Dinâmica populacional de leveduras na fermentação de mostos vínicos

Mestrado em Biologia Molecular e CelularEm ecossistemas microbianos complexos, com diferentes espécies e estirpes, há interacções intra- e interespecíficas que influenciam a estrutura das comunidades. Este é o cenário que se pode encontrar na fermentação de mostos de uva, que são um nicho ecológico complexo onde existem leveduras, fungos filamentosos e bactérias. De facto, a fermentação de mostos vínicos é um processo bioquímico complexo, onde a fermentação alcoólica, levada a cabo pelas leveduras, é a reacção mais importante. Este trabalho teve como objectivo a identificação das leveduras selvagens da região da Bairrada, e a monitorização das leveduras presentes ao longo do processo de fermentação. Pretendeu-se, ainda, caracterizar as leveduras associadas às diversas castas, especialmente à casta Baga. Procedeu-se ao isolamento de leveduras, de mosto inicial, do início da fermentação e do final de fermentação, de mostos de uvas de várias castas, nomeadamente Maria Gomes, Bical, Tinta Roriz, Touriga Nacional, Baga, e também de lagares. Posteriormente identificaram-se os isolados com técnicas de PCR por amplificação específica das regiões ITS, por RFLP do gene MET2, e análise das regiões inter-δ. As espécies que predominaram no mosto inicial e início de fermentação foram H. uvarum, I. terricola e M. pulcherrima. Relativamente ao final da fermentação, a grande maioria dos isolados foram identificados como sendo S. cerevisiae e H. uvarum. Quanto à análise dos perfis de bandas inter-δ foram obtidos 182 perfis diferentes em fermentações espontâneas e 150 em fermentações de lagares, sugerindo a existência de elevada variabilidade natural de S. cerevisiae. ABSTRACT: In complex microbial ecosystems there is intra- and interspecific interactions which influence the structure of the microbial community. This complexity is found in wine must fermentations where yeasts, filamentous fungi and bacteria are present. Indeed, the fermentation of wine musts is a complex biochemical process where the alcoholic fermentation, carried out by yeasts, is the most important reaction. The aims of this study were to identify and characterize the wild wine yeasts of the Bairrada region, to monitor the yeasts present throughout the fermentation process in both laboratorial and cellar fermentations, and to characterize the yeasts associated with the grapes Maria Gomes, Bical, Touriga Nacional, Tinta Roriz and especially Baga. For this, yeasts were isolated from several fermentation stages, namely from initial must, from the beginning of fermentation and from fermentation end points. The isolated yeasts where characterized by PCR through amplification of the ITS region, by RFLP of the MET2 gene, and by amplification of inter-δ regions. The dominant species in the initial must and beginning of fermentation were H. uvarum, I. terricola e M. pulcherrima, while S.cerevisiae dominated at the end of fermentation. As for the analysis of the inter-δ profiles, 182 different profiles were obtained in spontaneous fermentations and 150 in cellar fermentations, thus indicating significant natural diversity of S. cerevisiae strains

Estudo do stress proteotóxico no peixe zebra

Doutoramento em BiologiaA fidelidade da síntese proteica é fundamental para a estabilidade do proteoma e para a homeostasia celular. Em condições fisiológicas normais as células têm uma taxa de erro basal associada e esta muitas vezes aumenta com o envelhecimento e doença. Problemas na síntese das proteínas estão associados a várias doenças humanas e aos processos de envelhecimento. De facto, a incorporação de erros nas proteínas devido a tRNAs carregados pelas aminoacil-tRNA sintetases com o amino ácido errado causa doenças neurodegenerativas em humanos e ratos. Ainda não é claro como é que estas doenças se desenvolvem e se são uma consequência directa da disrupção do proteoma ou se são o resultado da toxicidade produzida pela acúmulação de proteínas mal traduzidas ao nível do ribossoma. Para elucidar como é que as células eucarióticas lidam com proteínas aberrantes e agregados proteicos (stress proteotóxico) desenvolvemos uma estratégia para destabilizar o proteoma. Para isso estabelecemos um sistema de erros de tradução em embriões de peixe zebra que assenta em tRNAs mutantes capazes de incorporar erradamente serina nas proteínas. As proteínas produzidas neste sistema despoletam as vias de resposta ao stress, nomeadamente a via da ubiquitina-proteassoma (UPP – “ubiquitin protesome pathway”) e a via do retículo endoplasmático (UPR – “unfolded protein response”). O stress proteotóxico gerado pelos erros de tradução altera a expressão génica e perfis de expressão de miRNAs, o desenvolvimento embrionário e viabilidade, aumenta a produção de espécies reactivas de oxigénio (ROS), leva ainda à acumulação de agregados proteicos e à disfunção mitocondrial. As malformações embrionárias e fenótipos de viabilidade que observámos foram revertidos por antioxidantes, o que sugere que os ROS desempenham papéis importantes nos fenótipos degenerativos celulares induzidos pela produção de proteínas aberrantes e agregação proteica. Estabelecemos ainda uma linha de peixe zebra transgénica para o estudo do stress proteotóxico. Este trabalho mostra que a destabilização do proteoma em embriões de peixe zebra com tRNAs mutantes é uma boa metodologia para estudar a biologia do stress proteotóxico visto que permite a agregação controlada do proteoma, mimetizando os processos de agregação de proteínas que ocorrem naturalmente durante o envelhecimento e em doenças conformacionais humanas.Protein synthesis fidelity is pivotal for proteome stability and cellular homeostasis. Even though normal physiologic conditions have an associated low protein synthesis error rate, this is frequently increased with aging and disease and the resulting protein misfolding is associated with various human diseases and aging processes. Importantly, gene mistranslations produced by tRNA misreading in the ribosome or tRNA mischarging by aminoacyl-tRNA synthetases cause neurodegenerative diseases in humans and mice. It is not yet clear how such diseases develop and whether they are a direct consequence of proteome disruption or a result of the toxicity produced by accumulation of mistranslated proteins. To elucidate how eukaryotic cells cope with protein misfolding and aggregation (proteotoxic stress) we have developed a strategy to destabilize the proteome in a regulated manner. We engineered transfer RNA (tRNAs) to misincorporate serine into proteins in zebrafish embryos. The mutant proteins misfold, trigger the unfolded protein response (UPR), up-regulate the ubiquitin proteasome pathway (UPP) and down-regulate protein synthesis. Proteotoxic stress generated by these gene mistranslations has strong effects on gene expression and miRNA profiles, embryo development and viability, increases the production of reactive oxygen species (ROS), accumulation of protein aggregates and mitochondrial dysfunction. Interestingly, embryo malformations and viability phenotypes could be reversed by ROS scavengers, suggesting that ROS play major roles in the cell degeneration phenotypes induced by protein misfolding and aggregation. We have also established a transgenic zebrafish line for proteotoxic stress studies. Hence, proteome mutagenesis by misreading tRNAs in zebrafish embryos is a powerful methodology to destabilize the proteome to study the biology of proteotoxic stress, it allows for controlled proteome aggregation and mimicking protein aggregation processes that occur naturally during aging and in human conformational diseases

Polymerase III transcription is necessary for T cell priming by dendritic cells

Exposure to microbe-associated molecular patterns (MAMPs) causes dendritic cells (DCs) to undergo a remarkable activation process characterized by changes in key biochemical mechanisms. These enhance antigen processing and presentation, as well as strengthen DC capacity to stimulate naïve T cell proliferation. Here, we show that in response to the MAMPS lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (Poly I:C), RNA polymerase III (Pol lII)-dependent transcription and consequently tRNA gene expression are strongly induced in DCs. This is in part caused by the phosphorylation and nuclear export of MAF1 homolog negative regulator of Poll III (MAF1), via a synergistic casein kinase 2 (CK2)- and mammalian target of rapamycin-dependent signaling cascade downstream of Toll-like receptors (TLRs). De novo tRNA expression is necessary to augment protein synthesis and compensate for tRNA degradation driven by TLR-dependent DC exposure to type-I IFN. Although protein synthesis is not strongly inhibited in absence of RNA Pol III activity, it compromises the translation of key DC mRNAs, like those coding for costimulatory molecules and proinflammatory cytokines, which instead can be stored in stress granules, as shown for CD86 mRNA. TLR-dependent CK2 stimulation and subsequent RNA Pol III activation are therefore key for the acquisition by DCs of their unique T cell immune-stimulatory functions.publishe

Conserved and highly expressed tRNA derived fragments in zebrafish

Background: Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identification by next generation sequencing (NGS) we are still far from understanding their full diversity and functional repertoire. Results: Here we report the identification of tRNA derived fragments (tRFs) by NGS of the sncRNA fraction of zebrafish. The tRFs identified are 18–30 nt long, are derived from specific 5′ and 3′ processing of mature tRNAs and are differentially expressed during development and in differentiated tissues, suggesting that they are likely produced by specific processing rather than random degradation of tRNAs. We further show that a highly expressed tRF (5′tRF-ProCGG) is cleaved in vitro by Dicer and has silencing ability, indicating that it can enter the RNAi pathway. A computational analysis of zebrafish tRFs shows that they are conserved among vertebrates and mining of publicly available datasets reveals that some 5′tRFs are differentially expressed in disease conditions, namely during infection and colorectal cancer. Conclusions: tRFs constitute a class of conserved regulatory RNAs in vertebrates and may be involved in mechanisms of genome regulation and in some diseases. Keywords: tRNA derived fragments, Zebrafish, Small non coding RNAs, tRNAspublishe

Dre-miR-2188 Targets Nrp2a and Mediates Proper Intersegmental Vessel Development in Zebrafish Embryos

BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos

BAD-LAMP controls TLR9 trafficking and signalling in human plasmacytoid dendritic cells

International audienceToll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1+ late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3+/LAMP2+/LAMP1− endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues

SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry

International audienceThe rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied ex vivo. Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction. This article has an associated First Person interview with the first author of the paper

Differences in the expression pattern of HCN isoforms among mammalian tissues: sources and implications

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in a broad range of cell types, but the expression of the various HCN isoforms is still poorly understood. In the present study we have compared the expression of HCN isoforms in rat excitable and non-excitable tissues at both the mRNA and protein levels. Real-time PCR and Western blot analysis revealed distinct expression patterns of the four HCN isoforms in brain, heart, pituitary and kidney, with inconsistent mRNA-protein expression correlation. The HCN2 was the most abundant mRNA transcript (95.6, 78.0 and 59.0 % in kidney heart and pituitary, respectively) except in the brain (42.0 %) whereas HCN4 was the most abundant protein isoform. Our results suggest that HCN channels are mostly produced by the HCN4 isoform in heart, which contrasts with the sharp differences in the isoform stoichiometry in pituitary (15 HCN4:2 HCN2:1 HCN1:1 HCN3), kidney (24 HCN4:2 HCN3:1 HCN2:1 HCN1) and brain (3 HCN4:2 HCN2:1 HCN1:1 HCN3). Moreover, deviations of the electrophoretic molecular weight (MW) of the HCN isoforms relative to the theoretical MW were observed, suggesting that N-glycosylation and enzymatic proteolysis influences HCN channel surface expression. We hypothesize that selective cleavage of HCN channels by membrane bound metalloendopeptidases could account for the multiplicity of properties of native HCN channels in different tissues

MOESM3 of Conserved and highly expressed tRNA derived fragments in zebrafish

Additional file 3: Table S1. Sequencing data relative to zebrafish tRFs found in publicly available GEO datasets. For each experiment the sequences of conserved tRFs identified and corresponding number of reads are displayed. ID corresponds to the tRF identification; “Query” corresponds to the zebrafish tRF that was used as a template to search for similar molecules in the datasets; “Sequence” corresponds to the sequence found in the datasets that is related to the “query”; “Count” gives the total number of reads retrieved for each “sequence”; “Source tag” is the GEO dataset sample ID number; “description” depicts the type of sample where the tRF was found

MOESM4 of Conserved and highly expressed tRNA derived fragments in zebrafish

Additional file 4: Table S2. Target predictions for 5′tRF-ProCGG. Seed match between nucleotides 2 and 7 were maintained, and up to 6 mismatches in the remaining sequence were allowed. Two of the target genes predicted play roles in embryonic patterning, cartilage and skeletal development, namely Sec23b and Myst3, which correlates with the expression pattern of 5′tRF-ProCGG (high expression in bone)