Are polyamines detected by the TAAR receptor family in the human Choroid Plexus?

Choroid plexuses (CPs) are highly vascularized structures located at the ventricular system of the brain constituted by a monolayer of choroid plexus epithelial cells (CPEC) located on a basement membrane which form a barrier between the blood and the cerebrospinal fluid (CSF) (BCSFB). CPECs through their interconnections restrict the passage of different substances and pathogens between the blood and the CSF. Moreover, the microvilli at the apical side and an extensive infolding at the basolateral side enhance the contact between the CP epithelium and CSF, and between the epithelium and stroma interstitial fluid, respectively, thus allowing one of its main functions which is to maintain cerebral homeostasis. Among other features, there is the chemical vigilance which is performed by different mechanisms as the taste signalling, the clearance of xenobiotics, the clearance of amyloid beta and finally but with more relevance to this work, the olfactory signalling. This olfactory cascade is activated when an odorant binds to an olfactory receptor (OR) which is a G protein-coupled receptor (GPCR) that exists in different tissues in mammalian´s body. The trace amine-associated receptors (TAARs) are a family of rhodopsin-like GPCRs which act as olfactory receptors with TAAR1 exception and are expressed in different tissues of different species. In humans there are 6 different types of TAARs and 3 pseudogenes. TAAR 13c, identified in zebrafish, can bind to cadaverine, a polyamine. Polyamines are polycations which appear naturally in cells and are closely related with neuronal cell biochemical activity at different points. Cadaverine is a diamine compound produced by the putrefaction of animal tissue and enable an aversive response to this odour. A previous data base research showed that TAAR1, 2 and 5 are the human receptors with more homology with TAAR13c and one of the more abundant TAARs in humans, which probably can trigger a response to a polyamine stimulus. TAAR1 was selected for this study because we could not amplify TAAR2 and 5 by RT-PCR in HIBCPP cell line. The HIBCPP cell line from choroid plexus carcinoma was used to the experiments and the expression of the mRNAs to the TAAR1 was demonstrated by the RT-PCR technique. In respect to polyamine´s biosynthesis and catabolic pathway enzymes identification, the RT-PCR technique enabled the confirmation of their mRNA expression after Sanger sequencing. The expression of key enzymes to these pathways was also demonstrated by immunocytochemistry and Western blot, in human CP cell line. To test the cell´s response to polyamines, calcium imaging (Ca2+ imaging) assays were performed and showed clear responses to cadaverine. After these tests, silencing of TAAR1 with a specific small interfering RNA (siRNA) was done and preliminary Ca2+ imaging responses to cadaverine enable to see a decrease in CP cells response, highlighting the potential role of TAAR1 in this polyamine response. In the closer future, more silencing assays will be done and Ca2+ imaging with transfected cells will be done too with cadaverine and other polyamines already tested as spermine, spermidine and putrescine. Western blot and immunocytochemistry techniques will enable the TAAR1 protein characterization and localization in the HIBCPP cells.No sistema nervoso central (SNC) é possível observar a presença de distintas barreiras que assumem um papel fundamental na proteção do cérebro contra as variações nas concentrações de determinadas substâncias ao nível do sangue, assim como contra a entrada de organismos patogénicos, toxinas e outras substâncias. Contudo, este nem sempre é um mecanismo de controlo favorável uma vez que estas podem funcionar como uma barreira à entrada de substâncias para o tratamento de patologias do SNC. De entre as barreiras existentes no cérebro salienta-se a barreira entre o sangue e o cérebro, a barreira aracnóideia e a barreira entre o sangue e o líquido cefalorraquidiano, o plexo coroide. O plexo coroide (CP) é uma estrutura altamente vascularizada localizada em cada um dos quatro ventrículos cerebrais. O CP é constituído por uma monocamada de células epiteliais localizada numa membrana basal e que por sua vez, formam uma barreira entre o sangue e o líquido cefalorraquidiano. Estas células, através das suas interconexões como é o caso das junções aderentes, junções apertadas e desmossomas, impedem a passagem de diferentes substâncias e organismos patogénicos, do sangue para o líquido cefalorraquidiano. Para além destas evidências, há a destacar o facto de as microvilosidades presentes na face apical assim como as invaginações existentes na superfície basal permitirem também uma boa interação entre o epitélio do plexo e o líquido cefalorraquidiano e entre o epitélio e o fluido intersticial, respetivamente, permitindo um conjugar de condições que vão assegurar uma correta manutenção da homeostase cerebral. De entre outras funções desempenhadas por esta estrutura, salienta-se a produção e secreção do líquido cefalorraquidiano e a vigilância química deste, que é assegurada por diferentes mecanismos como a sinalização gustativa, a remoção de xenobióticos, a remoção da proteína ß-amilóide, envolvida em doenças como a doença de Alzheimer, e por fim a sinalização olfativa. Esta cascata de sinalização olfativa é ativada quando uma molécula odorante se liga a um recetor olfativo, um recetor acoplado a uma proteína G. Esta ligação vai despoletar as cascatas de segundo mensageiro da adenosina monofosfato cíclica e do inositol 1,4,5-trifosfato. Há evidências de que a estimulação destes recetores olfativos ocorre pela ativação da proteína G olfativa levando a um aumento da adenosina monofosfato cíclica através da adenilato ciclase 3 de membrana, resultando num aumento do influxo de Ca2+ através de um canal nucleotídico cíclico, resultando na despolarização da membrana e consequentemente na geração de um potencial de ação nos neurónios olfativos. Os trace amine-associated receptors (TAARs) são uma família de recetores acoplados a proteína G que, à exceção do TAAR1, atuam como recetores olfativos, sendo expressos em diferentes tecidos em diferentes espécies. Diferentes estudos permitiram comprovar a expressão destes recetores em tecidos do epitélio olfativo e noutros como é o caso do cérebro, medula espinhal, coração, pulmões, fígado, baço, trato gastrointestinal, rins, testículos e células sanguíneas como os leucócitos. Em humanos é possível identificar 6 tipos diferentes de TAARs (1,2,5,6,8 e 9) e 3 pseudogenes (3, 4 e 7). Os genes que codificam para estes recetores encontram-se localizados no cromossoma 6 q23.1. O TAAR13c, expresso no peixe-zebra foi identificado como sendo o responsável por ligar à cadaverina, uma poliamina resultante da decomposição de tecidos mortos, gerada através da descarboxilação da lisina pela enzima lisina-descarboxílase, que ao ser detetada por este peixe, despoleta uma resposta aversiva por parte deste. As poliaminas são policatiões presentes em todas as células, que interagem com moléculas carregadas negativamente como é o caso do ácido desoxirribonucleico (DNA), ácido ribonucleico (RNA) ou ainda as proteínas. Estas substâncias como é o caso da cadaverina, espermina, espermidina e putrescina participam em diversas funções a nível fisiológico como o crescimento celular, proliferação, diferenciação, transcrição, tradução, regulação de canais iónicos, atividade enzimática, constituição das interações célula-célula e resposta ao stress oxidativo. Estas duas últimas funções conferem às poliaminas um papel fundamental no surgimento de diversas patologias do foro neurológico como é o caso da doença Parkinson, da esclerose lateral amiotrófica e isquemia, em que os valores destas substâncias surgem alterados. Contudo as poliaminas ao controlarem o stress oxidativo vão também ter um papel benéfico no controlo da doença de Alzheimer. Com o objetivo de estabelecer homologia entre o TAAR13c do peixe-zebra e os TAARs humanos foi realizada uma pesquisa com a ferramenta BlastX (NCBI) que revelou a existência de diferentes TAAR com maior homologia com o TAAR13c. Após uma pesquisa na base de dados Gene Expression Omnibus, foram selecionados para o estudo, os TAAR1, 2 e 5 por estes apresentarem maior abundância em humanos. Estes dados levaram-nos a supor que estes recetores teriam capacidade para despoletar uma resposta em células humanas do plexo coroide quando estimuladas por poliaminas como a cadaverina. Assim, o uso de uma linha de células humanas de carcinoma do plexo coroide, através da técnica de RT-PCR seguida de sequenciação pelo método de Sanger, permitiu identificar a expressão do mRNA para o TAAR1, o que não se verificou para os TAAR2 e 5. Um processo idêntico foi utilizado para a análise da expressão dos mRNAs para as enzimas das vias anabólica e catabólica das poliaminas, sendo a sua expressão confirmada por sequenciação. As enzimas chave destas vias, a espermina sintetase e a ornitina descarboxílase, foram também analisadas através das técnicas de Western blot e imunocitoquímica. Após a realização de um ensaio de MTT que permitiu comprovar que os estímulos de cadaverina aplicados não iriam comprometer a viabilidade celular, foram realizados ensaios de calcium imaging que possibilitaram verificar respostas por parte das células após estimulação com diferentes concentrações de cadaverina. Por forma e testar a especificidade da ligação do TAAR1 à cadaverina, foi realizado um ensaio preliminar (n=1) de silenciamento, transfetando as células com um pequeno RNA de interferência (siRNA) para o TAAR1. Os resultados demonstraram uma diminuição das respostas celulares à cadaverina quando em comparação com os controlos feitos apenas com o agente de transfeção ou com este último em conjunto com um scramble siRNA. Estes resultados, embora preliminares, são promissores quanto ao papel do TAAR1 na deteção da cadaverina. Futuramente, serão realizados novos ensaios de silenciamento do TAAR1 e estímulos com outras poliaminas como a espermina, espermidina e putrescina. Serão também efetuados ensaios de Western blot e imunocitoquímica, permitindo avaliar a expressão e localização do TAAR1

Evidence of a glocal marketing strategy: a case study in the Brazilian telecommunication market

Adaptation versus standardization dilemma has been coined as a major issue within the international marketing literature for the past fifty years, and academic research has showed that the extreme positions were unbearable and that the virtue lied in a middle position between these two positions. This opened space for another stream of research devoted to analyze and discuss the level of control and autonomy that Multinational Enterprises (MNEs) should grant to their subsidiaries. In this paper, we analyze the impact of subsidiaries’ capacity to innovate in marketing in an emerging economy. Thus, the main purpose of this study is to explore marketing autonomy in Multinational Enterprises’ subsidiaries to innovate and develop new products and services to the Brazilian telecommunications market. Using the grid proposed by Lasserre, several interviews to senior managers of Telefónica were conducted in order to evaluate the level of autonomy of Telefonica’s Brazilian subsidiary. The results show that Telefónica uses a glocal approach, since its subsidiary has autonomy to create and adapt its products when the headquarters recognize that global decisions might not meet local demands. Despite being a single case study, Telefóncia is a giant in the telecommunication sector, operating in numerous countries, thus the current insights on the way the company runs global operations may be helpful not only for researchers but also for other companies

Germinação de diferentes genótipos de feijão submetidos ao estresse hídrico pela diminuição do potencial osmótico.

O presente trabalho teve como objetivo simular condições padronizadas de déficit hídrico em laboratório para a avaliação da germinação de sementes de diferentes genótipos de feijão. O experimento foi realizado no Laboratório da Universidade Estadual de Goiás - Unidade Universitária de Ipameri

Yerba mate (Ilex paraguariensis St. Hil.) ministumps and minicuttings clonal propagation protocol

La domesticación de la yerba mate (Ilex paraguariensis St. Hil) es muy reciente, razón por la cual, las plantaciones son heterogéneas, presentan plantas de diferentes tamaños, crecimiento y vigor, resistencia a enfermedades y tipo de hojas entre otras. Esta variación se debe fundamentalmente a que las plantas son multiplicadas por semillas y en consecuencia ocurre una gran segregación de caracteres. A su vez, la producción de plantines de yerba mate desde semillas presenta una serie de limitantes: bajo porcentaje de germinación (13% a 14%), largo periodo para el comienzo de la germinación (100 y 365 días), las semillas de calidad genética son escasas y caras, se debe realizar el trasplante al contenedor final, elevado costo en mano de obra y largo período de viverización (12 a 15 meses). La técnica de propagación vegetativa por minicepas y miniestacas, desarrollada y descripta en el presente trabajo, es una alternativa a la producción de plantines por semillas, dado que permite aumentar la producción de propágulos (miniestacas) por unidad de superficie, con un menor tiempo de viverización. Además, esta técnica puede ser adaptada a la realidad de los pequeños y medianos productores. El objetivo del proyecto fue desarrollar una metodología de propagación vegetativa de yerba mate, que maximice el uso del espacio en el vivero, aumente la tasa de multiplicación y homogeneidad en el manejo de vivero, factible de ser implementada por pequeños y medianos viveristas.Domestication of yerba mate (Ilex paraguariensis St. Hil) is very recent, therefore, the plantations are heterogeneous, presenting plants of different sizes, growth and vigor, resistance to diseases and type of leaves among others. This variation is mainly due to the fact that plants are producedfrom seeds and consequently a high segregation of characters occurs. At the same time, plant production fromseedspresents low percentage of germination (13% to 14%), long period for the beginning of the germination (100 and 365 days), high labor cost and long period of nursing (12 to 15 months), andseed with genetic quality is scarce and expensive. The technique of vegetative propagation by ministumps and minicuttings, developed and described in the present work, is an alternative to the production of seedlings, since it allows to increase the production of propagules (minicuttings) per unit area, with a shorter time of nursing. In addition, this technique can be adapted to the reality of small and medium nurseries. The objective of the project was to develop a methodology of vegetative propagation of yerba mate, which maximizes the use of space in the nursery, increases the multiplication rate and homogeneity in nursery management; feasible to be implemented by small and medium nurserymen.Secretaría de Ciencia y Técnic

Yerba mate (Ilex paraguariensis St. Hil.) ministumps and minicuttings clonal propagation protocol

La domesticación de la yerba mate (Ilex paraguariensis St. Hil) es muy reciente, razón por la cual, las plantaciones son heterogéneas, presentan plantas de diferentes tamaños, crecimiento y vigor, resistencia a enfermedades y tipo de hojas entre otras. Esta variación se debe fundamentalmente a que las plantas son multiplicadas por semillas y en consecuencia ocurre una gran segregación de caracteres. A su vez, la producción de plantines de yerba mate desde semillas presenta una serie de limitantes: bajo porcentaje de germinación (13% a 14%), largo periodo para el comienzo de la germinación (100 y 365 días), las semillas de calidad genética son escasas y caras, se debe realizar el trasplante al contenedor final, elevado costo en mano de obra y largo período de viverización (12 a 15 meses). La técnica de propagación vegetativa por minicepas y miniestacas, desarrollada y descripta en el presente trabajo, es una alternativa a la producción de plantines por semillas, dado que permite aumentar la producción de propágulos (miniestacas) por unidad de superficie, con un menor tiempo de viverización. Además, esta técnica puede ser adaptada a la realidad de los pequeños y medianos productores. El objetivo del proyecto fue desarrollar una metodología de propagación vegetativa de yerba mate, que maximice el uso del espacio en el vivero, aumente la tasa de multiplicación y homogeneidad en el manejo de vivero, factible de ser implementada por pequeños y medianos viveristas.Domestication of yerba mate (Ilex paraguariensis St. Hil) is very recent, therefore, the plantations are heterogeneous, presenting plants of different sizes, growth and vigor, resistance to diseases and type of leaves among others. This variation is mainly due to the fact that plants are producedfrom seeds and consequently a high segregation of characters occurs. At the same time, plant production fromseedspresents low percentage of germination (13% to 14%), long period for the beginning of the germination (100 and 365 days), high labor cost and long period of nursing (12 to 15 months), andseed with genetic quality is scarce and expensive. The technique of vegetative propagation by ministumps and minicuttings, developed and described in the present work, is an alternative to the production of seedlings, since it allows to increase the production of propagules (minicuttings) per unit area, with a shorter time of nursing. In addition, this technique can be adapted to the reality of small and medium nurseries. The objective of the project was to develop a methodology of vegetative propagation of yerba mate, which maximizes the use of space in the nursery, increases the multiplication rate and homogeneity in nursery management; feasible to be implemented by small and medium nurserymen.Secretaría de Ciencia y Técnic

Atypical Phenotype in Two Patients with LAMA2 Mutations

Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene, which encodes the a2-chain of laminin. We report two patients with partial laminin-a2 deficiency and atypical phenotypes, one with almost exclusive central nervous system involvement (cognitive impairment and refractory epilepsy) and the second with marked cardiac dysfunction, rigid spine syndrome and limb-girdle weakness. Patients underwent clinical, histopathological, imaging and genetic studies. Both cases have two heterozygous LAMA2 variants sharing a potentially pathogenic missense mutation c.2461A>C (p.Thr821Pro) located in exon 18. Brain MRI was instrumental for the diagnosis, since muscular examination and motor achievements were normal in the first patient and there was a severe cardiac involvement in the second. The clinical phenotype of the patients is markedly different which could in part be explained by the different combination of mutations types (two missense versus a missense and a truncating mutation)

A cost–effective and eco-friendly treatment technology to remove realistic levels of mercury by means of the unmodified rice husk

In the present work the removal of mercury from aqueous solutions media and for realistic concentrations of this metal was evaluated, by using an inexpensive and highly available agricultural waste, rice husk. An economical method was developed based on the use of the natural/unmodified form of rice husk and on the amount of material required, which was minimized to produce the lowest amount of biological slurry. In order to study the kinetics of the process, batch stirred experiments were performed for initial concentrations of Hg 2+ of: 50 μg L -1 , representing the actual maximum value for Hg discharges from industrial sectors, and 500 μg L -1 , to simulate a situation of an accidental spill. The kinetic curves were described by a rapid removal of Hg 2+ by rice husk, in the first hours of contact, followed by a slower removal of Hg 2+ , until it reaches equilibrium. The removal of Hg 2+ using a rice husk dose of 0.50 g L -1 was ca. 83% for an initial concentration of 50 μg L -1 and 92% for 500 μg L -1 . Furthermore, the results show that is possible the reuse the rice husk in additional cleaning treatments, maintaining the efficiency of the removal process

Effects of physical exercise in biochemical parameters and dorsolateral prostate lesions: data from a rat model of prostate cancer

Background: Prostate cancer (PCa) is among the most prevalent cancers worldwide. Physical exercise is widely recognized due to its beneficial effects. This study aimed to evaluate the effects of physical exercise on biochemical pa- rameters and in dorsolateral prostate lesions in a rat model of PCa. Materials and Methods: Ninety-five male Wistar Unilever rats were randomly divided into eight groups sacrificed at 35 (groups I) or 61 weeks of age (groups II): control sedentary groups (Cont+Sed I (n = 10); Cont+Sed II (n = 10)); induced sedentary group (PCa+Sed I (n = 10); PCa+Sed II (n = 15)); control exercised groups (Cont+EX I (n = 10); Cont+EX II (n = 10)) and induced exercised groups (PCa+EX I (n = 10); PCa+EX II (n = 20)). All procedures were approved (DGAV, no. 021326). Animals from exercised groups started the exer- cise program in a treadmill at 8 weeks of age, for 28 weeks or 53 weeks. The animals were trained 5 days/week, 60 min per day. Prostate lesions were induced at 12 weeks of age, with sequential administration of flutamide, testosterone propion- ate and N-methyl-N-nitrosourea, and subcutaneous implants of crystalline testosterone. Animals were sacrificed at 35 or 61 weeks of age. Peripheral blood of all animals was col- lected by intracardiac puncture. A complete necropsy was performed. The dorsolateral prostate tissues sections were processed for histological analysis. Data were analysed using SPSS 25. p 0.05). Dorsolateral prostate lesions were classified as dysplasia, prostatic intraep- ithelial neoplasia (PIN) and microinvasive carcinoma. The number of prostate lesions was higher in animals from groups II than in those from groups I, mainly in PCa+Sed II animals when compared with PCa+Sed I (p 0.05). Conclusions: Overall, the animals sacrificed at 61 weeks of age developed more dorsolateral prostate lesions than ani- mals sacrificed at 35 weeks of age, which may be related to a longer testosterone exposure

High performance free-standing films by layer-by-layer assembly of graphene flakes and ribbons with natural polymers

In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) were developed through layer-by-layer assembly. First, GO was synthesized from graphite and multi-walled carbon nanotubes using a modified Hummer's method, yielding oxidized graphene flakes (o-GFs) and oxidized graphene nanoribbons (o-GNRs), respectively, which were then characterized. Then FS films were produced and their morphological, thermal and mechanical properties, as well as the o-GF and o-GNR dispersion along the films were assessed. Their degradation and swelling profiles as well as their biological behavior were evaluated. Graphite and nanotubes were successfully oxidized and exfoliated forming stable suspensions that could be combined with chitosan (CHI) and alginate (ALG) solutions by layer-by-layer processing. The addition of o-GFs and o-GNRs resulted in rougher, hydrophilic FS films with significantly improved mechanical properties relative to CHI/ALG films. The presence of o-GFs or o-GNRs did not affect the thermal stability and the addition of o-GFs resulted in films with enhanced cytocompatibility. The results demonstrate the high potential of the GO reinforced films for biomedical applications, in particular o-GF films, for wound healing, and cardiac and bone engineering applications.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) and the European program FEDER/ COMPETE for the financial support through project BioSeaGlue: EXPL/CTM-BIO/0646/2013 (FCOMP-01-0124-FEDER-041105) and for project PEst-C/CTM/LA0025/2013 (Strategic Project – LA 25 – 2013-2014). This work was also financially supported by FCT through the scholarships SFRH/BPD/96797/2013 granted to Sofia G. Caridade, SFRH/BD/97606/2013 granted to Maria Sousa, and SFRH/BD/87214/2012 granted to Eunice Cunha

Geochemistry, mineralogy, solid-phase fractionation and oral bioaccessibility of lead in urban soils of Lisbon

An urban survey of Lisbon, the largest city in Portugal, was carried out to investigate its environmental burden, emphasizing metallic elements and their public health impacts. This paper examines the geochemistry of lead (Pb) and its influence on human health data. A total of 51 soil samples were collected from urban recreational areas used by children to play outdoors. The semi-quantitative analysis of Pb was carried out by inductively coupled plasma mass spectrometry after an acid digestion. X-ray diffraction was used to characterize the soil mineralogy. The solid-phase distribution of Pb in the urban soils was investigated on a subset of 7 soils, out of a total of 51 samples, using a non-specific sequential extraction method coupled with chemometric analysis. Oral bioaccessibility measurements were obtained using the Unified BARGE Method developed by the Bioaccessibility Research Group of Europe. The objectives of the study are as follows: (1) investigation of Pb solid-phase distribution; (2) interpretation of Pb oral bioaccessibility measurements; (3) integration of metal geochemistry with human health data; and (4) understanding the influence of geochemistry and mineralogy on oral bioaccessibility. The results show that the bioaccessible fraction of Pb is lower when major metal fractions are associated with less soluble soil phases such as Fe oxyhydroxides, and more increased when the metal is in the highly soluble carbonate phase. However, there is some evidence that the proportion of carbonates in the soil environment is also a key control over the oral bioaccessibility of Pb, irrespective of its solid-phase fractionation