Desenvolvimento de pastilha potencialmente probiótica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência da espessura do substrato dental sobre a eficácia clareadora e citotoxicidade de diferentes protocolos de clareamento

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Exploring bioactive properties of marine cyanobacteria Isolated from the Portuguese coast: high potential as a source of anticancer compounds

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.

Enoxacin inhibits growth of prostate cancer cells and effectively restores microRNA processing

Prostate cancer (PCa) is one of the most incident malignancies worldwide. Although efficient therapy is available for early-stage PCa, treatment of advanced disease is mainly ineffective and remains a clinical challenge. microRNA (miRNA) dysregulation is associated with PCa development and progression. In fact, several studies have reported a widespread downregulation of miRNAs in PCa, which highlights the importance of studying compounds capable of restoring the global miRNA expression. The main aim of this study was to define the usefulness of enoxacin as an anti-tumoral agent in PCa, due to its ability to induce miRNA biogenesis in a TRBP-mediated manner. Using a panel of five PCa cell lines, we observed that all of them were wild type for the TARBP2 gene and expressed TRBP protein. Furthermore, primary prostate carcinomas displayed normal levels of TRBP protein. Remarkably, enoxacin was able to decrease cell viability, induce apoptosis, cause cell cycle arrest, and inhibit the invasiveness of cell lines. Enoxacin was also effective in restoring the global expression of miRNAs. This study is the first to show that PCa cells are highly responsive to the anti-tumoral effects of enoxacin. Therefore, enoxacin constitutes a promising therapeutic agent for PCa.

Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cells

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-β2, by quantitative methylation-specific PCR and RT-PCR, respectively. Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-β2 promoter hypermethylation levels, although mRNA re-expression was only attained GSTP1 and APC. Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.

Evaluation of the role of glutathione in the lead-induced toxicity in saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

Localization studies of the FemXAB protein family in Staphylococcus aureus

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

Bisphenol A at the reference level counteracts doxorubicin transcriptional effects on cancer related genes in HT29 cells

Human exposure to Bisphenol A (BPA) results mainly from ingestion of food and beverages. Information regarding BPA effects on colon cancer, one of the major causes of death in developed countries, is still scarce. Likewise, little is known about BPA drug interactions although its potential role in doxorubicin (DOX) chemoresistance has been suggested. This study aims to assess potential interactions between BPA and DOX on HT29 colon cancer cells. HT29 cell response was evaluated after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis of several cancer-associated genes (c-fos, AURKA, p21, bcl-xl and CLU) shows that BPA exposure induces slight up-regulation exclusively of bcl-xl without affecting cell viability. On the other hand, a sub-therapeutic DOX concentration (40nM) results in highly altered c-fos, bcl-xl, and CLU transcript levels, and this is not affected by co-exposure with BPA. Conversely, DOX at a therapeutic concentration (4μM) results in distinct and very severe transcriptional alterations of c-fos, AURKA, p21 and CLU that are counteracted by co-exposure with BPA resulting in transcript levels similar to those of control. Co-exposure with BPA slightly decreases apoptosis in relation to DOX 4μM alone without affecting DOX-induced loss of cell viability. These results suggest that BPA exposure can influence chemotherapy outcomes and therefore emphasize the necessity of a better understanding of BPA interactions with chemotherapeutic agents in the context of risk assessment.

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

Abstract Background: Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility. Results: A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation. Conclusions: Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

MicroRNA-375 plays a dual role in prostate carcinogenesis

Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, as well as with regional lymph nodes metastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.

Heat stress adaptation in hyperthermophiles: bosynthesis of inositol-containing compatible solutes

Dissertation presented to obtain the Ph.D. degree in Biochemistry