1H-NMR-based endometabolome profiles of Burkholderia cenocepacia clonal variants retrieved from a cystic fibrosis patient during chronic infection

During cystic fibrosis (CF) chronic lung infections, bacteria of the Burkholderia cepacia complex (Bcc) are exposed for several years to a stressful and changing environment. These environmental challenges results in genetic changes of the initial infecting strain with the consequent diversification of genotypes and phenotypes. The exploitation of functional and comparative genomic approaches has suggested that such diversification is associated with massive metabolic remodeling but these alterations are poorly understood. In the present work, we have explored a high resolution 1H-NMR-based metabolomic approach coupled to multivariate analysis to compare the endometabolome of three Burkholderia cenocepacia clonal variants retrieved from a CF patient from the onset of infection (IST439) until death with cepacia syndrome after 3.5 years (IST4113 and IST4134), to complement former proteomic and transcriptomic analyses. A fourth clonal variant (IST4129) retrieved from the same CF patient when the clinical condition worsened during the last months of life, was also examined since it was found to lack the third replicon. The metabolomic profiles obtained, based on the complete 1H-NMR spectra, highlight the separation of the four clonal variants examined, the most distinct profile corresponding to IST4129. Results indicate a variable content of several amino acids in the different isolates examined and suggest that glycolysis and the glyoxylate shunt are favored in late variants. Moreover, the concentration of two metabolites with demonstrated cellular protective functions against stress, glycine-betaine and trehalose, is different in the different isolates examined. However, no clear correlation could be established between their content and stress tolerance. For example, IST4113, previously found to be the most resistant variant to antimicrobials of different classes, exhibits low levels of trehalose and glycine-betaine but the highest resistance to heat and oxidative stress. Also, IST4129, with a high level of glycine-betaine but lacking the third replicon, previously associated with stress resistance and virulence, exhibits the highest susceptibility to all the stresses tested. Taken together, results from this study provide insights into the metabolic diversification of B. cenocepacia clonal variants during long-term infection of the CF airways

A new methodology for the analysis and validation of clusters and biclusters of genes

Tese de mestrado em Bioinformática, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2006A era da pós-genómica e das tecnologias de larga escala traz consigo a necessidade de desenvolver novos métodos para lidar com grandes quantidades de dados. Para tal, têm sido aplicados algoritmos de clustering e biclustering em bioinformática para descobrir padrões em dados biológicos. A validação dos resultados de clustering e de biclustering é essencial para a sua análise. Esta dissertação propõe uma nova metodologia para validar resultados de clustering e biclustering. A metodologia transpõe conceitos do algoritmo PageRank para ordenação dos termos da Gene Ontology associados a um cluster. O significado biológico de cada conjunto de genes é determinado pelos termos no topo da ordenação. A metodologia de validação foi concretizada numa nova ferramenta, denominada TermRank, e foi avaliada através da caracterização de um conjunto de clusters artificiais. A metodologia foi também utilizada para validar o resultado de um algoritmo de biclustering aplicado a dados reais de um estudo sobre a resposta global de Saccharomyces cerevisiae a um stress químico. A avaliação da ferramenta TermRank mostrou que esta produz caracterizações correctas dos clusters gerados artificialmente e que o algoritmo de biclustering gera biclusters compostos por genes relacionados entre si.The era of post-genomics and high-throughput technologies brings the need for developing new methods to cope with very large amounts of data. Clustering and biclustering algorithms have been used in bioinformatics to discover patterns in biological data. The validation of clustering and biclustering results is essential for their analysis. This dissertation presents a new methodology for validating and characterizing clustering and biclustering results, which uses PageRank concepts to rank Gene Ontology terms. The top ranked terms associated to each set of genes describe their biological interpretation. The validation methodology was implemented in a new tool, designated TermRank, and was evaluated through characterization of a set of artificial clusters. The methodology was also used to validate the output of a biclustering algorithm applied to real data from a study of the global response of Saccharomyces cerevisiae to a chemical stress. The evaluation showed that TermRank produces correct characterizations of the artificially generated clusters and that the biclusters generated by the validated biclustering algorithm are composed of related genes

The mitochondrial prohibitin complex, a context-dependent modulator of longevity, has broad effects in the C. elegans metabolome, suggesting a remodelling of the mitochondrial respiratory chain

Resumen del póster presentado al VI Spanish Worm Meeting, celebrado en Valencia del 9 al 10 de marzo de 2017.The mitochondrial prohibitin (PHB) complex, composed of two proteins, PHB-1 and PHB-2, is a context-dependent modulator of longevity. Specifically, PHB deficiency shortens the lifespan of otherwise wild type worms, while it dramatically extends lifespan under compromised metabolic conditions, as is the case of insulin-receptor daf-2(e1370) mutants. This extremely intriguing phenotype has been linked to alterations in mitochondrial function and in fat metabolism. However, the true function of the PHB complex remains elusive. To understand the impact that PHB depletion has on aging, we undertook mass spectrometry-based metabolomics and lipidomics approaches. These top-down approaches raised different working hypothesis that we are currently exploring. PHB depletion has a major effect on the metabolome, being the effect much more pronounced in wild type than in daf-2 mutant worms. In particularly, it highlighted that PHB depletion in either wild type or daf-2 mutant animal’s has a differential effect in triglycerides composition, in parallel with a diffferential effect on riboflavin, and nicotinate and nicotinamide metabolism. This led us to hypothesize that PHB has a direct or indirect effect on the mitochondrial respiratory chain (MRC), resulting in differential energy energy effciency. Preliminary results by blue-native gel analysis of enriched mitochondrial fractions indicate that PHB has a differential effect on the mitochondrial respiratory chain of wild type and daf-2 mutant animals. Consistently, oxygen consumption rate measurements using a Seahorse XFp Analyzer points to a differential effect of PHB depletion in the different genetic backgrounds during aging. These new findings will be discussed in the scope of the opposing longevity phenotype caused by PHB depletion.Peer reviewe

From the transcriptome to the metabolome: A systems view on the impact of the mitochondrial prohibitin complex during aging

Trabajo presentado en EMBO Workshop "C. elegans development, cell biology and gene expression and European Worm Meeting", celebrado en Barcelona (España) del 13 al 17 de junio de 2018

GSK-3 intestinal activity impacts mitochondrial function and ageing

Resumen del trabajo presentado en EMBO Workshop C. elegans development, cell biology and gene expression and European Worm Meeting, celebrado en Barcelona (España) del 13 al 17 de junio de 2018.Impaired mitochondrial function is a hallmark of ageing and age-related pathologies. The mitochondrial prohibitin complex, composed of PHB-1 and PHB-2, has emerged as a context-dependent modulator of lifespan. While PHB-1 or PHB-2 depletion shortens the lifespan of wild type worms, it dramatically extends lifespan under compromised metabolic conditions, as is the case of daf-2(e1370) mutants. To better understand the function of PHBs in ageing regulation, we performed a phenotype-based RNAi screening for prohibitin regulatory kinases. We identified the Glycogen Synthase Kinase -3 (GSK-3) as a suppressor of the reduced Nile Red phenotype in both phb-2(tm2998) and phb-2(tm2998);daf-2(e1370) mutants. GSK-3 is the worm orthologue of human GSK-3ß, a pleiotropic kinase involved in multiple cellular processes and linked to different diseases such as Alzheimer's, diabetes, cancer or neurodegeneration. Interestingly, GSK-3 depletion decreases the lifespan of wild-type worms and strongly suppresses the long-lived phenotypes of daf-2 and daf-2;phb-2, while it mildly affects phb-2 mutants. We used CRISPR/Cas9 to generate a gsk-3 endogenous gene tagging. GSK-3 is ubiquitously expressed and shows different cellular localization patterns depending on the tissue and prohibitin expression. We combine transcriptomic analysis and metabolic assays to demonstrate a role for GSK-3 as a global metabolic regulator. GSK-3 alters glycogen and fat stores and impacts mitochondrial lipid composition and respiration in a genotype-dependent manner. Moreover, we identify a gsk-3-dependent transcriptional blueprint related to ageing where intestinal specific genes are overrepresented. By SapTrap toolkit, we generated different gsk-3 tissue-specific endogenous knockouts and demonstrate that GSK-3 impact on ageing specifically relies on its intestinal activity

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved