P2 purinoceptors signaling in fibroblasts of rat subcutaneous tissue

Mestrado em Biologia Molecular e CelularO tecido conjuntivo parece estar envolvido na génese de diversas condições patológicas. O aumento da rigidez do tecido conjuntivo, resultante da fibrose, pode constituir um factor importante no mecanismo patogénico da dor crónica resistente a fármacos (Langevin & Sherman, 2007). Por outro lado, os nucleótidos extracelulares parecem estar envolvidos na fisiopatologia da dor crónica (Burnstock, 2001). Assim, este estudo teve como objectivo averiguar o efeito dos nucleótidos de adenina e uridina na proliferação e síntese de colagénio tipo I de fibroblastos do tecido subcutâneo de rato em cultura. Os resultados obtidos mostram que a incubação com UTP (0.3-100 M, n=5) induz um aumento da proliferação e da produção de colagénio tipo I, o qual é dependente da concentração. Contrariamente, o agonista selectivo dos receptores P2Y2, o MRS 2768 (10 μM, n=3), não teve qualquer efeito no que se refere à proliferação, mas diminuiu significativamente (P<0.05) a síntese de colagénio tipo I. Uma vez que o aumento da produção de colagénio induzida pelo UTP (100 μM) foi proporcional ao aumento do número de células (proliferação celular),podemos especular que este aumento se deve ao aumento do número de células per si do que a uma maior actividade sintética de cada célula. Assim, ao normalizar os valores do colagénio tipo I em relação aos valores obtidos do MTT para os mesmos momentos/dias, deixamos de observar diferenças estatisticamente significativas entre o controlo e as células expostas ao UTP. Uma vez que os receptores P2Y2 não parecem estar envolvidos nesta resposta do UTP (100 μM), esta poderá estar a ser mediada pela activação dos receptores P2Y4 e/ou P2Y6. Considerando que o RB-2 (10 μM, n=5), um antagonista não selectivo que actua preferencialmente no subtipo de receptores P2Y4, não foi capaz de modificar a resposta induzida pelo UTP (100 μM), os receptores P2Y4 parecem também não estar envolvidos. Por outro lado, o MRS 2578 (100 nM), um antagonista selectivo dos receptores P2Y6, atenuou de forma significativa o aumento induzido pelo UTP (100 μM). A corroborar os nossos resultados, uma análise imunocitoquímica mostrou uma imunorreactividade positiva contra os receptores P2Y2 e P2Y6, mostrando um padrão de marcação citoplasmático/membranar, o qual é típico para este tipo de receptores, ao contrário do padrão nuclear exibido pelo anticorpo contra os receptores P2Y4. Relativamente ao envolvimento dos receptores sensíveis ao ADP, os resultados obtidos mostraram que o ADPβS (10-100 μM, n=3-6), um análogo estável do ADP, não parece induzir efeitos significativamente diferentes (P>0.05) na proliferação celular. Contudo, a sua incubação continuada aumentou a produção de colagénio tipo I de forma dependente da concentração (P<0.05). De modo a identificar os receptores purinérgicos envolvidos neste efeito, testamos o ADPβS (100 μM) na presença do MRS 2179 (0.3 μM), do AR-C 66096 (0.1 μM), e do MRS 2211 (10 μM), os quais antagonizam selectivamente os receptores P2Y1, P2Y12 e P2Y13, respectivamente. O efeito facilitatório induzido pelo ADPβS (100 μM) foi atenuado de forma significativa na presença do antagonista dos receptores P2Y1, o MRS 2179 (0.3 μM, n=3), sem ser afectado pelo antagonista dos receptores P2Y12, o AR- C 66096 (0.1 μM, n=3). Pelo contrário, o MRS 2211 (10 μM, n=2) potenciou o aumento da produção de colagénio induzida pelo ADPβS (100 μM), indicando assim que a síntese de colagénio tipo I induzida pelo receptor P2Y1 pode estar a ser parcialmente influenciada por uma activação síncrona do receptor inibitório P2Y13. Por último, uma análise por imunocitoquímica mostrou que estas células apresentam imunorreactividade positiva para os receptores P2Y1 e P2Y13, exibindo um padrão citoplasmático/membranar, contrariamente ao padrão nuclear dos receptores ostentado pelo anticorpo contra os receptores P2Y12. Concluindo, a remodelação da fáscia superficial induzida pelos fibroblastos parece ser regulada por um balanço entre a activação dos receptores P2Y2 e P2Y6, assim como dos receptores P2Y13 e P2Y1. Clarificar as vias que conduzem ao processo de fibrose pode representar uma oportunidade para esclarecer o seu envolvimento na patogénese da dor crónica musculo-esquelética, bem como ser útil no desenvolvimento de novas estratégias terapêuticas.Connective tissue may be involved in the pathogenesis of a wide variety of disease conditions. Increased connective tissue stiffness due to fibrosis may be an important link to the pathogenic mechanism leading to drug-resistant chronic pain (Langevin & Sherman, 2007). In addition, extracellular nucleotides seem to be involved in the pathophysiology of chronic pain (Burnstock, 2001). Therefore, we aimed at investigating the effect of adenine and uridine nucleotides on the proliferation and synthesis of type I collagen by rat fibroblasts from subcutaneous connective tissue. The results showed that continuous incubation of UTP (0.3-100 M, n=5) concentration-dependently increased fibroblasts proliferation, as also increased the synthesis of type I collagen above the control levels. Conversely, the selective P2Y2 agonist, MRS 2768 (10 μM, n=3), was devoid of effect in what concerns proliferation, but significantly (P<0.05) decreased type I collagen synthesis. Since the increase in type I collagen synthesis induced by UTP (100 μM) was proportional to the increase in the amount of cells in the culture (fibroblasts proliferation), we speculated that such an increase could be related to the increase in the cell number rather than a higher synthetic activity. Thus, we performed a more detailed data analysis, in which we normalized type I collagen production taking into consideration the MTT values obtained at the same time points, and we observed no longer significant differences between control and UTP-exposed cells. Discounting the contribution of MRS 2768-sensitive P2Y2 receptors, UTP (100 μM)-induced increase in cells proliferation could be due to P2Y4 and/or P2Y6 receptor activation. Since RB-2 (10 μM, n=5), a non-selective antagonist that acts preferentially on the P2Y4 subtype, did not modify the effect of UTP (100 μM), P2Y4 does not seem to be involved. In turn, MRS 2578 (100 nM), which is a selective P2Y6 antagonist, significantly attenuated UTP (100 μM)-induced increase. To corroborate our results, an immunocytochemistry analysis showed a positive immunoreactivity against the P2Y2 and P2Y6 receptors exhibiting a cytoplasmic/membrane labeling pattern, which is typical for those receptors in many different cells, conversely to the nuclear labeling pattern exhibited by the antibody against the P2Y4. To investigate the involvement of ADP-sensitive P2 receptors on cell proliferation and extracellular matrix production, fibroblast cultures were continuously incubated with the stable ADP analogue, ADPβS (10-100 μM). Results obtained with ADPβS (10-100 μM, n=3-6) showed no significant (P>0.05) differences in fibroblast cells proliferation. However, a continuous incubation with ADPβS (10-100 μM, n=2-5) concentration-dependently increased type I collagen production by fibroblasts (P<0.05). In order to identify which purinoceptor(s) that could be mediating this effect, we tested ADPβS (100 μM) in the presence of MRS 2179 (0.3 μM), AR-C 66096 (0.1 μM), and MRS 2211 (10 μM), which antagonize selectively ADP-sensitive P2Y1, P2Y12 and P2Y13 receptors, respectively. The facilitatory effect of ADPβS (100 μM) was significantly attenuated in the presence of the P2Y1 antagonist, MRS 2179 (0.3 μM, n=3), without being affected by the P2Y12 antagonist, AR- C 66096 (0.1 μM, n=3). In contrast, MRS 2211 (10 μM, n=2) potentiated the effect of ADPβS (100 μM) on type I collagen synthesis, thus indicating that the P2Y1-receptor-induction of type I collagen synthesis may be partially counteracted by synchronous activation of the inhibitory P2Y13 receptor. Finally, an immunocytochemistry analysis showed that these cells exhibit immunoreactivity to P2Y1 and P2Y13 receptors with a cytoplasmic/membrane staining pattern, conversely to the nuclear pattern of P2Y12. Concluding, a delicate balance between the activation of P2Y2 and P2Y6, as well as P2Y13 and P2Y1 purinoceptors, might regulate fibroblast’s induced superficial fascia remodeling. Targeting the pathways leading to fibrosis may represent an opportunity to clarify its involvement in the pathogenesis of musculoskeletal chronic pain and it may be useful for designing novel therapeutic strategies to overcome this disease

Sharp mixed norm spherical restriction

Let $d\geq 2$ be an integer and let $2d/(d-1) < q \leq \infty$. In this paper we investigate the sharp form of the mixed norm Fourier extension inequality \begin{equation*} \big\|\widehat{f\sigma}\big\|_{L^q_{{\rm rad}}L^2_{{\rm ang}}(\mathbb{R}^d)} \leq {\bf C}_{d,q}\, \|f\|_{L^2(\mathbb{S}^{d-1},{\rm d}\sigma)}, \end{equation*} established by L. Vega in 1988. Letting $\mathcal{A}_d \subset (2d/(d-1), \infty]$ be the set of exponents for which the constant functions on $\mathbb{S}^{d-1}$ are the unique extremizers of this inequality, we show that: (i) $\mathcal{A}_d$ contains the even integers and $\infty$; (ii) $\mathcal{A}_d$ is an open set in the extended topology; (iii) $\mathcal{A}_d$ contains a neighborhood of infinity $(q_0(d), \infty]$ with $q_0(d) \leq \left(\tfrac{1}{2} + o(1)\right) d\log d$. In low dimensions we show that $q_0(2) \leq 6.76\,;\,q_0(3) \leq 5.45 \,;\, q_0(4) \leq 5.53 \,;\, q_0(5) \leq 6.07$. In particular, this breaks for the first time the even exponent barrier in sharp Fourier restriction theory. The crux of the matter in our approach is to establish a hierarchy between certain weighted norms of Bessel functions, a nontrivial question of independent interest within the theory of special functions.Comment: 21 page

THE USE of CONSORTIA for the INTERNATIONALIZATION of FIRMS – MOTA-ENGIL CASE STUDY

Internationalization has been widely studied throughout the years. Broadly, it has been predicted as irrevocable and having increasing impact on firm-related strategy. Within entry modes, consortium, has not received as much attention as others. Hence, it seems important to understand how this specific entry mode allows the entrance of firms in the international markets. This study intends to answer the question of “how” to internationalize, anticipating the consortium as the most feasible way for construction firms to enter certain markets. The reasons that determine its choice concern the specificness of the projects, markets and of the firm. In the first part of the study, we review the existent literature on consortia as an entry mode and as a tool of internationalization used by construction firms. Through this review we build a framework that reveals the motivations that lead to this choice. In the second part, we present the case study of Mota-Engil, as a potential source of valuable information which may contribute to the understanding of the phenomenon under study. This case study corroborates the motivations found to create consortia. The paper closes with its contributions, limitations and suggestions for future researches.consortia, internationalization, cooperation, construction

Heuristic Backtracking Algorithms for SAT

In recent years backtrack search SAT solvers have been the subject of dramatic improvements. These improvements allowed SAT solvers to successfully replace BDDs in many areas of formal verification, and also motivated the development of many new challenging problem instances, many of which too hard for the current generation of SAT solvers. As a result, further improvements to SAT technology are expected to have key consequences in formal verification. The objective of this paper is to propose heuristic approaches to the backtrack step of backtrack search SAT solvers, with the goal of increasing the ability of the SAT solver to search different parts of the search space. The proposed heuristics to the backtrack step are inspired by the heuristics proposed in recent years for the branching step of SAT solvers, namely VSIDS and some of its improvements. The preliminary experimental results are promising, and motivate the integration of heuristic backtracking in state-of-the-art SAT solvers. 1

A joint replenishment competitive location problem

Competitive Location Models seek the positions which maximize the market captured by an entrant firm from previously positioned competitors. Nevertheless, strategic location decisions may have a significant impact on inventory and shipment costs in the future affecting the firm’s competitive advantages. In this work we describe a model for the joint replenishment competitive location problem which considers both market capture and replenishment costs in order to choose the firm’s locations. We also present an metaherusitic method to solve it based on the Viswanathan’s (1996) algorithm to solve the Replenishment Problem and an Iterative Local Search Procedure to solve the Location Problem.N/

Evaluation of analytical methodologies to derive vulnerability functions

The recognition of fragility functions as a fundamental tool in seismic risk assessment has led to the development of more and more complex and elaborate procedures for their computation. Although vulnerability functions have been traditionally produced using observed damage and loss data, more recent studies propose the employment of analytical methodologies as a way to overcome the frequent lack of post-earthquake data. The variation of the structural modelling approaches on the estimation of building capacity has been the target of many studies in the past, however, its influence in the resulting vulnerability model, impact in loss estimations or propagation of the uncertainty to the seismic risk calculations has so far been the object of restricted scrutiny. Hence, in this paper, an extensive study of static and dynamic procedures for estimating the nonlinear response of buildings has been carried out in order to evaluate the impact of the chosen methodology on the resulting vulnerability and risk outputs. Moreover, the computational effort and numerical stability provided by each approach were evaluated and conclusions were obtained regarding which one offers the optimal balance between accuracy and complexity

A regret model applied to the maximum coverage location problem with queue discipline

This article discusses issues related to the location and allocation problems where is intended to demonstrate, through the random number generation, the influence of congestion of such systems in the final solutions. It is presented an algorithm that, in addition to the GRASP, incorporates the Regret with the pminmax method to evaluate the heuristic solution obtained in regard to its robustness for different scenarios. To the well know Maximum Coverage Location Problem from Church and Revelle [1] an alternative perspective is added in which the choice behavior of the server does not only depend on the elapsed time from the demand point looking to the center, but also includes the waiting time for service conditioned by a waiting queue.N/