Innovation-based Nets as Collective Actors: A Heterarchization Case Study from the Automotive Industry

Cooperation and collaboration between companies represents a key issue within the conceptual framework developed by the IMP Group. However, little attention has been paid to a phenomenon which can result from such collaboration, i.e. collective action. This involves cooperative activities undertaken by a significant number of actors sharing a common aim. This research uses the concept of issue-based net to open new avenues to understand collective action in the context of innovation activities, specifically by analyzing a case study of an innovation-based net in the automotive industry. Two main objectives are addressed in this study: Related to this discussion of different development paths of collective actors, the case study analysis focuses on how issue-based nets emerge and evolve in situations of innovation, specifically, what kind of structure and process issues characterize a heterarchization development path. Furthermore, the analysis addressed how issue-based nets change the positioning of individual member firms, a well as that of the collective actor within the overall network.Innovation, collective actor, issue-based nets, heterarchization, case study, automotive industry

The effects of oil dependence on growth volatility

In this thesis, we analyze the relationship between dependence on oil exports and growth volatility, controlling for other determinants. We collect annual data from 1995 to 2015 on a sample of 42 oil net exporting countries and use the standard system generalized methods of moments (GMM) approach developed by Arellano and Bover (1995) and Blundell and Bond (1998). We also investigate the channels that moderate this effect through macroeconomic policies suggested by policymakers and we find evidence that supports the mitigating effect of financial development, institutional quality and human capital on the transmission of oil dependence on growth volatility

Classification approach for diagnosis of arteriosclerosis using B-mode ultrasound carotid images

Tese de mestrado. Engenharia Biomédica. Faculdade de Engenharia. Universidade do Porto. 201

Produção e purificação de biofármacos antileucémicos utilizando nanomateriais

Biopharmaceuticals are a natural alternative to chemically synthesized pharmaceuticals. They can be based on enzymes, hormones, monoclonal antibodies, cytokines, among others; and are already applied in the treatment of many diseases that do not reply to common therapies. The enzyme L-asparaginase (L-ASNase) is a biopharmaceutical already used in treatments of many leukemias, namely Acute Lymphoblastic Leukemia (ALL), or Hodgkin’s disease. Particularly, in ALL, type of leukemia that attacks mostly children and young people, L-ASNase acts as an inhibitor of the carcinogen cells growth. However, the L-ASNase commercially available, and from Escherichia coli, contains a high activity of L-glutaminase, causing side effects, for example edema, fever, diabetes and haemorrhages, in the patients. Therefore, the search for new microorganisms that produce L-ASNase with decreased secondary effects is of extreme importance. In this work, a recombinant L-ASNase was produced from Bacillus Subtilis. After that, as the conventional methods for the purification of this enzyme are very expensive, it was developed a new purification technique for L-ASNase, more economical, through the usage of supported ionic liquids (SIL). Three SILs were synthesized and characterized, and different experimental conditions were tested, in order to optimize the purification process of L-ASNase, namely the cell extract total protein concentration and the material/cell extract ratio. At last, a semi-continuous assay was performed with the optimized conditions. The results obtained demonstrate that optimum experimental conditions for L-ASNase purification occur at a cell extract concentration of 16.7 mg/mL (original total protein concentration after cell lysis) and 50 mg of material per each mL of cell extract, corresponding to a specific activity of L-ASNase of 0.0382 U/mg and a purification fold of 3.46.Os biofármacos são uma alternativa natural aos fármacos quimicamente sintetizados. Estes podem ter como base enzimas, hormonas, anticorpos monoclonais, citocinas, entre outros; e já são aplicados no tratamento de diversas doenças que não respondem às terapias comuns. A enzima L-asparaginase (L-ASNase) é um biofármaco atualmente usado no tratamento de diversas leucemias, nomeadamente a Leucemia Linfoblástica Aguda (LLA), ou doença de Hodgkin. No caso específico da LLA, tipo de leucemia que ataca maioritariamente crianças e jovens, a L-ASNase atua como um inibidor do crescimento das células cancerígenas. Porém, a L-ASNase atualmente comercializada, e proveniente de Escherichia coli, contém uma atividade elevada de L-glutaminase, provocando efeitos secundários, como edema, febre, diabetes e hemorragias, nos pacientes. Deste modo, a busca por novos microorganismos que produzam L-ASNase com efeitos colaterais diminuídos é de extrema importância. Neste trabalho, produziu-se uma L-ASNase recombinante por Bacillus Subtillis. Posteriormente, e uma vez que os métodos convencionais para a purificação desta enzima são muito dispendiosos, desenvolveu-se uma nova técnica de purificação para a L-ASNase mais económica através da utilização de líquidos iónicos suportados (LIS). Foram sintetizados e caracterizados três LIS e testaram-se diferentes condições experimentais de forma a otimizar o processo de purificação da L-ASNase, nomeadamente a concentração do extrato celular e a razão material/extrato celular. Por fim, efetuou-se um ensaio em semi-contínuo, com as condições já otimizadas. Os resultados obtidos indicam que as condições ótimas de purificação da L-ASNase ocorrem com uma concentração de proteína total do extrato celular de 16.7 mg/mL (concentração de proteína total obtida após a lise celular) e uma massa de 50 mg de material por cada mL de extrato celular, correspondentes a uma atividade específica de L-ASNase de 0.0382 U/mg e fator de purificação de 3.46.Mestrado em Engenharia Químic

understanding the situation of unaccompanied minors in Europe - challenges and possibilities

Menores não acompanhados requerentes de asilos que deixam o seu país de origem são confrontados com múltiplos desafios e situações de perigo quando estão a caminho do seu país de destino. No entanto, mesmo quando chegam a países tidos como sendo seguros e respeitadores de direitos humanos, estes menores sofrem ainda várias violações a este nível, perpetuadas pelos estados receptores. Esta tese tem em consideração este tópico em solo da União Europeia, focando-se no mais importante instrumento legal internacional de direitos humanos para a protecção de crianças, a Convenção sobre os Direitos da Criança, e a principal directiva para a recepção de requerentes de asilo, bem como tendo em conta outros documentos vinculativos e não vinculativos, doutrina e jurisprudência relevantes. Consagrado na Convenção está uma parte controversa e essencial desta, o princípio do melhor interesse da criança, sobre o qual esta dissertação irá reflectir ao analisar a detenção de um grupo vulnerável, os menores não acompanhados requerentes de asilo, em território pertencente à União Europeia. De forma a conseguir isto, a tese analisa as diferentes fases que levam e influenciam a detenção de menores - os processos à chegada, o procedimento de asilo e as garantias legais processuais. Por fim, elabora-se uma reflecção sobre o acto de detenção de menores não acompanhados requerentes de asilo através da analise dos princípios de “último recurso” e “pela mínima duração possível”. Assim sendo, a tese ambiciona clarificar a famosa, mas desconhecida, situação dos menores não acompanhados requerentes de asilo que chegam à Europa e as violações de direitos humanos de que são vítimas, olhando para como a ilegalidade dos actos perpetuados é provada através de documentos e princípios legais internacionais, regionais e nacionais, mesmo se estes são defendidos e perpetuados por certos Estados.Unaccompanied asylum seeking minors leaving their countries of origins face a serious of challenges and dangerous situations on the way to their countries of destination. However, even when they arrive at safe countries they still face a number of violations of human rights, perpetuated by the receiving states. This thesis approaches this matter in the European Union, focusing on the main international human rights instrument protecting children, the Convention on the Rights of the Child, and the central European directive on the reception of asylum seekers, alongside other relevant binding and non-binding legal documents, doctrine and jurisprudence. Enshrined in the Convention is the best interest of the child principle, a controversial and essential part of it, which this dissertation will reflect upon through the detention of unaccompanied asylum seeking minors, a vulnerable group, in European Union territory. In order to do this, the thesis analyses the different stages that lead and affect the detention of minors - arrival procedures, asylum granting processes and judicial guarantees. Finally, it seeks to reflect on the act of the detention of unaccompanied asylum seeking minors itself by its assessing its legality concerning the principle of last resort and the principle of minimum time of detention as possible. As such, this thesis aims to shine light on the numerous violations of human rights that unaccompanied asylum seeking minors often suffer upon arrival in Europe and how their illegality is proven through a series of international, regional and national legally binding articles and principles, even if these acts are continuously perpetuated and states attempt to justify them.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.FCT grants: (SFRH/BD/80717/2011, EXPL/BBB-IMG/0363/2013, PTDC/EBB-BIO/119243/2010, PTDC/EBB-BIO/112786/2009, SFRH/BD/52202/2013, SFRH/BD/78308/2011)

Immune microenvironment dynamics of HER2 overexpressing breast cancer under dual anti-HER2 blockade

IntroductionThe clinical prognosis of the HER2-overexpressing (HER2-OE) subtype of breast cancer (BC) is influenced by the immune infiltrate of the tumor. Specifically, monocytic cells, which are promoters of pro-tumoral immunosuppression, and NK cells, whose basal cytotoxic function may be enhanced with therapeutic antibodies. One of the standards of care for HER2+ BC patients includes the combination of the anti-HER2 antibodies trastuzumab and pertuzumab. This dual combination was a breakthrough against trastuzumab resistance; however, this regimen does not yield complete clinical benefit for a large fraction of patients. Further therapy refinement is still hampered by the lack of knowledge on the immune mechanism of action of this antibody-based dual HER2 blockade.MethodsTo explore how the dual antibody challenge influences the phenotype and function of immune cells infiltrating the HER2-OE BC microenvironment, we developed in vitro 3D heterotypic cell models of this subtype. The models comprised aggregates of HER2+ BC cell lines and human peripheral blood mononuclear cells. Cells were co-encapsulated in a chemically inert alginate hydrogel and maintained in agitation-based culture system for up to 7 days.ResultsThe 3D models of the HER2-OE immune microenvironment retained original BC molecular features; the preservation of the NK cell compartment was achieved upon optimization of culture time and cytokine supplementation. Challenging the models with the standard-of-care combination of trastuzumab and pertuzumab resulted in enhanced immune cytotoxicity compared with trastuzumab alone. Features of the response to therapy within the immune tumor microenvironment were recapitulated, including induction of an immune effector state with NK cell activation, enhanced cell apoptosis and decline of immunosuppressive PD-L1+ immune cells.ConclusionsThis work presents a unique human 3D model for the study of immune effects of anti-HER2 biologicals, which can be used to test novel therapy regimens and improve anti-tumor immune function

Heterotypic Tumor Spheroids in Agitation-Based Cultures: A Scaffold-Free Cell Model That Sustains Long-Term Survival of Endothelial Cells

Endothelial cells (ECs) are an important component of the tumor microenvironment, playing key roles in tumor development and progression that span from angiogenesis to immune regulation and drug resistance. Heterotypic tumor spheroids are one of the most widely used in vitro tumor microenvironment models, presenting improved recapitulation of tumor microenvironments compared to 2D cultures, in a simple and low-cost setup. Heterotypic tumor spheroid models incorporating endothelial cells have been proposed but present multiple limitations, such as the short culture duration typically obtained, the use of animal-derived matrices, and poor reproducibility; the diversity of culture conditions employed hinders comparison between studies and standardization of relevant culture parameters. Herein, we developed long-term cultures of triple heterotypic spheroids composed of the HCC1954 tumor cell line, human fibroblasts, and ECs. We explored culture parameters potentially relevant for EC maintenance, such as tumor cell line, seeding cell number, cell ratio, and agitation vs. static culture. In HCC1954-based spheroids, we observed maintenance of viable EC for up to 1 month of culture in agitation, with retention of the identity markers CD31 and von Willebrand factor. At the optimized tumor cell:fibroblast:EC ratio of 1:3:10, HCC1954-based spheroids had a higher EC area/total spheroid area at 1 month of culture than the other cell ratios tested. EC maintenance was tumor cell line-dependent, and in HCC1954-based spheroids it was also dependent on the presence of fibroblasts and agitation. Moreover, vascular endothelial growth factor (VEGF) supplementation was not required for maintenance of EC, as the factor was endogenously produced. ECs co-localized with fibroblasts, which accumulated preferentially in the core of the spheroids and secreted EC-relevant extracellular matrix proteins, such as collagen I and IV. This simple model setup does not rely on artificial or animal-derived scaffolds and can serve as a useful tool to explore the culture parameters influencing heterotypic spheroids, contributing to model standardization, as well as to explore molecular cross talk of ECs within the tumor microenvironment, and potentially its effects on drug response

Um espaço uterino num tempo de tensão : Psicossomática, depressão e imunidade : Estudo de caso – Um olhar sob o ponto de vista da psicossomática

Dissertação de Mestrado apresentada ao ISPA - Instituto UniversitárioO presente estudo procurou observar, descrever e integrar num sistema compreensivo, os elementos depressivos presentes em determinada forma de Patologia Psicossomática – Hipertensão e Cancro do Colo do Útero – recorrendo-se ao método do Estudo de Caso, observando um caso individual, através do material de dez sessões onde se utilizou a Entrevista Clínica Semi-Directiva, da aplicação dos Testes Projectivos, Rorschach e Thematic Apperception Test (TAT), e do Teste Instrumental, Figura de Rey-Osterrieth. É um estudo de cariz marcadamente exploratório, naturalmente subjectivo que entende o sujeito como único: o sujeito como unidade psicossomática. Observou-se no caso estudado a presença de diversos elementos psicopatológicos associados à patologia Psicossomática, além de uma posição depressiva não elaborada, produzindo do ponto de vista biológico uma desregulação e desorganização do sistema imunitário, que esteve na base da Hipertensão e do Cancro do Colo do Útero. Este, é essencialmente um trabalho que apela à relação e que como a maioria das investigações com este desenho metodológico, recorre à interpretação, assumindo toda a subjectividade científica que a encerra.This study observes, describes and integrates into a comprehensive system the depressive elements of a specific form of Psychosomatic Pathology – Hypertension and Uterine Cancer – which were found after analysis of an individual case study. In the ten sessions analysed were used the Semi-Directive Clinical Interview and several types of tests like Projective Tests, Rorschach and Thematic Apperception Test (TAT) and the Instrumental Test, Rey-Osterrieth Complex Figure. This work is an exploratory study, naturally subjective than perceive the subject as unique: the subject as a psychosomatic unit. Several psychopathological elements related with the psychosomatic pathology were observed, along with a non elaborated depressive position which, from a biological point of view, has produced a deregulation and disorganization of the immune system which formed the basis of Hypertension and Uterine Cancer. This work appeals essentially to relation and like most research using this methodology it makes use of interpretation, hence assuming all inherent scientific subjectivity