Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

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.

Immersiveness of ubiquitous computing environments prototypes: A case study

The development of ubiquitous computing (ubicomp) environments raises several challenges in terms of their evaluation. Ubicomp virtual reality prototyping tools enable users to experience the system to be developed and are of great help to face those challenges, as they support developers in assessing the consequences of a design decision in the early phases of development. Given the situated nature of ubicomp environments, a particular issue to consider is the level of realism provided by the prototypes. This work presents a case study where two ubicomp prototypes, featuring different levels of immersion (desktop-based versus CAVE-based), were developed and compared. The goal was to determine the cost/benefits relation of both solutions, which provided better user experience results, and whether or not simpler solutions provide the same user experience results as more elaborate one.

PDMS encasing system for integrated lab-on-chip Ag/AgCl reference electrodes

Poly(dimethylsiloxane) (PDMS) is an organosilicon polymer widely used in the fabrication of microfluidic systems to integrate biochips. In this study, we propose the use of an adapted PDMS mould for the creation of a miniaturized, reusable, reference electrode for in-chip electrochemical measurements. Through its integrated microfluidic system it is possible to replenish internal buffer solutions, unclog critical junctions and treat the electrode’s surface, assuring a long term reuse of the same device. Planar Ag/AgCl reference electrodes were microfabricated over a passivated p-type Silicon Wafer. The PDMS mould, containing an integrated microfluidic system, was fabricated based on patterned SU-8 mould, which includes a lateral horizontal inlet access point. Surface oxidation was used for irreversible permanent bondage between flat surfaces. The final result was planar Ag/AgCl reference electrode with integrated microfluidic that allows for electrochemical analysis in biochips

Dynamic wet etching of silicon through isopropanol alcohol evaporation

In this paper, Isopropanol (IPA) availability during the anisotropic etching of silicon in Potassium Hydroxide (KOH) solutions was investigated. Squares of 8 to 40 m were patterned to (100) oriented silicon wafers through DWL (Direct Writing Laser) photolithography. The wet etching process was performed inside an open HDPE (High Density Polyethylene) flask with ultrasonic agitation. IPA volume and evaporation was studied in a dynamic etching process, and subsequent influence on the silicon etching was inspected. For the tested conditions, evaporation rates for water vapor and IPA were determined as approximately 0.0417 mL/min and 0.175 mL/min, respectively. Results demonstrate that IPA availability, and not concentration, plays an important role in the definition of the final structure. Transversal SEM (Scanning Electron Microscopy) analysis demonstrates a correlation between microloading effects (as a consequence of structure spacing) and the angle formed towards the (100) plane.

Comparação de probabilidades de acontecimentos formulados de forma explícita e implícita

Neste artigo estuda-se a realização de alunos futuros educadores e professores dos primeiros anos de escolaridade na comparação de probabilidades de acontecimentos em diferentes contextos sociais, formulados de forma explícita e implícita, tendo em vista averiguar a influência do nível de explicitação na comparação de probabilidades. Participaram no estudo 51 alunos futuros educadores e professores dos primeiros anos de escolaridade, que responderam a um questionário contendo duas questões, com vários itens envolvendo a comparação de probabilidades de acontecimentos formulados explícita e implicitamente. Em termos de resultados, globalmente, verificou-se que os itens formulados implicitamente se revelaram mais difíceis para os alunos do que os itens formulados explicitamente.

Synthesis of new peptide derivatives that self-assemble into nanostructured hydrogels for biomedical applications

Tese de Doutoramento em Ciências (área de especialização em Química)

Micro- and mesoporous structures as drug delivery carriers for salicylic acid

The potential of salicylic acid (SA) encapsulated in porous materials as drug delivery carriers for cancer treatment was studied. Different porous structures, the microporous zeolite NaY, and the mesoporous SBA-15 and MCM-41 were used as hosts for the anti-inflammatory drug. Characterization with different techniques (FTIR, UV/vis, TGA, 1H NMR, and 13C CPMAS NMR) demonstrated the successful loading of SA into the porous hosts. The mesoporous structures showed to be very efficient to encapsulate the SA molecule. The obtained drug delivery systems (DDS) accommodated 0.74 mmol (341 mg/gZEO) in NaY and 1.07 mmol (493 mg/gZEO) to 1.23 mmol (566 mg/gZEO) for SBA-15 and MCM-41, respectively. Interactions between SA molecules and pore structures were identified. A fast and unrestricted liberation of SA at 10 min of the dissolution assay was achieved with 29.3, 46.6, and 50.1 µg/mL of SA from NaY, SBA-15, and MCM-41, respectively, in the in vitro drug release studies (PBS buffer pH 7.4, 37 °C). Kinetic modeling was used to determine the release patterns of the DDS. The porous structures and DDS were evaluated on Hs578T and MDA-MB-468 breast cancer cell lines viability. The porous structures are nontoxic to cancer cells. Cell viability reduction was only observed after the release of SA from MCM- 41 followed by SBA-15 in both breast cancer cell lines.

Green's relations, regularity and abundancy for semigroups of quasi-onto transformations

Let V be an infinite-dimensional vector space and for every infinite cardinal n such that n≤dimV, let AE(V,n) denote the semigroup of all linear transformations of V whose defect is less than n. In 2009, Mendes-Gonçalves and Sullivan studied the ideal structure of AE(V,n). Here, we consider a similarly-defined semigroup AE(X,q) of transformations defined on an infinite set X. Quite surprisingly, the results obtained for sets differ substantially from the results obtained in the linear setting.

Combined bioremediation and enzyme production by Aspergillus sp. in olive mill and winery wastewaters

Olive mill wastewaters (OMW) and vinasses (VS) are effluents produced respectively by olive mills and wineries, both sectors are of great economic importance in Mediterranean countries. These effluents cause a large environmental impact, when not properly processed, due to their high concentration of phenolic compounds, COD and colour. OMW may be treated by biological processes but, in this case, a dilution is necessary, increasing water consumption. The approach here in proposed consists on the bioremediation of OMW and VS by filamentous fungi. In a screening stage, three fungi (Aspergillus ibericus, Aspergillus uvarum, Aspergillus niger) were selected to bioremediate undiluted OMW, two-fold diluted OMW supplemented with nutrients, and a mixture of OMW and VS in the proportion 1:1 (v/v). Higher reductions of phenolic compounds, colour and COD were achieved mixing both residues; with A. uvarum providing the best results. In addition, the production of enzymes was also evaluated during this bioremediation process, detecting in all cases lipolytic, proteolytic and tannase activities. A. ibericus, A. uvarum and A. niger achieved the highest value of lipase (1253.7 ± 161.2 U/L), protease (3700 ± 124.3 U/L) and tannase (284.4 ± 12.1 U/L) activities, respectively. Consequently, this process is an interesting alternative to traditional processes to manage these residues, providing simultaneously high economic products, which can be employed in the same industries.

Magnetoliposomes based on manganese ferrite nanoparticles as nanocarriers for antitumor drugs

Publicado em "NanoPT2016 book of abstracts"

Biomimetic superhydrophobic surfaces patterned with wettable spots as implantable chips for in vivo high-content 3D biomaterials response assessment

"Tissue engineering: part A", vol. 21, suppl. 1 (2015)

Fundamental studies on natural deep eutectic solvents: physico-chemical, thermal and rheological properties

When combined at particular molar fractions, sugars, aminoacids or organic acids a present a high melting point depression, becoming liquids at room temperature. These are called Natural Deep Eutectic Solvents – NADES and are envisaged to play a major role on the chemical engineering processes of the future. Nonetheless, there is a significant lack of knowledge of its fundamental and basic properties, which is hindering their industrial applications. For this reason it is important to extend the knowledge on these systems, boosting their application development [1]. In this work, we have developed and characterized NADES based on choline chloride, organic acids, amino acids and sugars. Their density, thermal behavior, conductivity and polarity were assessed for different compositions. The conductivity was measured from 0 to 40 °C and the temperature effect was well described by the Vogel-Fulcher-Tammann equation. The morphological characterization of the crystallizable materials was done by polarized optical microscopy that provided also evidence of homogeneity/phase separation. Additionally, the rheological and thermodynamic properties of the NADES and the effect of water content were also studied. The results show these systems have Newtonian behavior and present significant viscosity decrease with temperature and water content, due to increase on the molecular mobility. The anhydrous systems present viscosities that range from higher than 1000Pa.s at 20°C to less than 1Pa.s at 70°C. DSC characterization confirms that for water content as high as 1:1:1 molar ratio, the mixture retains its single phase behavior. The results obtained demonstrate that the NADES properties can be finely tunned by careful selection of its constituents. NADES present the necessary properties for use as extraction solvents. They can be prepared from inexpensive raw materials and tailored for the selective extraction of target molecules. The data produced in this work is hereafter importance for the selection of the most promising candidates avoiding a time consuming and expensive trial and error phase providing also data for the development of models able to predict their properties and the mechanisms that allow the formation of the deep eutectic mixtures.

Spawning habitat selection by Octopus vulgaris: new insights for a more effective management of this resource

The selection of spawning habitat of a population of Octopus vulgaris that is subject to a small-scale exploitation was studied in the Cíes Islands within the National Park of the Atlantic Islands of Galicia (NW Spain). The technique used was visual censuses by scuba diving. We conducted 93 visual censuses from April 2012 to April 2014. The total swept area was 123.69 ha. Habitat features (season, depth, zone, bottom temperature, swept area, bottom substrate type, and creels fishing impact) were evaluated as predictors of the presence/absence of spawning dens using GAM models. O. vulgaris has a noteworthy preference for spawning in areas with hard bottom substrate and moderate depth (approximately 20 m). The higher density of spawning dens (1.08ha−1) was found in a surveyed area of 50.14ha located in the northeastern part of the northern Cíes Island. We propose to protect the area comprised from Punta Escodelo to Punta Ferreiro between 5 and 30 m depth. This area has a surface of 158 ha equivalent to 5.98% of the total marine area of the Cíes islands. The strengths and weaknesses of a management strategy based on the protection of the species’ spawning habitat are discussed.

Rheological characterization of EVA and HDPE polymer modified bitumens under large deformation at 20 º C

Large amplitude oscillatory shear (LAOS) coupled with Fourier transform rheology (FTR) was used for the first time to characterize the large deformation behavior of selected bituminous binders at 20 C. Two polymer modified bitumens (PMB) containing recycled EVA and HDPE and two unmodified bitumens were tested with LAOS-FTR. The LAOS-FTR response of all binders was compared at same frequency, at same Deborah number (by tuning the frequency to the relaxation time of each binder) and at same phase shift angle d (by tuning the frequency to the one corresponding to d = 50 in the SAOS response of each sample). In all the approaches, LAOS-FTR results allowed to differentiate between all the nonlinear mechanical characteristics of the tested binders. All binders show LAOS-FTR patterns reminiscent from colloidal dispersions and emulsions. EVA PMB was less prone to strain-induced microstructural changes when compared to HDPE PMB which showed larger values of nonlinear FTR parameters for the range of shear strains tested in LAOS.