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The results of a survey of 28 over-the-counter tablet preparations of isoflavones extracted from soy and 2 with black cohosh that are available in Portuguese pharmacies are presented. All were remedies for hot flushes and post-menopause related problems. Their isoflavone content was analysed by HPLC/DAD and LC/MS-MS. The results show that the isoflavonoids in soy extracts sold by the pharmaceutical companies in Portugal are mainly daidzin, glycitin and genistin.

In cardiac tissue two mitochondria subpopulations, the subsarcolemmal and the intermyofibrillar mitochondria, present different functional emphasis, although limited information exists about the underlying molecular mechanisms. Our study evidenced higher OXPHOS activity of intermyofibrillar compared to subsarcolemmal mitochondria, paralleled by distinct membrane proteins susceptibility to oxidative damage and not to quantitative differences of OXPHOS composition. Indeed, subsarcolemmal subunits of respiratory chain complexes were more prone to carbonylation while intermyofibrillar mitochondria were more susceptible to nitration. Among membrane protein targets to posttranslational modifications, ATP synthase subunits alpha and beta were notoriously more carbonylated in both subpopulations, although more intensely in subsarcolemmal mitochondria. Our data highlight a localization dependence of cardiac mitochondria OXPHOS activity and susceptibility to posttranslational modifications.

The product-ion spectra of the sodiated molecules of glycerol phosphatidylcholine phospholipids (GPC) were obtained, using fast-atom bombardment (FAB) as the ionization method, in a tandem mass spectrometer. The product-ion spectra of these sodiated molecules of GPCs were found to differ significantly from those of the protonated GPC molecules. This difference is due to the absence of the ion of m/z 184 (protonated-phosphocholine moiety) and to the presence of an ion resulting from the loss of trimethylamine (m = 59 Da) from the polar head group, which is the dominant fragmentation. This characteristic neutral loss provides a means of identification of this class of phospholipids and of differentiation from other phospholipid classes in complex mixtures by performing a constant-neutral-loss scan of 59.

With the completion of the genome sequence of Drosophila melanogaster the importance of constructing a proteome map is to be considered. Therefore, with the application of recent advances in proteomic analysis approaches, a protein map of D. melanogaster larvae hemolymph proteins was obtained using 2-DE in the range of pH 3-10. After Coomassie colloidal detection of 289 spots, a total of 105 were excised from the gel and digested with trypsin. Identification was done based on a combination of MALDI-TOF/TOF MS and MS/MS spectra. The 99 proteins identified using this approach include a large number of metabolic enzymes, translational apparatus components, and structural proteins. Among these we emphasize the identification of proteins with molecular chaperone properties (heat shock proteins and PPIases) and protein spots involved in defense responses such as antioxidant and immunological defense mechanisms (thioredoxin, prophenoloxidase, and serine proteases), as well as in signal transduction pathways.

Caries onset and progression is influenced by diverse bacterial, dietary, environmental, socioeconomic and physiological risk factors. The most significant markers include caries experience, the concentrations of mutans-group streptococci and lactobacilli, as well as protective factors, such as the buffering capacity of saliva. The aim of this study was to evaluate and compare caries risk factors. The salivary parameters determined include: buffer capacity, cariogenic bacteria counts, pH, flow rate and total protein content. Individual aspects including hygiene and diet habits were also considered. Results showed that the value corresponding to the difference of stimulated saliva pH and resting saliva pH is positively correlated with DMFT index. Lactobacilli were positively correlated with mutans streptococci, as previously described for caries diagnostics. However, the results of this study were inconclusive, showing that salivary tests parameters either as single test or even in combination are incapable of predicting caries emphasising the importance of saliva composition.

At the present, the term “glycoxidation” is recognized as the synergistic interaction between glycation and oxidative processes which, with the help of redox-active metals, consequently leads to the production of deleterious tissue modifications. The association between glycation and oxidation events is considered one of the major factors in the accumulation of non-functional damaged proteins, enhancing the oxidative damage at the cellular level. Because of the central role of insulin in the biology of diabetes, we investigated the site-specific oxidation of native and glycated insulin (mono, di, and tri-glycated forms), through metal-catalyzed oxidation, with a combination of liquid chromatography and mass spectrometry. With this approach we were able to identify the residues that were mainly oxidized, and peptide sequences resulting from oxidative cleavage of insulin. Tyrosine, phenylalanine, and cysteine were the main affected residues. Time-course analysis (0–48 h) of the oxidative damage enabled to detect more pronounced and earlier oxidative modifications in the case of glycated insulin. We also observed more severe oxidative damage as the number of glycation sites increased in insulin. These oxidative modifications included other oxidized residues, namely proline, histidine, valine, leucine, and glycine, which were shown to be carbonylated. In addition, we identified new sites of peptide cleavage with the formation of new fragments, derived mainly from chain B, which were both glycated and oxidatively modified. Peptide fragmentation occurred mainly between the residues phenylalanine, glycine, leucine, and tyrosine. Moreover, for diglycated and triglycated forms we observed further oxidative cleavage occurring in both chains, with oxidation and fragmentation of residues occurring near cysteine bridges, especially in chain A.

Dissertação de Mestrado em Engenharia Biomédica

The objective of this thesis is to develop an algorithm that co-registers, in a single volume, whole-body Human PET/CT Scans taken at different instants in time, to facilitate the comparison of studies in situations where the patient had to multiple PET/CT exams. The idea is to isolate, from the initials CT volumes, the rigid body parts that can be transformed with a rigid transformation – the bone structures. Bone structures were obtained with segmentation (the process of dividing a volume into subsections, here based on intensity values) and a further labeling, taking into account the connectivity between neighboring voxels. Once the bone structures were labeled in both volumes, the next step was the establishment of its correspondences between the regions in the two volumes. This was based on the number of voxels associated with each region (size of region) and with the Euclidian distance between the centers of mass of regions from different volumes. Once this correspondence was achieved, the rigid transformation model is constructed and applied to each regions of the volume, which was defined as the Target volume. The results of the developed method are still far from those required for clinical applications. We hope that, with future developments of the presented algorithm, will lead to the improvement of medical image analysis in this field