Action Recognition in Videos: from Motion Capture Labs to the Web

This paper presents a survey of human action recognition approaches based on visual data recorded from a single video camera. We propose an organizing framework which puts in evidence the evolution of the area, with techniques moving from heavily constrained motion capture scenarios towards more challenging, realistic, "in the wild" videos. The proposed organization is based on the representation used as input for the recognition task, emphasizing the hypothesis assumed and thus, the constraints imposed on the type of video that each technique is able to address. Expliciting the hypothesis and constraints makes the framework particularly useful to select a method, given an application. Another advantage of the proposed organization is that it allows categorizing newest approaches seamlessly with traditional ones, while providing an insightful perspective of the evolution of the action recognition task up to now. That perspective is the basis for the discussion in the end of the paper, where we also present the main open issues in the area.Comment: Preprint submitted to CVIU, survey paper, 46 pages, 2 figures, 4 table

Portuguese and Brazilian family business: in between urgency and delay perceptions in the succession process

Family business has been the focus of several studies over the last two decades and its relevance has been supported by the interdisciplinary perspectives in the fields of management, entrepreneurship, economics, psychology, and sociology. Despite that, there is still insufficient knowledge about the key role of family influences in the business, namely the intergenerational management succession, its planning and effectiveness. According to a recent research focused on the entrepreneurial succession in Portugal (AEP, 2011), 50% of family businesses are not passed on to the second generation and only 20% reach the third generation. In fact, business succession planning has been identified as one of the most challenging steps in the life of the family firm, both in maintaining the competiveness of the business, and in overcoming intra/ inter family conflicts. Nonetheless, resistance to succession, relationship founder/ successor, planning of succession, and type of organisational culture, among others, explain how executive succession is one of the most important and hardest tasks in organisational life (Zahra, 2005). This paper will be supported mainly by qualitative data, taking into account the main results from the project “Roadmap for Portuguese Family Businesses” (NORTE2020/FEDER) developed in Portugal (Marques, 2018) and in Brazil (Silva, 2018), which analyses in-depth interviews conducted to Portuguese (N 23) and Brazilian (N 11) founders/managers/owners. In the present article we wish to discuss the main management challenges of a family business, particularly the importance of succession preparation and the role of the family in the socialisation of the second (third or subsequent) generation.NORTE-02-0853-FEDER-00001

Final Conference Program

List of talks during the conference

Decoding human cardiac stem cells regenerative potential in acute myocardial infarction

Acute Myocardial Infarction (AMI) remains a leading cause of death worldwide. After AMI, clinical restauration of blood flow aggravates tissue damage (Ischemia/Reperfusion, I/R injury), critically decreasing the number of viable cardiomyocytes (CMs). Human myocardium harbors a population of endogenous cardiac stem/progenitor cells (CSCs) that is activated upon I/R injury, contributing to myocardial repair through the establishment of an auto/paracrine molecular crosstalk between CSCs and CMs in stress. Transplantation of CSCs is currently being tested in several clinical trials, and although some improvements have been reported regarding decrease of the infarcted area, it is still not enough to show benefit over pharmacological standard-of-care. Our work aims at combining the development of relevant I/R in vitro human cell models with implementation of advanced mass spectrometry (MS)-based proteomic tools to further characterize hCSC and unveil associated regenerative mechanisms upon AMI. hCSCs employed in the phase I/II clinical trial CARE-MI (NCT02439398) were used (allogeneic therapy). Different strategies were explored to recapitulate both phases of I/R injury in the human adult heart, including: the use of human adult/mature cells, 3D culture system and stirred-tank bioreactor technology. Firstly, we developed a transwell co-culture cell based I/R model, with human CSCs and human induced pluripotent stem cell derived CMs (hiPSC-CMs). Following this work, and aiming at further improving the relevance of the I/R injury in vitro setup, 3D hiPSC-CM aggregate cultures and bioreactors were combined, allowing the control/monitoring of environmental parameters such as pH and dissolved oxygen, critical in the context of I/R physiology. Important features of I/R injury were successfully captured in the two models, including hiPSC-CM death upon reperfusion, disruption of cell ultra-structure organization, as well as increased release of angiogenic and inflammatory cytokines, consistent with the described pathophysiology of AMI. hCSCs response to I/R was further probed using whole proteome analysis (including quantitative SWATH methodology), allowing us to propose new pathways in the hCSCs-mediated regenerative process along the different phases of I/R injury through the identification of more than 3800 proteins and quantification of 714 proteins. Our data shows that our AMI-setup up-regulates hCSC proteins associated with several pro-migratory, proliferation and stress response-related pathways. Moreover, our results reinforce the idea that paracrine-mediated mechanisms are a central response in hCSC activation, with the enrichment of several paracrine signaling and pro-angiogenic pathways. We also show for the first time increased CXCL6 secretion by hCSCs upon injury, suggesting a relevant role of this angiogenic cytokine in hCSC mediated myocardial regeneration. Overall, multiple strategies were used to develop novel and robust I/R injury in vitro models, recapitulating several features of the human adult myocardium. The systems established allowed to better characterize hCSC mechanisms of action in response to AMI contexts. The knowledge generated has the potential to be used in the development of novel strategies excelling endogenous and transplanted hCSCs regenerative potential

Unveiling human Cardiac Stem Cells regenerative potential in Ishemia/Reperfusion Injury

After an Acute Myocardial Infarction (AMI), Ischemia-Reperfusion (I/R) injury is responsible for a critical decrease in the number of viable cardiomyocytes (hCMs). Human myocardium harbors a population of endogenous cardiac stem cells (hCSCs) that is activated upon I/R injury, contributing to myocardial repair through the establishment of an auto/paracrine molecular crosstalk between hCSCs and hCMs in stress. Clinical trials involving transplantation of hCSCs into the infarcted myocardium have demonstrated the potential of these cells. Although some improvements have been reported regarding increase in viable myocardium and improved tissue contractility, extensive data indicates that transplanted cells do not survive in the myocardium and this has led to the postulation of a paracrine mechanism for the observed beneficial effects. Using the same cells currently employed in the allogenous hCSCs transplantation clinical trial CARE-MI (EUDRA 2013-001358-81), our work aims at setting up the first in vitro human I/R injury model in order to better decipher the mechanisms of action of hCSCs upon AMI using proteomic tools. Mono-cultures of donor derived hCSCs, hCMs and co-cultures with the two cell types were established using human donor derived CSCs and cardiomyocytes derived from human induced pluripotent stem cells at different maturation stages (hiPSC-CMs). Ischemia was mimicked by substituting growth media by Ischemia Mimetic Solution and placing the cells at 0% O2 for 5 hours. In the reperfusion step, cells were placed back in their physiological culture conditions (3% O2). The effect of I/R injury in hCSCs was accessed by total proteome analysis (using LC-MS) at different time points. Growth factor secretion, cells’ viability, as well as hCSCs proliferation was also monitored in both mono- and co-culture systems. More than 2000 proteins were identified in hCSCs exposed to injury including proteins associated with mitochondrial dysfunction and oxidative stress. Important features of I/R injury were successfully captured, namely hCSCs proliferation activation upon insult, increase in key growth factors secretion, and the protective effect of hCSCs on hiPSC-CMs. This system will allow further understanding on the molecular landscape of the myocardium during AMI, namely regarding hCSCs regenerative response and hCMs survival. The knowledge generated in this work will hopefully potentiate the development of novel molecular and cell-based therapies for myocardium regeneration. The authors acknowledge the projects CARE-MI (HEALTH-2009-242038), CARDIOSTEM (MITP-TB/ECE/0013/2013, the Portuguese Foundation for Science and Technology- FCT (PTDC/BBB-BIO/1414) and iNOVA4Health (UID/Multi/04462/2013) for financial support. MJCS is a recipient of the FCT fellowship SFRH/BPD/52339/2013

hiPSC and hiPSC-cardiomyocytes are alternative EV biofactories for cardiac regeneration

In cardiac regenerative medicine, there is a growing interest in using EV as cell-mimetic therapeutics due to their potential superior efficacy and overall advantages over cell transplantation: i) absence of oncogenic risk, ii) low immunogenicity [1], iii) easier large-scale manufacturing and iv) consistent product profile. However, most studies have focused particularly on the potential of either mesenchymal or cardiac progenitor cell-derived EV to promote cardiac repair [2]. Here, we study the potential of human induced pluripotent stem cells (hiPSC) and hiPSC-derived cardiomyocytes (hiPSC-CM) as alternative cell factories for the production of a high yield of therapeutic EV for cardiac regeneration. Due to their high self-renewal ability and capacity to differentiate into functional cardiomyocytes, these cells can provide an unlimited source of EV for application in cardiac regeneration. We generated and characterized EV derived from key stages of hiPSC-CM differentiation and maturation, i.e. from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the goal of studying their potential role as therapeutics, and whether their yield and function was influenced by the state of their parent cell. Two hiPSC lines were differentiated into hiPSC-CM and cultured as 3D spheroids in a fatty acid supplemented medium to improve CM maturation [3,4]. EV isolation was performed based on density separation on an iodixanol discontinuous gradient, and EV were characterized in terms of particle size and particle size distribution, presence of EV-specific markers, and imaging through transmission electron microscopy. Functional studies were performed using human umbilical vein endothelial cells (HUVECs) to evaluate EV-uptake, migration and angiogenesis. EV yield varied along CM differentiation stages, with a minimum for CPC, for both cell lines. Bioactivity assays with HUVECs showed that uptake of PKH26-labelled EV could be blocked by dynasore, an inhibitor of dynamin-2, a GTPase that plays a crucial role in clathrin and caveolin-dependent endocytosis. Increased migration was observed in HUVECs treated with hiPSC, CPC and CM-derived EV (92.25 ± 14.69% wound closure at 24h for hiPSC, 77.13 ± 13.64 % for CPC, 74.71 ± 19.86% for CMi, 69.2 ± 19.12% for CMm versus 45.65 ± 7.26% for control), but angiogenic properties were found only for hiPSC-EV (fold change of 11.2 ± 4.59 in total segment length vs. control, p\u3c0.001). Current efforts towards the characterization of EV small RNA cargo aim at understanding the correlation between cargo composition and in vitro activity, to identify the optimal cell factory for scalable therapeutic EV production. Funding: FCT PhD fellowship PD/BD/139078/2018; IC&TD Projects MetaCardio” (PTDC/BTM-SAL/32566/2017) and NETDIAMOND (SAICTPAC/0047/2015), and iNOVA4Health Research Unit (UIDB/04462/2020). 1. Zhu, X., et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. Journal of Extracellular Vesicles 6, 1, 2017. 2. El Harane, et al. Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors. European Heart Journal 39, 20, 2018. 3. Correia, C., et al. Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Scientific reports 7, 1, 2017. 4. Correia, C., et al. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. Biotechnology and Bioengineering 115, 3, 2018

Négociation de succession intergénérationnelle aux “mains de la famille”: témoignages d’entreprises familiales portugaises et brésiliennes

O processo de sucessão intergeracional constitui-se numa das etapas mais desafiadoras na vida de uma empresa familiar tanto na competitividade do negócio, quanto na superação de conflitos intra/interfamiliares. Dada a insuficiência do conhecimento sobre a influência familiar nos negócios, pretende-se ampliar os resultados do projeto “Roadmap para Empresas Familiares Portuguesas”, tomando como referência empresas familiares portuguesas e brasileiras. A partir de um desenho de pesquisa qualitativo, analisam-se as estratégias de negociação de (potenciais) conflitos no processo de sucessão e respetivas perceções de empresários/gestores, aprofundando o conhecimento sobre os principais desafios na passagem do “legado” empresarial.The succession process of intergenerational management is one of the most challenging steps in the life of a family firm, both in terms of business competitiveness and in overcoming intra/ interfamily conflicts. Given the insufficiency of knowledge regarding family influence on business, the purpose of this paper is to expand the main findings of the project “Roadmap para Empresas Familiares Portuguesas”, taking into account Portuguese and Brazilian family businesses. Based on a qualitative research design, the negotiation strategies of (potential) conflicts in the succession process and respective perceptions of founders/managers are analysed, deepening the knowledge about the main challenges in the passage of the business “legacy”.Le processus de succession intergénérationnelle constitue l’une des étapes les plus difficiles de la vie d’une entreprise familiale, tant en matière de compétitivité de l’entreprise que de dépassement des conflits intra/interfamiliaux. Compte tenu du manque de connaissances sur l’influence familiale sur les affaires, nous avons l’intention d’élargir les résultats du projet « Roadmap para Empresas Familiares Portuguesas » en prenant comme référence des entreprises familiales portugaises et brésiliennes. Sur la base d’un plan de recherche qualitative, les stratégies de négociation des conflits (potentiels) dans le processus de succession et les perceptions respectives des fondateurs/dirigeants sont analysées, en approfondissant les connaissances sur les principaux défis dans le passage de « l’héritage » d’entreprise

Monitoring the production of AAV vectors in insect cells by fluorescence spectroscopy

Adeno-associated viruses (AAV) are among the most promising viral vectors for gene therapy, since they can transduce non-dividing cells from several tissues while maintaining a long-term gene expression. Besides, AAVs possess low immunogenicity compared to other viral vectors, and are physically resistant, which makes them resilient to industrial manufacturing conditions, long-term storage, and in vivo administration. One of the systems available for large scale production of AAVs is the insect cell-baculovirus expression vector system (IC-BEVS). Insect cells grow in suspension to high cell densities with modest growth requirements and without the need of serum supplementation. Consequently, scaling up the production in order to achieve the large number of AAV needed for clinical trials is more straight‑forward than with transfection-based systems. However, methods for online monitoring of AAV production are still lacking. Such methods would allow determination of the best time of harvest in real-time, thus allowing recovery of AAV as soon as its concentration medium was higher. Here we apply Fluorescence Spectroscopy to baculovirus-infected insect cell cultures producing adeno‑associated virus vectors, correlating the spectra to critical process parameters like cell concentration, viability and AAV concentration. Sf9 cells were co-infected with two baculovirus (expressing AAV rep and cap and a CMV-GFP transgene) at low or high multiplicities of infection (MOI), and the culture was followed by Fluorescence Spectroscopy in situ through a bioreactor probe. After an exploratory calibration using data from only one bioreactor, we attested the aptitude of this technique to capture overall data trend: using a 3 component PLS model, we have obtained a calibration NRMSE of 2.9% for total AAV particles per cell, 5.9% for viable cell density and 0.9% for viability). Additional bioreactor productions using different infection parameters (CCI, MOI, time of infection) allowed testing the robustness of fluorescence monitoring to process variability. With this dataset, we tested several pre-treatment methods for the raw spectra, as well as different regression algorithms in order to establish a good predictive model. Ultimately, fluorescence spectroscopy provides a simple tool for online monitoring of key process variables in baculovirus-infected insect cell cultures. Acknowledgments: Funding from Fundação para a Ciência e a Tecnologia, projects EXPL/BBBBIO/1129/2013 and Daniel Pais’ PhD research grant PD/BD/105873/2014

A novel β-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharification

ABSTRACT: An intracellular β-xylosidase (AbXyl), fromthe thermoalkaline Anoxybacillus sp. 3M,was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by β-mercaptoethanol. In the presence of p-nitrophenyl-β-D-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s−1, KcatKm−1 of 2322 s−1mMandwas moderately influenced by xylose (Ki 21.25mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.info:eu-repo/semantics/publishedVersio