Size-Control Regulation during Regeneration

Functional regeneration of organs upon injury is a key process for animals survival. Contrary to humans, some vertebrates are remarkably competent in regenerating after acute organ or appendage lesions. This advantageous skill allows overcoming limitations in repair even in adult stages, when tissues are fully developed, via a process of epimorphic regeneration. One such organism is the zebrafish, which can regenerate several organs, namely its heart, retina, spinal cord and fins. (...)

Glia-Motoneuron dialogue in ALS onset and progression in SOD1G93A-mice model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the pro-gressive loss of motoneurons (MN). Increasing evidence points glial cells as key players for ALS onset and progression. Indeed, MN-glia signalling pathways involving either neuroprotection or inflammation are likely to be altered in ALS. We aimed to study the molecules related with glial function and/or reactivity by evaluating glial markers and hemichannels, mainly present in astrocytes. We also studied molecules involved in mi-croglia-MN dialogue (CXCR3/CCL21; CX3CR1/CX3CL1; MFG-E8), as well as proliferation (Ki-67) and inflammatory-related molecules (TLR2/4, NLRP3; IL-18) and alarming/calming signals (HMGB1/autotaxin). We used lumbar spinal cord (SC) homogenates from mice expressing a mutant human-SOD1 protein (mSOD1) at presymptomatic and late-symptomatic ALS stages. SJL (WT) mice at same ages were used as controls. We observed decreased expression of genes associated with astrocytic (GFAP and S100B) and microglial (CD11b) markers in mSOD1 at the presymptomatic phase, as well as diminished levels of gap junction components pannexin1 and connexin43 and expression of Ki-67 and decreased autotax-in. In addition, microglial-MN communication was negatively affected in mSOD1 mice as well as in-flammatory response. Interestingly, we observed astrocytic (S100B) and microglial (CD11b) reactivity, increased proliferation (Ki-67) and increased autotaxin expression in symptomatic mSOD1 mice. In-creased MN-microglial dialogue (CXCR3/CCL21; CX3CR1/CX3CL1; MFG-E8) and hemichannel activ-ity, namely connexin43 and pannexin1, were also observed in mSOD1 at the symptomatic phase, along with an elevated inflammatory response as indicated by increased levels of HMGB1 and NLRP3. Our results suggest that decreased autotaxin expression is a feature of the presymptomatic stage, and precede the network of pro-inflammatory-related symptomatic determinants, including HMGB1, CCL21, CX3CL1, and NLRP3. The identification of the molecules and signaling pathways that are dif-ferentially activated along ALS progression will contribute for a better design of therapeutic strategies for disease onset and progression.

Proteomics based approach to understand tissue regeneration

Dissertation presented to obtain the PhD degree in Biochemistry

Unraveling intrinsic mechanisms of Nerve Regeneration

Unlike injury to the peripheral nervous system (PNS), where injured neurons can trigger a regenerative program that leads to axonal elongation and in some cases proper reinnervation, after injury to the central nervous system (CNS) neurons fail to produce the same response. The regenerative program includes the activation of several injury signals that will lead to the expression of genes associated with axonal regeneration. As a consequence, the spawned somatic response will ensure the supply of molecular components required for axonal elongation. The capacity of some neurons to trigger a regenerative response has led to investigate the mechanisms underlying neuronal regeneration. Thus, non-regenerative models (like injury to the CNS) and regenerative models (such as injury to the PNS) were used to understand the differences underlying those two responses to injury. To do so, the regenerative properties of dorsal root ganglion (DRG) neurons were addressed. This particular type of neurons possesses two branches, a central axon, that has a limited capacity to regenerate; and a peripheral axon, where regeneration can occur over long distances. In the first paradigm used to understand the neuronal regeneration mechanisms, we evaluated the activation of injury signals in a non-regenerative model. Injury signals include the positive injury signals, which are described as being enhancers of axonal regeneration by activating several transcription factors. The currently known positive injury signals are ERK, JNK and STAT3. To evaluate whether the lack of regeneration following injury to the central branch of DRG neurons was due to inactivation of these signals, activation of the transcription factors pELK-1, p-c-jun (downstream targets of ERK and JNK, respectively) and pSTAT3 were examined. Results have shown no impairment in the activation of these signals. As a consequence, we further proceed with evaluation of other candidates that could participate in axonal regeneration failure. By comparing the protein profiles that were triggered following either injury to the central branch of DRG neurons or injury to their peripheral branch, we were able to identify high levels of GSK3-β, ROCKII and HSP-40 after injury to the central branch of DRG neurons. While in vitro knockdown of HSP-40 in DRG neurons showed to be toxic for the cells, evaluation of pCRMP2 (a GSK3-β downstream target) and pMLC (a ROCKII downstream target), which are known to impair axonal regeneration, revealed high levels of both proteins following injury to the central branch when comparing with injury to their peripheral one. Altogether, these results suggest that activation of positive injury signals is not sufficient to elicit axonal regeneration; HSP-40 is likely to participate in the cell survival program; whereas GSK3-β and ROCKII activity may condition the regenerative capacity following injury to the nervous system.(...)

What hides behind the fixed-term contracts wage gap?

In Portugal, about 20% of full-time workers are employed under a fixed-term contract. Using a rich longitudinal matched employer-employee dataset for Portugal, with more than 20 million observations and covering the 2002-2012 period, we confirm the common idea that fixed-term contracts are not desirable when compared to permanent ones, by estimating a conditional wage gap of -1.7 log points. Then, we evaluate the sources of that wage penalty by combining a three way high-dimensional fixed effects model with the decomposition of Gelbach (2014), in which the three dimensions considered are the worker’s unobserved ability, the firm’s compensation wage policy and the job title effect. It is shown that the average worker with a fixed-term contract is less productive than his/her permanent counterparts, explaining -3.92 log points of the FTC wage penalty. Additionally, the sorting of workers into lower-paid job titles is also responsible for -0.59 log points of the wage gap. Surprisingly, we found that the allocation of workers among firms mitigates the existing wage penalty (in 4.23 log points), as fixed-term workers are concentrated into firms with a more generous compensation policy. Finally, following Figueiredo et al. (2014), we further control for the worker-firm match characteristics and reach the conclusion that fixed-term employment relationships have an overrepresentation of low quality worker-firm matches, explaining 0.65 log points of the FTC wage penalty.

Building a thinner gap in a gas-gap heat switch

25th International Cryogenic Engineering Conference and the International Cryogenic Materials Conference in 2014, ICEC 25–ICMC 2014

8-Phase Ring oscillator for modern receivers

The evolution of receiver architectures, built in modern CMOS technologies, allows the design of high efficient receivers. A key block in modern receivers is the oscillator. The main objective of this thesis is to design a very low power and low area 8-Phase Ring Oscillator for biomedical applications (ISM and WMTS bands). Oscillators with multiphase outputs and variable duty cycles are required. In this thesis we are focused in 12.5% and 50% duty-cycles approaches. The proposed circuit uses eight inverters in a ring structure, in order to generate the output duty cycle of 50%. The duty cycle of 1/8 is achieved through the combination of the longer duty cycle signals in pairs, using, for this purpose, NAND gates. Since the general application are not only the wireless communications context, as well as industrial, scientific and medical plans, the 8-Phase Oscillator is simulated to be wideband between 100 MHz and 1 GHz, and be able to operate in the ISM bands (447 MHz-930 MHz) and WMTS (600 MHz). The circuit prototype is designed in UMC 130 nm CMOS technology. The maximum value of current drawn from a DC power source of 1.2 V, at a maximum frequency of 930 MHz achieved, is 17.54 mA. After completion of the oscillator layout studied (occupied area is 165 μm x 83 μm). Measurement results confirm the expected operating range from the simulations, and therefore, that the oscillator fulfil effectively the goals initially proposed in order to be used as Local Oscillator in RF Modern Receivers.

Corporate performance and the pay gap

Increasing disparity between executive compensation and that of the average worker (the pay gap) has generated a fierce debate about its causes and effects. This paper studies the determinants and performance effects of the pay gap through the prism of Tournament Incentives and the Equity Fairness Theory. Results show that the size of the pay gap is caused primarily by the size of the firm and by the standards of its industry and also by the unionization rate and whether the Chairman is also the CEO. The paper Concludes by showins that the pay gap has a positive effect on firm performance in the United States Keywords:

The role of Hedgehog signaling during zebrafish larvae fin fold regeneration

Several studies have demonstrated that although the structure of the adult and larval zebrafish caudal fin is different, there are similarities at the cellular and molecular level that turn larval zebrafish fin fold a useful model to study the basic principles of regeneration. In this process, while the essential role for Hedgehog (Hh) signaling is well established in the adult zebrafish caudal fin system, its involvement in juvenile tissue regeneration is still unknown. The aim of this Master thesis was therefore to evaluate the contribution of the Hh signaling pathway to the larval zebrafish fin fold regeneration process. Accordingly, we analyzed the expression of several Hh signaling components through in situ hybridization. Here, we showed that several of these genes are effectively expressed in the larval regenerating fin tissue, suggesting a role for Hh signaling also during larval regeneration. However, divergence in the regulation of few Hh signaling components appears to exist between the adult and larval zebrafish fin regeneration processes. Nevertheless, similarly to adult caudal fin regeneration, when Hh signaling was blocked, by using cyclopamine, the larval fin fold regenerative outgrowth is severely impaired. Since larval zebrafish fin fold is ciliated, and primary cilia are closely related to Hh signaling regulation in vertebrate systems, we further addressed the role of primary cilia during larval fin fold regeneration process. To this end, we used the zebrafish iguana mutant, in which primary cilia are not formed, to study the modulation of Hh signaling expression during larval fin fold regeneration in the absence of primary cilia. Here, we found that several genes were expressed with a delay, coincident with the delay in the mutant fin fold regeneration observed in previous work. We show that Hh signaling in the fin fold is crucial to promote cell proliferation. When Hh signaling is blocked using cyclopamine there is a strong blockage of cell proliferation and regeneration is also blocked. Surprisingly, in iguana mutants where Hh signaling is impaired but not totally blocked, cell proliferation is not detected but regeneration still occurs. This raises the question about the requirement of cell proliferation in larvae fin fold regeneration. By blocking the cell cycle using aphidicolin we demonstrate that cell proliferation is not necessary for zebrafish larvae fin fold regeneration.

Decisions of technology innovation: the role of indicators

This work presents research conducted to understand the role of indicators in decisions of technology innovation. A gap was detected in the literature of innovation and technology assessment about the use and influence of indicators in this type of decision. It was important to address this gap because indicators are often frequent elements of innovation and technology assessment studies. The research was designed to determine the extent of the use and influence of indicators in decisions of technology innovation, to characterize the role of indicators in these decisions, and to understand how indicators are used in these decisions. The latter involved the test of four possible explanatory factors: the type and phase of decision, and the context and process of construction of evidence. Furthermore, it focused on three Portuguese innovation groups: public researchers, business R&D&I leaders and policymakers. The research used a combination of methods to collect quantitative and qualitative information, such as surveys, case studies and social network analysis. This research concluded that the use of indicators is different from their influence in decisions of technology innovation. In fact, there is a high use of indicators in these decisions, but lower and differentiated differences in their influence in each innovation group. This suggests that political-behavioural methods are also involved in the decisions to different degrees. The main social influences in the decisions came mostly from hierarchies, knowledge-based contacts and users. Furthermore, the research established that indicators played mostly symbolic roles in decisions of policymakers and business R&D&I leaders, although their role with researchers was more differentiated. Indicators were also described as helpful instruments to conduct a reasonable interpretation of data and to balance options in innovation and technology assessments studies, in particular when contextualised, described in detail and with discussion upon the options made. Results suggest that there are four main explanatory factors for the role of indicators in these decisions: First, the type of decision appears to be a factor to consider when explaining the role of indicators. In fact, each type of decision had different influences on the way indicators are used, and each type of decision used different types of indicators. Results for policy-making were particularly different from decisions of acquisition and development of products/technology. Second, the phase of the decision can help to understand the role indicators play in these decisions. Results distinguished between two phases detected in all decisions – before and after the decision – as well as two other phases that can be used to complement the decision process and where indicators can be involved. Third, the context of decision is an important factor to consider when explaining the way indicators are taken into consideration in policy decisions. In fact, the role of indicators can be influenced by the particular context of the decision maker, in which all types of evidence can be selected or downplayed. More importantly, the use of persuasive analytical evidence appears to be related with the dispute existent in the policy context. Fourth and last, the process of construction of evidence is a factor to consider when explaining the way indicators are involved in these decisions. In fact, indicators and other evidence were brought to the decision processes according to their availability and capacity to support the different arguments and interests of the actors and stakeholders. In one case, an indicator lost much persuasion strength with the controversies that it went through during the decision process. Therefore, it can be argued that the use of indicators is high but not very influential; their role is mostly symbolic to policymakers and business decisions, but varies among researchers. The role of indicators in these decisions depends on the type and phase of the decision and the context and process of construction of evidence. The latter two are related to the particular context of each decision maker, the existence of elements of dispute and controversies that influence the way indicators are introduced in the decision-making process.

Terapia celular com células mesenquimais por via intracoronária: contributos do estudo invasivo da microcirculação coronária

RESUMO: Introdução - A utilização de células e das suas propriedades para o tratamento das doenças cardiovasculares, é uma promessa para o futuro e talvez a única forma de ultrapassar algumas das insuficiências das terapêuticas atuais. A via de entrega das células mais utilizada na investigação tem sido a intracoronária, ganhando a microcirculação especial relevância, por ser onde ocorre a primeira interação com o tecido nativo. As células estaminais mesenquimais (CEM) têm propriedades que as tornam particularmente aptas para a Terapia Celular, mas as suas dimensões, superiores ao diâmetro dos capilares, tem motivado controvérsia quanto à sua entrega intracoronária. A cardiologia de intervenção tem atualmente técnicas que permitem a avaliação em tempo real e in vivo do estado da microcirculação coronária. A determinação do índice da resistência da microcirculação (IRM) fornece informação sobre a circulação dos pequenos vasos, de forma independente da circulação coronária e do estado hemodinâmico, mas a aplicabilidade clínica deste conhecimento encontra-se ainda por definir. Objectivos Esclarecer o potencial do IRM no estudo dos efeitos do transplante de CEM por via intracoronária. População e Métodos . Estudo pré-clínico com modelo animal (suíno) desenvolvido em 3 fases. Na Primeira Fase foram utilizados 8 animais saudáveis para estudar e validar a técnica de determinação de estudo da microcirculação. Efetuou-se a determinação do IRM com duas doses diferentes de papaverina para a indução da resposta hiperémica máxima (5 e 10 mg) e após a disfunção da microcirculação com injeção intracoronária de microesferas de embozene com 40 μm de diâmetro. Na Segunda Fase foram utilizados 18 animais saudáveis, randomizados em grupo controlo e grupo recetor de 30 x 106 CEM por via intracoronária. Foram avaliados de forma cega o IRM, a pressão aórtica, o fluxo coronário epicárdico e a ocorrência de alterações electrocardiográficas. Na Terceira Fase foram utilizados 18 animais, com enfarte agudo do miocárdio provocado (EAM), randomizados em grupo controlo, grupo recetor de CEM expandidas de forma convencional e grupo recetor de CEM expandidas com metodologia inovadora e de menores dimensões. Foi realizada uma exploração da dose/efeito com infusão faseada de 10 x 106, 15 x 106 e 20 x 106 CEM, com determinação do IRM, da pressão aórtica, do fluxo coronário epicárdico e da ocorrência de alterações eletrocardiográficas. Quatro semanas após a entrega das células foi novamente avaliado o IRM e foi efetuado o estudo anatomopatológico dos animais na procura de evidência de neoangiogénese e de regeneração miocárdica, ou de um efeito positivo da resposta reparadora após o enfarte. Resultados Nas 3 fases todos os animais mantiveram estabilidade hemodinâmica e eletrocardiográfica, com exceção da elevação de ST de V1-V3 verificada após a injeção das microesferas. Na Primeira Fase as duas doses de papaverina induziram uma resposta hiperémica eficaz, sem tradução com significado na determinação do IRM (variação da pressão distal de - 11,4 ± 5 e de - 10,6± 5 mmHg com as doses de 5 e 10 mg respetivamente (p=0,5). Com a injeção das microesferas o IRM teve uma elevação média de 310 ± 190 %, para um valor médio de 41,3 ± 16 U (p = 0,001). Na Segunda Fase não houve diferenças significativas dos parâmetros hemodinâmicos, do fluxo epicárdico e da avaliação eletrocardiográfica entre os dois grupos. O IRM de base foi semelhante e após a infusão intracoronária observou-se uma elevação expressiva do IRM nos animais que receberam células em comparação com o grupo controlo (8,8 U ± 1 vs. 14,2 U ± 1,8, P=0,02) e quanto ao seu valor de base (aumento de 112%, p=0,008). Na terceira Fase não houve novamente diferenças significativas dos parâmetros hemodinâmicos, do fluxo epicárdico e da avaliação eletrocardiográfica entre os três grupos. Houve uma elevação do IRM nos animais que receberam células a partir da 2ª dose (72% nas células convencionai e 108% nas células inovadoras) e que se manteve com a 3ª dose (100% nas células convencionais e 88% nas inovadoras) com significado estatístico em comparação com o grupo controlo (p=0,034 com a 2ªdose e p=0,024 com a 3ª dose). Quatro semanas após a entrega das CEM observou-se a descida do IRM nos dois grupos que receberam células, para valores sobreponíveis aos do grupo controlo e aos valores pós-EAM. Na avaliação anatomopatológica e histológica dos corações explantados não houve diferenças entre os três grupos. Conclusões O IRM permite distinguir alterações da microcirculação coronária motivadas pela entrega intracoronária de CEM, na ausência de alterações de outros parâmetros clínicos da circulação coronária utilizados em tempo real. As alterações do IRM são progressivas e passíveis de avaliar o efeito/dose, embora não tenha sido possível determinar diferenças com os dois tipos de CEM. No nosso modelo a injeção intracoronária não se associou a evidência de efeito benéfico na reparação ou regeneração miocárdica após o EAM.---------------------------- ABSTRACT: ABSTRACT Introduction The use of cells for the treatment of cardiovascular disease is a promise for the future and perhaps the only option to overcome some of the shortcomings of current therapies. The strategy for the delivery of cells most often used in current research has been the intracoronary route and due to this microcirculation gains special relevance, mainly because it is the first interaction site of transplanted cells with the native tissue. Mesenchymal stem cells (MSC) have properties that make them suitable for Cell Therapy, but its dimensions, larger than the diameter of capillaries, have prompted controversy about the safety of intracoronary delivery. The interventional cardiology currently has techniques that allow for real-time and in vivo assessment of coronary microcirculation state. The determination of the index of microcirculatory resistance index (IMR) provides information about small vessels, independently of the coronary circulation and hemodynamic status, but the clinical applicability of this knowledge is yet to be defined. Objectives To clarify the potential use of IMR in the study of the effects of MSC through intracoronary transplantation. Population and Methods Preclinical study with swine model developed in three phases. In Phase One 8 healthy animals were used to study and validate the IMR assessment in our animal model. IMR was assessed with two different doses of papaverine for inducing the maximal hyperaemic response (5 and 10 mg) and microcirculation dysfunction was achieved after intracoronary injection with embozene microspheres with 40 μm in diameter. In Phase Two we randomized 18 healthy animals divided between the control group and the one receiving 30 x 106 MSC through an intracoronary infusion. There we blindly evaluated IMR, the aortic pressure, the epicardial coronary flow and the occurrence of ECG changes. In Phase Three we used 18 animals with a provoked acute myocardial infarction (AMI), randomized into a control group, a MSC expanded conventionally receiver group and a MSC expanded with an innovative methodology receiver group. There was a stepwise infusion with doses of 10 x 106, 15 x 106 and 20 x 106 MSC with determination of IMR, the aortic pressure, the epicardial coronary flow and occurrence of electrocardiographic abnormalities. Four weeks after cell delivery we again measured the IMR and proceeded with the pathological study of animals in the search for evidence of neoangiogenesis and myocardial regeneration, or a positive effect in the reparative response following the infarction. Results All animals remained hemodynamically stable and with no electrocardiographic abnormalities, except for the ST elevation in V1-V3 observed after injection of the microspheres. In Phase One the two doses of papaverine achieved an hyperemic and effective response without significant differences in IMR (variation of the distal pressure -11.4 ± 5 and -10.6 ± 5 mmHg with the doses of 5 and 10 mg respectively (p = 0.5). With the injection of the microspheres the IMR had an average increase of 310 ± 190% for an average value of 41.3 ± 16 U (p = 0.001). In the second phase there were no significant differences in hemodynamic parameters, epicardial flow and electrocardiographic assessment between the two groups. The baseline IMR was similar and after intracoronary infusion there was a significant increase in animals receiving cells compared with the control group (8.8 ± U 1 vs. 14.2 ± 1.8, p = 0.02) and with their baseline (112% increase, p = 0.008). In the third phase again there were no significant differences in hemodynamic parameters, the epicardial flow and electrocardiographic evaluation between the three groups. There was a significant increase in IMR in animals that received cells from the 2nd dose (72% in conventional cells and 108% in the innovative cells) that remained with the 3rd dose (100% in conventional cells and 88% in the innovative) with statistical significance compared with the control group (p = 0.034 with 2nd dose, p = 0.024 with 3rd dose). Four weeks after delivery of the MSC we observed the fall of the IMR in the two groups that received cells with values overlapping those of the control group. In pathological and histological evaluation of removed hearts there were no differences among the three groups. Conclusions The IMR allows for the differentiation of changes in coronary microcirculation motivated by intracoronary delivery of MSC in the absence of modification in other clinical parameters. IMR changes are progressive and enable the evaluation of the effect / dose, though it has not been possible to determine differences in the two types of MSC. In our model, intracoronary injection of MSC was not associated with evidence of repair or myocardial regeneration after AMI.