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

Tese de Doutoramento em Biologia de Plantas

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.

Assessment of liposome disruption to quantify drug delivery in vitro

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

In vitro mechanical fatigue behavior of poly-ε-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and subjected to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behaviour of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow’s criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.

Cinco para ouvir: discursos de ex-reclusos/as sobre a Prisão

Dissertação de mestrado integrado em Psicologia

Estudo de validação do Partner Interaction Questionnaire para fumantes e ex-fumantes

O presente artigo tem por objetivo validar uma versão adaptada do Partner Interaction Questionnaire (PIQ), instrumento que avalia o suporte para deixar de fumar dado pelo parceiro. É apresentada uma versão adaptada do PIQ para fumantes e abstinentes. Participaram no estudo 224 fumantes (52.7% do sexo feminino e 47.3% do sexo masculino; média de idades=28.6 anos; DP=8.69) e 169 ex-fumantes (68.6% do sexo masculino e 31.4% do sexo feminino; média de idades= 43.1 anos; DP=13.02). O instrumento apresentou boa fidelidade (alfa de Cronbach: 0.73) para a subescala positiva e para a subescala negativa (alfa de Cronbach: 0.77). O estudo de validade revelou a presença de dois fatores que explicam 43.36% da variância. O instrumento apresenta boa validade de construto, correlacionando-se positivamente com o ajustamento de casais e a qualidade de vida mental, e negativamente com a depressão. Esta versão adaptada do PIQ reuniu as condições necessárias para a sua aplicação em fumantes e ex-fumantes, podendo ser uma escala útil em programas de intervenção de cessação tabágica e de manutenção da abstinência.

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drugs resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

The Warburg effect in mycobacterial granulomas is dependent on the recruitment and activation of macrophages by interferon-γ

Granulomas are the hallmark of mycobacterial disease. Here, we demonstrate that both the cell recruitment and the increased glucose consumption in granulomatous infiltrates during Mycobacterium avium infection are highly dependent on interferon-y (IFN-y). Mycobacterium avium-infected mice lacking IFN-y signalling failed to developed significant inflammatory infiltrations and lacked the characteristic uptake of the glucose analogue fluorine-18-fluorodeoxyglucose (FDG). To assess the role of macrophages in glucose uptake we infected mice with a selective impairment of IFN-y signalling in the macrophage lineage (MIIG mice). Although only a partial reduction of the granulomatous areas was observed in infected MIIG mice, the insensitivity of macrophages to IFN-y reduced the accumulation of FDG. In vivo, ex vivo and in vitro assays showed that macrophage activated by IFN-y displayed increased rates of glucose uptake and in vitro studies showed also that they had increased lactate production and increased expression of key glycolytic enzymes. Overall, our results show that the activation of macrophages by IFN-y is responsible for the Warburg effect observed in organs infected with M. avium.

Ex situ collection of Limonium spp.: cytogenetic and reproductive characterization for the conservation of rare species

Dissertação mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

O efeito da insulina na placentação: estudos in vitro com células HTR8-SVneo

Dissertação de mestrado em Bioquímica Aplicada

The powerful in vitro bioactivity of Euterpe oleracea Mart. seeds and related phenolic compounds

The Euterpe oleracea Mart. (açaí) is a plant from the Amazon region, classified as "super fruit" because of its various functional properties. However, limited investigation has been performed on açaí by-products, such as seeds. Therefore, the aim of this work was to characterized the phenolic compounds of the aqueous extract of açaí seeds and further evaluate its bioactivity (antioxidant and cytotoxic activities. Only proanthocyanidins were detected, being a B-type (epi)catechin tetramer the most abundant; however, procyanidin trimmers were the most predominant form. Açaí seeds extract revealed a high antioxidant (EC50 ranging from 3.6 to 19.4 μg/mL) and cytotoxic activity, being more effective in the cervical carcinoma cell line (HeLa; GI50 = 18 μg/mL); it did not show toxicity for non-tumor cells. Açaí seeds are considered a waste and could have an added economic benefit, through the extraction of natural antioxidants, particularly proanthocyanidins, that could find applications in food and pharmaceutical industries.

Adipogenic cell sheets to recreate in vitro adipose tissue microenvironments

Cell sheet (CS) engineering, taking advantage of cellular self-matrix organized as in native tissue, has been largely explored, including by us, for different purposes [1â 3]. Herein we propose for the ï¬ rst time, the use of human adipose stem cells (hASCs)-derived CS to create adipose tissue analogues with different levels of maturation. hASCs were cultured on UpCellTM thermo-responsive dishes for 1, 3 and 5 days under basal conditions previously established by us [3]. The inï¬ uence of pre-differentiation time and respective cell number, over CS stability and differentiation was assessed. Mechanically robust CS were only obtained with 5 days pre-differentiation period. Adipogenesis was followed along the culture assessing the variation of expression of mesenchymal (CD73, CD105 but not CD90) and adipogenic (PPARg, FABP4 and LPL) markers by ï¬ ow cytometry, immunocytochemistry and RT-PCR. Increased ratio of differentiated cells was achieved for longer pre-differentiation periods, while maturation degree was modulated by the maintenance medium. Independently of the overall CS differentiation/maturation level, 3D constructs were fabricated by stacking and further culturing 3 CS. Thus, by varying the culture conditions, different 3D adipose tissue-like microenvironments were recreated, enabling future development of new tissue engineering strategies, as well as further study of adipose tissue role in the regeneration of different tissues.

In vitro and in vivo studies of temozolomide loading in zeolite structures as drug delivery systems for glioblastoma

Zeolites Y (faujasite) and MOR (mordonite) were used as hosts for temozolomide (TMZ), a current good-standard chemotherapeutic agent used in the treatment of glioblastoma brain tumors. TMZ was loaded into zeolites by liquid-phase adsorption at controlled pH. FTIR, 1H NMR, MS, SEM, UV/vis and chemical analysis demonstrated the successful loading of TMZ into zeolite hosts. The hydrolysis of TMZ in MTIC (TMZ metabolite) after the preparation of drug delivery systems (DDS) was observed in simulated body fluid. The effect of zeolites and DDS were evaluated on the viability of glioblastoma cell lines. Unloaded Y zeolite presented toxicity to cancer cells in contrast to MOR. In accordance, the best results in potentiation of the TMZ effect was obtained with MOR. We found that mordonite loaded with 0.026 mmol of TMZ was able to decrease the half maximal inhibitory concentrations (IC50) at least 3-fold in comparison to free temozolomide both in vitro and in vivo.

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for Monocarboxylate Transporters as metabolic targets for therapy.

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia-inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors.

Avaliação da política de incentivo à leitura Ex-'Porte-Pago' - Relatório final

(Excerto) O futuro do apoio à imprensa, em geral, e à imprensa regional, em particular, tem sido objecto de profunda discussão. Para uns, a imprensa é um pilar estrutural e estruturante dos sistemas democráticos e, nesse quadro, os estados têm responsabilidades no sentido de garantir a pluralidade de vozes e a diversidade de olhares. Para outros, estes mecanismos de apoio promovem o imobilismo e distanciam os jornais da realidade objectiva dos mercados e dos interesses dos leitores. Qualquer que venha a ser a política futura nesta matéria, em Portugal, é notório que a imprensa regional é uma área com reconhecida importância social e à qual o Estado tem dado atenção: produzindo legislação específica, implementando decisões e despendendo, no processo, verbas do erário público.