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

Genetic algorithms for the non emergency patients transport service

The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the paper. The authors would like to thank Dr. Elaine DeBock for reviewing the manuscript.

Improvement of in silico strain engineering methods in Saccharomyces cerevisiae

PhD thesis in Bioengineering

Combining analytical methodologies for the identification of Aspergillus section Flavi strains isolated from Brazilian agricultural commodities

The use of chemical analysis of microbial components, including proteins, became an important achievement in the 80’s of the last century to the microbial identification. This led a more objective microbial identification scheme, called chemotaxonomy, and the analytical tools used in the field are mainly 1D/2D gel electrophoresis, spectrophotometry, high-performance liquid chromatography, gas chromatography, and combined gas chromatography-mass spectrometry. The Edman degradation reaction was also applied to peptides sequence giving important insights to the microbial identification. The rapid development of these techniques, in association with knowledge generated by DNA sequencing and phylogeny based on rRNA gene and housekeeping genes sequences, boosted the microbial identification to an unparalleled scale. The recent results of mass spectrometry (MS), like Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF), for rapid and reliable microbial identification showed considerable promise. In addition, the technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with other biological techniques. At present, MALDI-TOF MS adds an additional step for polyphasic identification which is essential when there is a paucity of characters or high DNA homologies for delimiting very close related species. The full impact of this approach is now being appreciated when more diverse species are studied in detail and successfully identified. However, even with the best polyphasic system, identification of some taxa remains time-consuming and determining what represents a species remains subjective. The possibilities opened with new and even more robust mass spectrometers combined with sound and reliable databases allow not only the microbial identification based on the proteome fingerprinting but also include de novo specific proteins sequencing as additional step. These approaches are pushing the boundaries in the microbial identification field.

Public service culture collections of microorganisms: are they important for biotechnology?

Microbiology as a scientific discipline recognised the need to preserve microorganisms for scientific studies establishing from its very beginning research culture collections (CC). Later on, to better serve different scientific fields and bioindustries with the increasing number of strains of scientific, medical, ecological and biotechnological importance public service CC were established with the specific aims to support their user communities. Currently, the more developed public service CC are recognised as microBiological Resources Centres (mBRC). mBRC are considered to be one of the key elements for sustainable international scientific infrastructure, which is necessary to underpin successful delivery of the benefits of biotechnology, whether within the health sector, the industrial sector or other sectors, and in turn ensure that these advances help drive economic growth. In more detail, mBRCs are defined by Organisation for Economic Co-operation and Development (OECD) as service providers and repositories of the living cells, genomes of organisms, and information relating to heredity and functions of biological systems. mBRCs contain collections of culturable organisms (e.g., microorganisms, plant, animal cells), replicable parts of these (e.g. genomes, plasmids, virus, cDNAs), viable but not yet culturable organisms, cells and tissues, as well as database containing molecular, physiological and structural information relevant to these collections and related bioinformatics. Thus mBRCs are fundamental to harnessing and preserving the world’s microbial biodiversity and genetic resources and serve as an essential element of the infrastructure for research and development. mBRCs serve a multitude of functions and assume a range of shapes and forms. Some are large national centres performing a comprehensive role providing access to diverse organisms. Other centres play much narrower, yet important, roles supplying limited but crucial specialised resources. In the era of the knowledge-based bio-economy mBRCs are recognised as vital element to underpinning the biotechnology.

Use of a polyphasic approach including MALDI-TOF MS for identification of Aspergillus section Flavi strains isolated from food commodities in Brazil

Brazil is one the largest producers and exporters of food commodities in the world. The evaluation of fungi capable of spoilage and the production mycotoxins in these commodities is an important issue that can be of help in bioeconomic development. The present work aimed to identify fungi of the genus Aspergillus section Flavi isolated from different food commodities in Brazil. Thirty-five fungal isolates belonging to the section Flavi were identified and characterised. Different classic phenotypic and genotypic methodologies were used, as well as a novel approach based on proteomic profiles produced by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Type or reference strains for each taxonomic group were included in this study. Three isolates that presented discordant identification patterns were further analysed using the internal transcribed spacer (ITS) region and calmodulin gene sequences. The data obtained from the phenotypic and spectral analyses divide the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus, and Aspergillus tamarii. Final polyphasic fungal identification was achieved by joining data from molecular analyses, classical morphology, and biochemical and proteomic profiles generated by MALDI-TOF MS.

PTX3-based genetic testing for risk of aspergillosis after lung transplant

PTX3-based genetic testing for risk of aspergillosis after lung transplant

Exploring female mice interstrain differences relevant for models of depression

Depression is an extremely heterogeneous disorder. Diverse molecular mechanisms have been suggested to underlie its etiology. To understand the molecular mechanisms responsible for this complex disorder, researchers have been using animal models extensively, namely mice from various genetic backgrounds and harboring distinct genetic modifications. The use of numerous mouse models has contributed to enrich our knowledge on depression. However, accumulating data also revealed that the intrinsic characteristics of each mouse strain might influence the experimental outcomes, which may justify some conflicting evidence reported in the literature. To further understand the impact of the genetic background, we performed a multimodal comparative study encompassing the most relevant parameters commonly addressed in depression, in three of the most widely used mouse strains: Balb/c, C57BL/6, and CD-1. Moreover, female mice were selected for this study taken into account the higher prevalence of depression in women and the fewer animal studies using this gender. Our results show that Balb/c mice have a more pronounced anxious-like behavior than CD-1 and C57BL/6 mice, whereas C57BL/6 animals present the strongest depressive-like trait. Furthermore, C57BL/6 mice display the highest rate of proliferating cells and brain-derived neurotrophic factor (Bdnf) expression levels in the hippocampus, while hippocampal dentate granular neurons of Balb/c mice show smaller dendritic lengths and fewer ramifications. Of notice, the expression levels of inducible nitric oxide synthase (iNos) predict 39.5% of the depressive-like behavior index, which suggests a key role of hippocampal iNOS in depression. Overall, this study reveals important interstrain differences in several behavioral dimensions and molecular and cellular parameters that should be considered when preparing and analyzing experiments addressing depression using mouse models. It further contributes to the literature by revealing the predictive value of hippocampal iNos expression levels in depressive-like behavior, irrespectively of the mouse strain.

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae

To be or not to be a pseudogene: a molecular epidemiological approach to the mclx genes and its impact in tuberculosis

Tuberculosis presents a myriad of symptoms, progression routes and propagation patterns not yet fully understood. Whereas for a long time research has focused solely on the patient immunity and overall susceptibility, it is nowadays widely accepted that the genetic diversity of its causative agent, Mycobacterium tuberculosis, plays a key role in this dynamic. This study focuses on a particular family of genes, the mclxs (Mycobacterium cyclase/LuxR-like genes), which codify for a particular and nearly mycobacterial-exclusive combination of protein domains. mclxs genes were found to be pseudogenized by frameshift-causing insertion(s)/deletion(s) in a considerable number of M. tuberculosis complex strains and clinical isolates. To discern the functional implications of the pseudogenization, we have analysed the pattern of frameshift-causing mutations in a group of M. tuberculosis isolates while taking into account their microbial-, patient- and disease-related traits. Our logistic regression-based analyses have revealed disparate effects associated with the transcriptional inactivation of two mclx genes. In fact, mclx2 (Rv1358) pseudogenization appears to be primarily driven by the microbial phylogenetic background, being mainly related to the Euro-American (EAm) lineage; on the other hand, mclx3 (Rv2488c) presents a higher tendency for pseudogenization among isolates from patients born on the Western Pacific area, and from isolates causing extra-pulmonary infections. These results contribute to the overall knowledge on the biology of M. tuberculosis infection, whereas at the same time launch the necessary basis for the functional assessment of these so far overlooked genes.

Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Polyphasic approach including MALDI-TOF MS/MS analysis for identification and characterisation of Fusarium verticillioides in Brazilian corn kernels

Fusarium verticillioides is considered one of the most important global sources of fumonisin contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG)5 and (GACA)4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol) and B2 (705.83 g/mol)

A genetic perspective on African prehistory

The various genetic systems (mitochondrial DNA, the Y-chromosome and the genome-wide autosomes) indicate that Africa is the most genetically diverse continent in the world and the most likely place of origin for anatomically modern humans. However, where in Africa modern humans arose and how the current genetic makeup within the continent was shaped is still open to debate. Here, we summarize the debate and focus especially on the maternally inherited mitochondrial DNA (mtDNA) and a recently revised chronology for the African mtDNA tree. We discuss the possible origin of modern humans in southern, eastern or Central Africa; the possibility of a migration from southern to eastern Africa more than 100 ka, carrying lineages within mtDNA haplogroup L0; the evidence for a climate-change-mediated population expansion in eastern Africa involving mtDNA haplogroup L3, leading to the “out-of-Africa” migration around 70–60 ka; the re-population of North Africa from the Near East around 40–30 ka suggested by mtDNA haplogroups U6 and M1; the evidence for population expansions and dispersals across the continent at the onset of the Holocene ; and the impact of the Bantu dispersals in Central, eastern and southern Africa within the last few millennia.