou mesmo amplificações e alterações do número de cromossomas. A hibridação comparativa do genoma (aCGH, array Comparative Genome Hybridization) é uma técnica molecular de elevada aplicabilidade na identificação de variações polimórficas e na procura de diferenças genéticas que justifiquem variações fenotípicas. O objectivo deste trabalho consistiu em detectar variações genéticas, usando a técnica aCGH, entre estirpes de Saccharomyces cerevisiae isoladas de diferentes ambientes: estirpes autóctones de vinhas e lagares de fermentação de duas localizações geográficas distintas de Portugal, e estirpes clínicas isoladas de indivíduos imuno-deprimidos. Para tal, foram usados microarrays de DNA contendo sondas para todos os genes da levedura S. cerevisiae S288C, que foi usada como estirpe de referência neste estudo. A variabilidade genética entre as estirpes estudadas foi analisada, recorrendo a vários programas como CGH-Miner e SAM. Grupos de genes e variações cromossómicas característicos de determinadas estirpes foram encontrados. Os genes associados à maior variabilidade genética das estirpes estudadas pertencem às categorias funcionais de transportadores, resposta ao stress ou a drogas, factores de transcrição e tradução. Foi também possível identificar um conjunto de genes que poderão ser utilizados para criar uma futura assinatura genética das leveduras vínicas. A técnica de aCGH permitiu uma abordagem global do genoma de leveduras possibilitando diferentes análises de resultados que respondem a diferentes questões. ABSTRACT: The relationship between genotype and phenotype is highly complex. Accumulation of mutations and adaptation of microorganisms to different environmental conditions result in important genetic diversity, which can be detected as single nucleotide polymorphisms (SNPs), indels (deletions and insertions), or even amplifications or chromosome copy number changes. Comparative Genome Hybridization (aCGH) is a powerful molecular tool to identify polymorphic variation of wild type organisms and to unravel genome differences associated to phenotypic variation. In this study, we have used DNA microarrays of the complete orfeome of Saccharomyces cerevisiae S288C, to identify genetic variability present in wild type yeast strains and to correlate such variability with ecological adaptation. For this, yeast strains isolated from 2 different geographical locations in Portugal and also yeast clinical isolates and wine commercial strains were studied. The S. cerevisiae S288C strain, whose genome is completely sequenced, was used as reference for the comparative genomics study. Genome variations was analysed with CGM-Miner and SAM algorithms. Several characteristic gene groups were found within some wild-type strains, as well as unique genome alterations. Transporters, stress and drug response genes, transcription and translation factors were related to these strains’ genetic variability. Part of a wine yeast signature was found and the genes were characterized. Overall, aCGH proved to be an excellent tool to access yeast genome diversity

Histamine stimulates neurogenesis in the rodent subventricular zone

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell-based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic-co-glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine-loaded microparticles facilitated neuronal differentiation of SVZ-GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial-derived vehicles may represent a new tool for brain repair strategies. STEM CELLS 2012; 30:773784Fundacao para a Ciencia e a Tecnologia-Portugal [SFRH/BD/42848/2008, SFRH/BPD/34841/2007, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, PTDC/CTM/099659/2008]; Fundacao Calouste Gulbenkian [96542]info:eu-repo/semantics/publishedVersio

Migration and differentiation of neuronal precursors in the postnatal brain: insights from the subventricular zone and cerebellum

Fundação para a Ciência e a Tecnologia - SFRH/BD/42848/2008, através do Programa MIT_Portugal em Sistemas de Bioengenharia; projectos PTDC/SAUNEU/104415/2008 e Projecto ref. 96542 da Fundação Caloust Gulbenkia

Comparative genomics of yeast strains isolated from diverse ecological niches unveils important genome diversity

Background: Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate. Results: In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates. Conclusion: We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in subtelomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome.The authors wish to thank Adega Cooperativa da Bairrada, Cantanhede, Portugal, for providing the commercial strains. The clinical strains were a kind gift of Prof. Mick Tuite from the University of Kent-UK. This work was funded by Fundação para a Ciência e Tecnologia through projects FEDER/FCT, POCI/AGR/56102/2004 and CONC-REEQ/737/2001