INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index). Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

Communication strategy for Continente online store: improving the Ecommerce experience

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA ��� School of Business and Economics

Ultrastructural alterations of intracellular stages and effects on blood forms of Trypanosoma cruzi induced in vivo by 2-amino-5-(1-methyil-5-nitro-2-imidazolyl)-1,3,4 - Thiadiazole

An electron microscopy study shows that the administration of a single dose (500 mg/kg, p.o.) of 2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1, 3, 4-thiadiazole induces in mice infected with Trypanosoma cruzi results in degenerative lesions of the intracellular stages. Ultrastructural alterations are detected as early as 6 hours after the drug administration and destruction of the parasites occurs within 18 - 36 hours. Trypomastigotes are cleared from the bloodstream 4 to 6 hours after treatment. The combined effect on both developmental stages is apparently responsible for the in vivo ejfects of this drug which is the most active drug ever tested in our laboratory in experimental Chagas' disease.

Which consumer capabilities are actually triggered by children in-store?

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA ��� School of Business and Economics

Contribui����o para o estudo da anexina V na apoptose celular em concentrados de eritr��citos

A hemoterapia moderna baseia-se na utiliza����o correcta dos diversos componentes sangu��neos, associados a um maior controle de qualidade do sangue, o que a torna mais segura e, actualmente, muitos doentes sao beneficiados pois, a transfus��o de componentes sanguineos, em situa��oes v��rias, est�� na linha da frente na manuten����o da vida e em casos extremos, o ��ltimo recurso que salva vidas. A qualidade e a seguran��a nas transfus��es de sangue s��o grandes preocupa����es da ��rea m��dica, autoridades de sa��de e doente1. O sangue obtido pelos Centros de Sangue provem de dadores volunt��rios, dotados de uma enorme sensibilidade social, que periodicamente assumem uma postura benevola e altruista e consequentemente mant��m os bancos de sangue providos de um produto imprescindivel no tratamento de diversas patologias. O produto final dispon��vel ��� concentrado de eritr��citos (CE��s), plasma e concentrado plaquet��rio ��� tem de assumir um car��cter seguro e vi��vel de modo a que os riscos para o doente sejam diminutos2. O controlo de qualidade aplicado a todo o sangue doado realiza provas de conformidade nas unidades com especifica����es previamente definidas, sendo a h��molise um dos par��metros importantes na avalia����o da qualidade dos concentrados de eritr��citos, pois, pode ocasionar implica����es clinicas para o receptor. Para al��m disso a avalia����o da concentra����o de hemoglobina (Hg) no sangue doado mostra-se um controlo imprescindivel que salvaguarda a qualidade e seguran��a do componente a transfundir3;4.At�� se obter um CE h�� todo um processo moroso e de responsabilidade vital. Todo o sangue obtido passa por v��rias etapas fundamentais at�� �� obten����o do componente pretendido (analise, produ����o e armazenamento). Os CE���s obtidos quando armazenados, num ambiente de refrigera����o, t��m uma vida ��til de 42 dias. Ap��s este per��odo, o sangue deve ser inutilizado por se verificar altera����es bioqu��micas, biomec��nicas, e imunol��gicas nos CE���s e por consequ��ncia a sua instabilidade vital no que ao tratamento de patologias, para as quais este componente est�� indicado, diz respeito5. Foi realizado um estudo experimental com o objetivo de avaliar a contribui����o da Anexina V na apoptose celular nos concentrados de eritr��citos, constatando a degrada����o dos mesmos ao longo de todo o per��odo de armazenamento e validar o paradigma que a ci��ncia preconiza: ���Os CE���s ap��s os 42 dias armazenados, em condi����es espec��ficas (2 a 6�� cent��grados), s��o inviaveis para transfundir���6;7. A avalia����o dos n��veis de apoptose por citometria de fluxo �� geralmente realizada por m��todos que utilizam Anexina V como marcador vital, que se associa aos res��duos de fosfatidilserina, externalizados no in��cio do processo apopt��tico. A Anexina V �� uma prote��na humana end��gena dependente do i��o Ca+2, amplamente distribu��da intracelularmente em altas concentra����es na placenta e em concentra����es mais baixas nos eritr��citos, plaquetas e mon��citos. Apresenta como principal caracter��stica a capacidade de se ligar �� fosfatidilserina, um fosfolip��do presente na camada interna da bicamada lip��dica, que durante a apoptose celular �� translocada para a camada externa da membrana celular. A determina����o da Anexina V �� normalmente utilizada para verificar se as c��lulas s��o vi��veis, apopt��ticas ou necr��ticas por meio de diferen��as na integridade da membrana plasm��tica. Assim, ao conjugar a Anexina V ao FITC (Isotiocianato de fluoresce��na) �� poss��vel identificar e quantificar as c��lulas apopt��ticas por citometria de fluxo7. Numa amostra de 15 CE���s, a qual foi induzida a hem��lise, verificou-se, por citometria de fluxo, que a viabilidade deste componente se desvanesce ao longo do tempo, confirmando assim que o tratamento, manuseamento e armazenamento do sangue compromete a vitalidade terapeutica deste insubstituivel produto vital.

How can clusters help in the product type choice? The case of a specialized retail store

This work project (WP) is a study about a clustering strategy for Sport Zone. The general cluster study���s objective is to create groups such that within each group the individuals are similar to each other, but should be different among groups. The clusters creation is a mix of common sense, trial and error and some statistical supporting techniques. Our particular objective is to support category managers to better define the product type to be displayed in the stores��� shelves by doing store clusters. This research was carried out for Sport Zone, and comprises an objective definition, a literature review, the clustering activity itself, some factor analysis and a discriminant analysis to better frame our work. Together with this quantitative part, a survey addressed to category managers to better understand their key drivers, for choosing the type of product of each store, was carried out. Based in a non-random sample of 65 stores with data referring to 2013, the final result was the choice of 6 store clusters (Figure 1) which were individually characterized as the main outcome of this work. In what relates to our selected variables, all were important for the distinction between clusters, which proves the adequacy of their choice. The interpretation of the results gives category managers a tool to understand which products best fit the clustered stores. Furthermore, as a side finding thanks to the clusterization, a STP (Segmentation, Targeting and Positioning) was initiated, being this WP the first steps of a continuous process.

Mechanisms underlying intracellular delivery

AuNPs are versatile systems used for different biomedical application including imaging, drug and gene delivery. These systems support the intracellular transport of active molecules, a step that is considered one of the crucial problems in drug delivery. Nevertheless, in order to design optimal multifunctional AuNPs for specific and efficient nanomedicine applications, the mechanism by which AuNPs interact with living cells must be fully understand. The main goal of this work consisted in the assessment of the cellular uptake mechanism of 14 nm spherical AuNPs by A549 cells, through fluorescent spectroscopy and microscopy, in combination with quantitative analysis by ICP-MS. TAMRA labeled AuNPs were characterized by UV-visible and fluorescent spectroscopy and the final hydrodynamic diameter of 22.5 �� 0.33 nm was obtained by DLS. Regarding the cellular uptake studies, the AuNPs presented a fast cellular uptake kinetics reaching a saturation point after 6 hours of incubation in A549 cells. Further investigation concerning the internalization mechanism of this AuNPs was evaluated using specific inhibitors for different endocytic pathways. Optimal inhibition was achieved using chlorpromazine, inhibitor of clathrin-mediated endocytosis, resulting in a 23.5 % inhibition of AuNPs after 1 hour of incubation. This preliminary result obtained by fluorescent spectroscopy suggests that these AuNPs were predominantly uptake by clathrin-mediated endocytosis, meaning that other endocytic pathways must be involved in the cellular uptake of this AuNPs. In what cell viability is concern, the prepared AuNPs and the endocytic inhibitors revealed no significant effect on the cell viability in A549 cell line.

The impact of in-store activations (communication) in the consumer's emotions

Consumer behavior: Sport Zone. The analysis of "The impact of in-store activations (communication) in the consumer's emotions" Several studies have been conducted on the consumer behavior. This study aims to analyze and understand which factors are important to consumers��� emotions when the purchase decision occurs, the brand awareness, brand loyalty and the campaigns/activations��� impact in the above factors. Two research surveys were conducted to realize this study, the first online and the other was an interview to the Agency Up Partner who conceived and put into practice this Fitness campaign. First of all, was the consumer���s survey, a survey with 100 answers, to understand which factors are taken into account when a campaign in-store is held, in which the atmosphere is mainly used to arouse consumer���s desire to purchase, and also emotions. Second, the interview with the agency was realized to find out on what they were based on when they delineate it, and if the raise of emotions was taken into account in the origin of it. Concluding, emotions have a significant impact on formation of consumer in-store behavior, satisfaction and loyalty. As we could assay through of how this Fitness campaign was carried out as well as the optimal feedback received by consumers, improved attention over in-store marketing activity strongly influences consumer behavior at the point of purchase. ���Sport Zone: A new store concept where the love for sports is combined with functionality���

The consumption of beauty products on famale young adults in Portugal. Recommenrations for the brand Lanc��me to improve its store trafic

This project aims to explore the Portuguese Beauty and Cosmetics market, and to discuss the usage and purchase behaviour of young female adults, between 18 and 26 years old. After a market analysis based on secondary data, it evaluates the results of qualitative and quantitative research based on 9 interviews and 126 online questionnaires to explore the consumers��� reasoning when choosing products from this category ��� fragrances, skin care or make-up ��� as well as their attitude towards brands, with a special focus on the premium cosmetics brand Lanc��me. Contrary to our expectations there was no statistically significant positive influence of the online touchpoints within this age segment���s purchase intention. However, results indicate that Lanc��me is already being perceived by some as young and modern, but is still suffering the threat of Mass Market brands that are valued by this target, mainly due to a price sensitivity towards premium beauty brands.

Epidemic store for massive scale systems

The MAP-i doctoral program of the Universities of Minho, Aveiro and Porto

IL-17A promotes intracellular growth of Mycobacterium by inhibiting apoptosis of infected macrophages

The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

Gellan gum-coated gold nanorods: an intracellular nanosystem for bone tissue engineering

Gold nanorods (AuNRs) have emerged as an exceptional nanotool for a myriad of applications ranging from cancer therapy to tissue engineering. However, their surface modification with biocompatible and stabilizing biomaterials is crucial to allow their use in a biological environment. Herein, low-acyl gellan gum (GG) was used to coat AuNRs surface, taking advantage of its stabilizing, biocompatible and gelling features. The layer-by-layer based strategy implied the successive deposition of poly(acrylic acid), poly(allylamine hydrochloride) and GG, which allowed the formation of a GG hydrogel-like shell with 7 nm thickness around individual AuNRs. Stability studies in a wide range of pH and salt concentrations showed that the polysaccharide coating can prevent AuNRs aggregation. Moreover, a reversible pH-responsive feature of the nanoparticles was observed. Cytocompatibility and osteogenic ability of GG-coated AuNRs was also addressed. After 14 days of culturing within SaOS-2, an osteoblast-like cell line, in vitro studies revealed that AuNRs-GG exhibit no cytotoxicity, were internalized by the cells and localized inside lysosomes. AuNRs-GG combined with osteogenic media enhanced the mineralization capacity two-fold, as compared to cells exposed to osteogenic media alone. The proposed system has shown interesting features for osteogenesis, and further insights might be relevant for drug delivery, tissue engineering and regenerative medicine.

Forecasting human entrances at a commercial store using facial recognition data

Disserta����o de mestrado integrado em Engenharia e Gest��o de Sistemas de Informa����o

Importancia del Ca2+ en la fisiolog��a esperm��tica: modulaci��n de su actividad por progesterona y prote��nas del plasma seminal. Importance of calcium in sperm physiology: its regulation by progesterone and proteins from the seminal plasma.

El objetivo general del proyecto es estudiar el efecto de la progesterona y de algunas prote��nas del plasma seminal sobre la actividad del Ca2+ en diferentes procesos fisiol��gicos que ocurren en el espermatozoide, los cuales est��n estrechamente relacionados con la capacidad fertilizante de esta c��lula. La progesterona, principal esteroide secretado por las c��lulas del cumulus oophorus, ejerce su efecto a trav��s de un receptor no-gen��mico provocando aumento en el calcio intracelular de los espermatozoides y, consecuentemente, promoviendo la capacitaci��n, la respuesta quimiot��ctica y la exocitosis acrosomal. Pese a estas observaciones, los mecanismos a trav��s de los cuales la progesterona estimula fen��menos tan diversos en el espermatozoide son a��n desconocidos. Tampoco se conoce con exactitud el papel funcional y los mecanismos de acci��n de algunas prote��nas del plasma seminal que interaccionan y se unen a los espermatozoides, con alta especificidad, durante la eyaculaci��n. Por lo tanto, resulta altamente interesante profundizar los estudios sobre las propiedades funcionales de las prote��nas caltrin (calcium transport inhibitor) y ��-microseminoprotein (MSP) del plasma seminal de mam��feros, las cuales responden a las caracter��sticas mencionadas. Los estudios hasta ahora realizados han dado cuenta de que caltrin inhibe la incorporaci��n de Ca2+ extracelular, previene la exocitosis acrosomal espont��nea y promueve la uni��n espermatozoide-zona pel��cida. Tambi��n hay datos preliminares que sugieren un efecto inhibitorio sobre la movilidad hiperactivada de los espermatozoides. Respecto a MSP, s��lo se sabe que inhibe la exocitosis acrosomal espont��nea y que su contenido, en el plasma seminal, guarda una relaci��n inversa con la fertilidad. Por todo lo expuesto, se propone estudiar los mecanismos de acci��n de la progesterona y las prote��nas caltrin y MSP sobre los procesos fisiol��gicos antes indicados. Para ello, se estudiar��n las variaciones de Ca2+ intracelular en espermatozoides individuales sometidos a diferentes tratamientos (gradientes de progesterona, capacitaci��n en presencia y ausencia de caltrin y/o MSP, etc.), usando video microscop��a de fluorescencia y an��lisis computarizado de im��genes. Tambi��n se examinar�� la influencia de estas mol��culas sobre la interacci��n de gametas y la fertilizaci��n.