A Carvalho was an MSc student of University of Trás-os-Montes and Alto Douro in collaboration with Hospitalar Centre of Trás-os-Montes and Alto Douro. She has a degree in Genetics and Biotechnology and an MSc in Molecular Genetics, both from the University of Trás-os-Montes and Alto Douro. She published 1 paper in a European conference proceeding with scientific refereeing and also presented oral and panel communications in other conferences.
Studies showed that about 20% of all recognized clinical pregnancies end in a spontaneous abortion mainly in the first semester. Risk factors associated with the occurrence of a sudden miscarriage have been established and genetic factors are the most prevalent; as a problem that affects innumerable couples, it is important to increase the quality of prognosis and diagnosis. Genomics, bioinformatics and proteomics techniques can be used as powerful tools to provide an integrated molecular analysis of genotypic and phenotypic factors potentially related to sudden miscarriage. A genomic phase of investigation was based on the study of intronic, exonic and untranslated regions of certain genes using PCR amplification followed by sequencing and in silico analysis. The genes of interest are involved in angiogenesis and apoptosis processes. The proteomic phase was divided into three stages. First, protein extraction using the SDS-PAGE technique was optimized. Second, proteins were separated using the SDS-PAGE×IEF technique. Finally, spots extracted from the electrophoresis gels were identified by a MALDI-TOF/MS technique. Some of spontaneous abortions samples had a normal karyotype, wherein the most prevalent alterations found were single nucleotide polymorphisms (SNPs). Using the software Human Splice Finder, it was estimated that 75% and 23% of these differences were in intronic and exonic regions respectively. Considering the exonic regions, 54% of the amino acid substitutions encoded by SNPs could lead to an alteration in the function of some protein domains and or cause damage to some protein structures according to ProtFun 2.2 and PolyPhen-2 software predictions. In the MALDI-TOF/MS analysis, it was possible to identify 23 different proteins related to glycolysis, regulation of the cell cycle, transcription and angiogenic mechanisms and stress responses. HSP 70 and 90 were also identified. From these results we can hypothesize the presence of an undetected bacterial infection. Anti-HSPs produced in excess may have affected fetal growth as the balance between HSPs and their respective antibodies is essential for healthy embryonic development.
P Magalhães has completed his degree in Genetics and Biotechnology and MSc Biotechnology for Health Sciences, in the University of Trás-os-Montes and Alto Douro. In his Master thesis, he cooperated and worked in Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro and Bioscope Group, University of Nova de Lisboa, working on Proteomics and Mass Spectrometry. He has 3 poster presentations and 1 oral communication in national and international conferences.
Microbial resistance to antibiotics is a worldwide problem in human and veterinary medicine. Escherichia coli are commensal microorganism of the gastrointestinal tract of animals and humans. The knowledge about the transfer of genes and specific features of mechanisms such as horizontal gene transfer allows us to understand the acquisition of resistance mechanisms in different organisms. The presence of pMdT1 plasmid containing a gene that encodes a variant of the AAC (6\')-lb-cr protein which confers resistance to kanamycin and tobramycin and decreases the susceptibility to ciprofloxacin and norfloxacin has great importance in the study of antibiotic resistance. Two E. coli samples were analyzed. Electromax DH10B is a transformation-ready strain and TF-Se20 is a strain that contains the pMdT1 plasmid expressing the acetylase gene. After extraction, fractionation and quantification, proteins were separated by one dimensional and after by two-dimensional electrophoresis and subsequent analysis by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS). To determine the sequence of the protein of interest, liquid chromatography-mass spectrometry was performed (LC-MS).From gels containing TF-Se20 strain it was possible to identify 76 distinct proteins, 71 of which had a known function and from Electromax DH10B strain 72 different proteins were identified of which 71 being associated with a biological process. Proteins identified were related to biological processes such as glycolysis and biosynthesis of proteins. Aminoglycoside N (6’)-acetyltransferase type 1 was sequenced.The application of proteomics helped to clarify and to obtain more information about the mechanisms of resistance to determine the total proteome of two samples and analyzing the sequence of acetylase mediated by pMdT1 plasmid.