Aging and Alzheimer's disease: Searching for novel molecular cues

Tese de Doutoramento em Ciências da Saúde

The effects of solvent composition on the affinity of a peptide towards hair keratin: experimental and molecular dynamics data

The study of the interaction between hair filaments and formulations or peptides is of utmost importance in fields like cosmetic research. Keratin intermediate filaments structure is not fully described, limiting the molecular dynamics (MD) studies in this field although its high potential to improve the area. We developed a computational model of a truncated protofibril, simulated its behavior in alcoholic based formulations and with one peptide. The simulations showed a strong interaction between the benzyl alcohol molecules of the formulations and the model, leading to the disorganization of the keratin chains, which regress with the removal of the alcohol molecules. This behavior can explain the increase of peptide uptake in hair shafts evidenced in fluorescence microscopy pictures. The model developed is valid to computationally reproduce the interaction between hair and alcoholic formulations and provide a robust base for new MD studies about hair properties. It is shown that the MD simulations can improve hair cosmetic research, improving the uptake of a compound of interest.

On line pre-concentration for simultaneous determination of low molecular weight organic acids and inorganic anions in Amazonian river water samples employing ion chromatography with conductivity detection

An ion chromatography procedure, employing an IonPac AC15 concentrator column was used to investigate on line preconcentration for the simultaneous determination of inorganic anions and organic acids in river water. Twelve organic acids and nine inorganic anions were separated without any interference from other compounds and carry-over problems between samples. The injection loop was replaced by a Dionex AC15 concentrator column. The proposed procedure employed an auto-sampler that injected 1.5 ml of sample into a KOH mobile phase, generated by an Eluent Generator, at 1.5 mL min-1, which carried the sample to the chromatographic columns (one guard column, model AG-15, and one analytical column, model AS15, with 250 x 4mm i.d.). The gradient elution concentrations consisted of a 10.0 mmol l-1 KOH solution from 0 to 6.5 min, gradually increased to 45.0 mmol l-1 KOH at 21 min., and immediatelly returned and maintained at the initial concentrations until 24 min. of total run. The compounds were eluted and transported to an electro-conductivity detection cell that was attached to an electrochemical detector. The advantage of using concentrator column was the capability of performing routine simultaneous determinations for ions from 0.01 to 1.0 mg l-1 organic acids (acetate, propionic acid, formic acid, butyric acid, glycolic acid, pyruvate, tartaric acid, phthalic acid, methanesulfonic acid, valeric acid, maleic acid, oxalic acid, chlorate and citric acid) and 0.01 to 5.0 mg l-1 inorganic anions (fluoride, chloride, nitrite, nitrate, bromide, sulfate and phosphate), without extensive sample pretreatment and with an analysis time of only 24 minutes.

Influence of climate on clinical diagnostic dry eye tests: pilot study

Purpose. To analyze dry eye disease (DED) tests and their consistency in similar nonsymptomatic population samples living in two geographic locations with different climates (Continental vs. Atlantic). Methods. This is a pilot study including 14 nonsymptomatic residents from Valladolid (Continental climate, Spain) and 14 sex-matched and similarly aged residents from Braga (Atlantic climate, Portugal); they were assessed during the same season (spring) of two consecutive years. Phenol red thread test, conjunctival hyperemia, fluorescein tear breakup time, corneal and conjunctival staining, and Schirmer test were evaluated on three different consecutive visits. Reliability was assessed using the intraclass correlation coefficient and weighted kappa (J) coefficient for quantitative and ordinal variables, respectively. Results. Fourteen subjects were recruited in each city with a mean (TSD) age of 63.0 (T1.7) and 59.1 (T0.9) years (p = 0.08) in Valladolid and Braga, respectively. Intraclass correlation coefficient and J values of the tests performed were below 0.69 and 0.61, respectively, for both samples, thus showing moderate to poor reliability. Subsequently, comparisons were made between the results corresponding to the middle and higher outdoor relative humidity (RH) visit in each location as there were no differences in mean temperature (p Q 0.75) despite RH values significantly differing (p e 0.005). Significant (p e 0.05) differences were observed between Valladolid and Braga samples on tear breakup time (middle RH visit, 2.76 T 0.60 vs. 5.26 T 0.64 seconds; higher RH visit, 2.61 T 0.32 vs. 5.78 T 0.88 seconds) and corneal (middle RH, 0.64 T 0.17 vs. 0.14 T 0.10; higher RH, 0.60 T 0.22 vs. 0.0 T 0.0) and conjunctival staining (middle RH, 0.61 T 0.17 vs. 0.14 T 0.08; higher RH, 0.57 T 0.15 vs. 0.18 T 0.09). Conclusions. This pilot study provides initial evidence to support that DED test outcomes assessing the ocular surface integrity and tear stability are climate dependent. Future large-sample studies should support these outcomes also in DED patients. This knowledge is fundamental for multicenter clinical trials. Lack of consistency in diagnostic clinical tests for DED was also corroborated. (Optom Vis Sci 2015;92:e284Ye289)

A Gß protein and the TupA Co-Regulator bind to protein kinase A Tpk2 to act as antagonistic molecular switches of fungal morphological changes

A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

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.

Widespread environmental contamination with Mycobacterium tuberculosis complex revealed by a molecular detection protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

Clear cell adenocarcinoma of the cervix in a young woman: a diagnostic challenge

Clear cell adenocarcinoma of the cervix is a rare tumor, classically related with in utero diethylstilbestrol (DES) exposure. The authors report a rare case of clear cell adenocarcinoma of the cervix in a 21-yearold woman who had no history of in utero DES exposure, presenting with intermittent vaginal bleeding. It stresses the relevance to always clarify the etiology of abnormal genital bleeding and consider the possibility of cervicovaginal tumors.

The role of liquid-based cytology and ancillary techniques in pleural and pericardic effusions: An institutional experience

BACKGROUND: Fine-needle aspiration cytology (FNAC) of serous membrane effusions may fulfil a challenging role in the diagnostic analysis of both primary and metastatic disease. From this perspective, liquid-based cytology (LBC) represents a feasible and reliable method for empowering the performance of ancillary techniques (ie, immunocytochemistry and molecular testing) with high diagnostic accuracy. METHODS: In total, 3171 LBC pleural and pericardic effusions were appraised between January 2000 and December 2013. They were classified as negative for malignancy (NM), suspicious for malignancy (SM), or positive for malignancy (PM). RESULTS: The cytologic diagnoses included 2721 NM effusions (2505 pleural and 216 pericardic), 104 SM effusions (93 pleural and 11 pericardic), and 346 PM effusions (321 pleural and 25 pericardic). The malignant pleural series included 76 unknown malignancies (36 SM and 40 PM effusions), 174 metastatic lesions (85 SM and 89 PM effusions), 14 lymphomas (3 SM and 11 PM effusions), 16 mesotheliomas (5 SM and 11 SM effusions), and 3 myelomas (all SM effusions). The malignant pericardic category included 20 unknown malignancies (5 SM and 15 PM effusions), 15 metastatic lesions (1 SM and 14 PM effusions), and 1 lymphoma (1 PM effusion). There were 411 conclusive immunocytochemical analyses and 47 molecular analyses, and the authors documented 88% sensitivity, 100% specificity, 98% diagnostic accuracy, 98% negative predictive value, and 100% positive predictive value for FNAC. CONCLUSIONS: FNAC represents a primary diagnostic tool for effusions and a reliable approach with which to determine the correct follow-up. Furthermore, LBC is useful for ancillary techniques, such as immunocytochemistry and molecular analysis, with feasible diagnostic and predictive utility.

Molecular regulation of flowering induction in Quercus suber

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepeneurship

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

Validity of the Brazilian Portuguese version of the bipolar spectrum diagnostic scale

OBJECTIVE: Bipolar spectrum disorders (BSDs) are prevalent and frequently unrecognized and undertreated. This report describes the development and validation of the Brazilian version of the bipolar spectrum diagnostic scale (B-BSDS), a screening instrument for bipolar disorders, in an adult psychiatric population. METHOD: 114 consecutive patients attending an outpatient psychiatric clinic completed the B-BSDS. A research psychiatrist, blind to the B-BSDS scores, interviewed patients by means of a modified version of the mood module of the Structured Clinical Interview for DSM-IV ("gold standard"). Subthreshold bipolar disorders were defined as recurrent hypomania without a major depressive episode or with fewer symptoms than those required for threshold hypomania. RESULTS: The internal consistency of the B-BSDS evaluated with Cronbach's alpha coefficient was 0.89 (95% CI; 0.86-0.91). On the basis of the modified SCID, 70 patients (61.4%) of the sample received a diagnosis of BSDs. A B-BSDS screening score of 16 or more items yielded: sensitivity of 0.79 (95% CI; 0.72-0.85), specificity of 0.77 (95% CI; 0.70-0.83), a positive predictive value of 0.85 (95% CI; 0.78-0.91) and a negative predictive value of 0.70 (95% CI; 0.63-0.75). CONCLUSION: The present data demonstrate that the B-BSDS is a valid instrument for the screening of BSDs.

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas