Blocking tumor exosome release using nanovectorization systems

Exosomes are small membrane vesicles secreted by most cell types, either normal or malignant and are found in most body fluids such as saliva, plasma and breast milk. In the past decade, the interest in these vesicles has been growing more and more since it was found that besides their beneficial functions such as the removal of cellular debris and unnecessary proteins during cell maturation process, they can also interact with other cells and transfer information between them, thus helping diseases like cancer to progress. The present work intended to use gold nanoparticles as vehicles for gene silencing in an attempt to reduce the tumor-derived exosome secretion, regulated by Rab27a protein, and also aimed to compare the exosome secretion between two breast cell lines, MCF7 and MDA. Changes in RAB27A gene expression were measured by Real-time Quantitative PCR and it was revealed a decreased in RAB27A gene expression, as expected. Exosomes were isolated and purified by two different methods, ultracentrifugation and the commercial kit ExoQuick™ Solution, and further characterized using Western Blot analysis. ExoQuick™ Solution was proven to be the most efficient method for exosome isolation and it was revealed that MDA cells secrete more exosomes. Furthermore, the isolated MCF7-derived exosomes were placed together with a normal bronchial/tracheal epithelial cell line (BTEC) for an additional assay, which aimed to observe the uptake of exosomes by other cells and the exosomes’ capability of promoting cell-cell communication. This observation was made based on alterations in the expression levels of c-Myc and miR-21 genes and the fact that they both have an increased expression in BTEC cells incubated with tumor-derived exosomes when compared to control cells (without incubation with the exosomes) lead us to the conclusion that the exosome uptake and exchange of information between the exosomes and the normal cells did occurred.

Analysis of chromium behaviour and speciation during the electrodialytic process

The interest in chromium (Cr) arises from the widespread use of this heavy metal in various industrial processes that cause its release as liquid, solid and gaseous waste into the environment. The impact of Cr on the environment and living organisms primarily depends on its chemical form, since Cr(III) is an essential micronutrient for humans, other animals and plants, and Cr(VI) is highly toxic and a known human carcinogen. This study aimed to evaluate if the electrodialytic process (ED) is an appropriate treatment for Cr removal, through a critical overview of Cr speciation, before and after the ED experiments, to assess possible Cr(III)-Cr(VI) interconversions during the treatment. ED was the treatment technique applied to two types of matrices containing Cr: chromate copper arsenate (CCA) contaminated soil and municipal solid waste incineration (MSWI) fly ash. In order to study Cr remediation, three EDR set-ups were used: a new set-up, the combined cell (2/3C or 3/2C), with three compartments, alternating current between two anodes and different initial experimental conditions, one set-up with three compartments (3C cell) and the other set-up with two compartments (2C cell). The Cr removal rates obtained in this study were between 10-36% for the soil, and 1-13% for the fly ash. The highest Cr removal rates were achieved in the 26 days experiments: 36% for the soil, 13% for the fly ash. Regarding the 13 days experiments, the highest Cr removal rates were attained with the 2/3C set-up: 24% for the soil, 5% for the fly ash. The analysis of Cr(VI) was performed before and after ED experiments to evaluate eventual changes in Cr speciation during the treatment. This analysis was conducted by two methods: USEPA Method 3060A, for the extraction of Cr(VI); and Hach Company Method 8023, for the detection of Cr(VI). Despite the differences in Cr total concentration, both matrices presented a similar speciation, with Cr(III) being the main species found and Cr(VI) less than 3% of Cr total, before and after the treatment. For fly ash, Cr(VI) was initially below the detection limit of the method and remained that way after the treatment. For soil, Cr(VI) decreased after the treatment. Oxidation of Cr(III) to Cr(VI) did not occur during the ED process since there was no increase in Cr(VI) in the matrices after the treatment. Hence, the results of this study indicate that ED is an appropriate technique to remediate matrices containing Cr because it contributes to Cr removal, without causing Cr(III)-Cr(VI) interconversions.

Development of process control strategies exploiting knowledge from systems biology: application to MDCK suspension cells

Madine Darby Canine Kidney (MDCK) cell lines have been extensively evaluated for their potential as host cells for influenza vaccine production. Recent studies allowed the cultivation of these cells in a fully defined medium and in suspension. However, reaching high cell densities in animal cell cultures still remains a challenge. To address this shortcoming, a combined methodology allied with knowledge from systems biology was reported to study the impact of the cell environment on the flux distribution. An optimization of the medium composition was proposed for both a batch and a continuous system in order to reach higher cell densities. To obtain insight into the metabolic activity of these cells, a detailed metabolic model previously developed by Wahl A. et. al was used. The experimental data of four cultivations of MDCK suspension cells, grown under different conditions and used in this work came from the Max Planck Institute, Magdeburg, Germany. Classical metabolic flux analysis (MFA) was used to estimate the intracellular flux distribution of each cultivation and then combined with partial least squares (PLS) method to establish a link between the estimated metabolic state and the cell environment. The validation of the MFA model was made and its consistency checked. The resulted PLS model explained almost 70% of the variance present in the flux distribution. The medium optimization for the continuous system and for the batch system resulted in higher biomass growth rates than the ones obtained experimentally, 0.034 h-1 and 0.030 h-1, respectively, thus reducing in almost 10 hours the duplication time. Additionally, the optimal medium obtained for the continuous system almost did not consider pyruvate. Overall the proposed methodology seems to be effective and both proposed medium optimizations seem to be promising to reach high cell densities.

Primary cilia and the regulation of hedgehog signaling: link to cardiac development

RESUMO: As células eucarióticas evoluíram um sistema de sinalização complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinalização são essenciais para a sobrevivência da célula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transdução de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a proteína Smoothened (Smo) na ausência dos seus ligandos, as proteínas Sonic hedgehog (Shh). Estas proteínas são reguladores fundamentais do desenvolvimento embrionário, como ilustrado pelas malformações drásticas observadas em embriões humanos e de murganho com perturbações da transdução de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malformações ósseas. Igualmente importantes são as consequências da ativação inapropriada da via de sinalização Hh na formação de tumores. Curiosamente, os componentes desta via localizam-se nos cílios primários. Além disso, demonstrou-se que esta localização é crucial para a sinalização através da via Hh. Na presença dos ligandos, Ptch1 é internalizado e destinado a degradação ou sequestrado num compartimento da célula de onde não pode desempenhar o seu papel inibitório. A proteína Arl13b é uma pequena GTPase pertencente à família Arf/Arl da superfamília Ras de pequenas GTPases e foi implicada no síndrome de Joubert, uma ciliopatia caracterizada por ataxia congénita cerebelar, hipotonia, atrso mental e cardiopatia congénita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gestação (E13,5) e exibem anomalias morfológicas nos cílios que levam à interrupção da sinalização Hh. Além disso, a Arl13b está diretamente envolvida na regulação da via Hh, controlando a localização de vários componentes desta via nos cílios primários. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que são estruturas de actina envolvidas em macropinocitose e internalização de recetores, e que regula a sua formação. Além disso, aprofundámos o conhecimento do processo de ativação da via de sinalização Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos após ativação da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir daí, sugerindo uma indução contínua da reorganização do citoesqueleto de actina quando a via está ativada. Observámos ainda que a inibição da formação de CDRs através do silenciamento de WAVE1, uma proteína necessária para a formação destas estruturas, resulta na diminuição da ativação da via de sinalização Hh. Além disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, através do silenciamento de uma proteína necessária para a cisão de macropinossomas, nomeadamente a proteína BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose são necessárias para a ativação da via de sinalização Hh e indicam que esta via de internalização controla os níveis de sinal Hh. Durante o desenvolvimento, as células proliferativas dependem do cílio primário para a transdução de várias vias de sinalização. A via Hh induz a diferenciação do músculo cardíaco. Por conseguinte, os murganhos deficientes na via de sinalização Hh exibem uma variedade de defeitos de lateralidade, incluindo alteração do looping do coração, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investigámos o papel da Arl13b no desenvolvimento do coração. Mostramos que a Arl13b é altamente expressa no coração de embriões de murganho e de murganhos adultos ao nível do mRNA e da proteína. Além disso, o perfil de distribuição da Arl13b no coração segue o dos cílios primários, que são essenciais para o desenvolvimento cardíaco. Corações de murganhos hnn no estadio E12,5 mostram um canal átrio-ventricular aberto, espessamento da camada compacta ventricular e aumento do índice mitótico no ventrículo esquerdo. Além disso, um atraso de 1 a 2 dias no desenvolvimento é observado em corações de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b é necessária para o desenvolvimento embrionário do coração e que defeitos cardíacos podem contribuir para a letalidade embrionária de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regulação dos níveis de superfície do recetor Ptch1, que envolve a remodelação do citoesqueleto de actina e a formação de CDRs após a ativação da via de sinalização Hh. Este mecanismo permite um feedback negativo que evita a repressão excessiva da via através da remoção de Ptch1 da superfície da célula. Além disso, determinou-se que uma mutação de perda de função na Arl13b causa defeitos cardíacos durante o desenvolvimento, possivelmente relacionados com a associação dos defeitos em cílios primários e na sinalização Hh, existentes em murganhos deficientes para Arl13b. A via de sinalização Hh tem tido um papel central entre as vias de sinalização, uma vez que a sua regulação é crucial para o funcionamento apropriada da célula. Assim, a descoberta de um novo mecanismo de tráfego através de macropinocitose e CDRs que controla a ativação e repressão da via de sinalização Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir à identificação de novos alvos e estratégias terapêuticas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.

Predictive value of the ankle-brachial index in the evaluation of intermittent claudication

The purpose of this study was to determine whether the ankle-brachial index (ABI) could be used to predict the prognosis for a patient with intermittent claudication (IC). We studied 611 patients prospectively during 28 months of follow-up. We analyzed the predictive power of using various levels of ABI - 0.30 to 0.70 at 0.05 increments - in terms of the measure's specificity (association with a favorable outcome after exercise rehabilitation therapy) and sensitivity (association with a poor outcome after exercise rehabilitation therapy). We found that using an ABI of 0.30 as a cut-off value produced the lowest margin of error overall, but the predictive power was still low with respect to identifying the patients with a poor prognosis after non-aggressive therapeutic treatment. Further study is needed to perhaps identify a second factor that could increase the sensitivity of the test.

Implementation of membrane processes for improvement of the detection of food contaminants

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities (http://www.em3e.eu)

On the labor share the long-run equilibrium and its adjustment process

This research computes an Equilibrium Labor Share using a VECM for a panel of 19 countries, analyzes what determines the speed at which the labor share adjusts towards that equilibrium and decomposes this adjustment in terms of real wages and employment. Results suggest that the speed at which a country adjusts decreases with employment protection legislation and labor taxes. Most countries’ labor shares adjustment is made through real wages changes instead of changing employment, suggesting that wage moderation policies may play an important role on the adjustment process without harming employment. Keywords: Equilibrium

Sexing the human skull through the mastoid process

OBJECTIVE: The purpose of this study was to evaluate the significance for sex determination of the measurement of the area formed by the xerographic projection of 3 craniometric points related to the mastoid process: the porion, asterion, and mastoidale points. METHOD: Sixty skulls, 30 male and 30 female, were analyzed. A xerographic copy of each side of the skull was obtained. On each xerographic copy, the craniometric points were marked to demarcate a triangle. The area (mm²) of the demarcated triangle for each side of the skull (right (D) and left (E) sides) was determined, and the total value of these measures (T) was calculated. RESULTS: Concerning the right area of the male and female skulls, 60% of the values overlapped; for the left area, 51.67% overlapped, and for the total area, 36.67% overlapped. The analysis of the differences between the sexes in the areas studied was significant for the 3 areas. Regarding the total area, which is the preferred measurement because of the asymmetry between the sides of the skull, the value of the mean was 1505.32 mm² for male skulls, which was greater than the maximum value obtained in the female skulls. The value of the mean for female skulls was 1221.24 mm², less than the minimum value obtained for the male skulls. CONCLUSIONS: This study demonstrates a significant result in the 3 studied areas, (D), (E), and (T). The total area values show less overlapping of values between the sexes, and therefore can be used for sexing human skulls. For the population studied, values of the total area that were greater than or equal to 1447.40 mm² belonged to male crania (95% confidence). Values for this area that were less than or equal to 1260.36 mm² belonged to female crania (95% confidence).

On the labor share the long-run equilibrium and its adjustment process

This research computes an Equilibrium Labor Share using a VECM for a panel of 19 countries, analyzes what determines the speed at which the labor share adjusts towards that equilibrium and decomposes this adjustment in terms of real wages and employment. Results suggest that the speed at which a country adjusts decreases with employment protection legislation and labor taxes. Most countries’ labor shares adjustment is made through real wages changes instead of changing employment, suggesting that wage moderation policies may play an important role on the adjustment process without harming employment.

Analysis of a controversial decision process: the case of the pumped hydro storage power plant Atdorf in Germany

Based on the report for the unit “Métodos Interactivos de Participação e Decisão A” (Interactive methods of participation and decision A), coordinated by Prof. Lia Maldonado Teles de Vasconcelos and Prof. Nuno Miguel Ribeiro Videira Costa. This unit was provided for the PhD Program in Technology Assessment in 2015/2016.

Systemic hypertension, diabetes mellitus, and dyslipidemia in relation to body mass index: evaluation of a Brazilian population

OBJECTIVE: To determine the prevalence of systemic hypertension, diabetes mellitus, hypercholesterolemia, and hypertriglyceridemia in a Brazilian population in relation to body mass index. METHOD: Retrospective evaluation of 1213 adults (mean age: 45.2 ± 12.8; 80.6% females) divided into groups according to body mass index [normal (18.5 - 24.4 kg/m²); overweight (25 - 29.9 kg/m²); grade 1 obesity (30 - 34.9 kg/m²); grade 2 obesity (35 - 39.9 kg/m²), and grade 3 obesity (> 40 kg/m²)]. The prevalence of hypertension, diabetes mellitus, hypercholesterolemia, and hypertriglyceridemia were analyzed in each group. The severity of cardiovascular risk was determined. High-risk patients were considered those reporting 2 or more of the following factors: systemic hypertension, HDL < 35 mg/dL, total cholesterol > 240 mg/dL, triglycerides > 200 mg/dL when HDL < 35 mg/dL, and glycemia > 126 mg/dL. Moderate-risk patients were those reporting 2 or more of the following factors: systemic hypertension, HDL < 45, triglycerides > 200 mg/dL, and total cholesterol > 200 mg/dL. RESULTS: The prevalence of systemic hypertension, diabetes mellitus, hypertriglyceridemia, and low HDL-cholesterol levels increased along with weight, but the prevalence of hypercholesterolemia did not. The odds ratio adjusted for gender and age, according to grade of obesity compared with patients with normal weight were respectively 5.9, 8.6, and 14.8 for systemic hypertension, 3.8, 5.8, and 9.2 for diabetes mellitus and 1.2, 1.3, and 2.6 for hypertriglyceridemia. We also verified that body mass index was positively related to cardiovascular high risk (P < .001) CONCLUSION: In our population, cardiovascular risk increased along with body mass index.

Trading the changes of stock market index composition: Evidence from European markets

This paper studies the changes in European stock market indexes composition from 1995 to 2015. It was found that there are mixed price effects producing abnormal returns around the effective replacement of added and deleted stocks. The price pressure hypothesis seems to hold for added stocks in some indexes but not for deleted stocks as there is not a clear inversion of behaviour after the replacement. Finally, the building and back testing of a trading strategy aiming to capture some of those abnormal returns shows it yields a Sharpe Ratio of 1.4 and generates an annualised alpha of 11%.

The interception process in tropical rain forests: a literature review and critique (*).

A review of the literature on measurements of rain water interception processes by forests is made. Information on Africa, Central and South America, and Asia is given. A general analysis is made and the need to further the measurements under field conditions is stressed.