Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

Role of Notch signaling on the differentiation of early lymphoid progenitors cells: a view throughout the development of the embryonic chicken thymus and spleen

Dissertação para a obtenção do grau de Mestre em Genética Molecular e Biomedicina

Genetic and functional analysis of the bacillus subtilis BSP1 gam cluster

Mannans (linear mannan, glucomannan, galactomannan and galactoglucomannan) are the major constituents of the hemicellulose fraction in softwoods and show great importance as a renewable resource for fuel or feedstock applications. As complex polysaccharides, mannans can only be degraded through a synergistic action of different mannan-degrading enzymes, mannanases. Microbial mannanases are mainly extracellular enzymes that can act in wide range of pH and temperature, contributing to pulp and paper, pharmaceutical, food and feed, oil and textile successful industrial applications. Knowing and controlling these microbial mannan-degrading enzymes are essential to take advantage of their great biotechnological potential. The genome of the laboratory 168 strain of Bacillus subtilis carries genes gmuA-G dedicated to the degradation and utilization of glucomannan, including an extracellular -mannanase. Recently, the genome sequence of an undomesticated strain of B. subtilis, BSP1, was determined. In BSP1, the gmuA-G operon is maintained, interestingly, however, a second cluster of genes was found (gam cluster), which comprise a second putative extracellular β-mannanase, and most likely specify a system for the degradation and utilization of a different mannan polymer, galactoglucomannan. The genetic organization and function of the gam cluster, and whether its presence in BSP1 strain results in new hemicellulolytic capabilities, compared to those of the laboratory strain, was address in this work. In silico and in vivo mRNA analyses performed in this study revealed that the gam cluster, comprising nine genes, is organized and expressed in at least six different transcriptional units. Furthermore, cloning, expression, and production of Bbsp2923 in Escherichia coli was achieved and preliminary characterization shows that the enzyme is indeed a β-mannanase. Finally, the high hemicellulolytic capacity of the undomesticated B. subtilis BSP1, demonstrated in this work by qualitative analyses, suggests potential to be used in the food and feed industries.

Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency

RESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de superfície celular ancoradas a GPI apresentam várias funções essenciais para a manutenção celular. A deficiência na síntese de GPI é o que caracteriza principalmente a deficiência hereditária em GPI, um grupo de doenças autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento, impondo a compactação da cromatina, associada à hipoacetilação de histonas, e consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência de hemólise intravascular e anemia, sendo que estas duas últimas características definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular. Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA variam entre células primárias hematopoiéticas normais. Adicionalmente, a configuração dos nucleossomas no promotor de PIGM é mais compacta em células B do que em células eritróides e tal está correlacionado com os níveis de expressão de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expressão de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrição de PIGM em ambas as células B e eritróide. Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC. Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com IGD associada a PIGM, uma das principais características que define a HPN. Por último, resultados preliminares mostraram que a repressão da transcrição de PIGM devida à mutação patogénica -270C>G está associada com a diminuição da frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”, sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, específico-detecido, por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

Leadership paradoxes in Angolan organizations: Emic paradoxes, etic paradoxes, and paradox work

The study departs from two assumptions. First, it considers that organizations and their leadership are inherently paradoxical and that, in that sense, dealing with paradox is a necessary component of the leadership process. Second, it explores whether the paradoxes of leadership may manifest differently in different contexts. We explore the emergence of paradox in the leadership of Angolan organizations. Angola is an economy transitioning from a centrally-planned to a market mode, and this makes it a rich site for understanding the specificities of paradoxical processes in an under-researched, “rest of the world”, context. The findings of our inductive study led to the emergence of four interrelated paradoxes and highlight the importance of paradoxical work as a management requirement.

A medição de eventos adversos hospitalares através dos dados administrativos

RESUMO - A qualidade e segurança são pilares essenciais dos sistemas de saúde modernos. A sua monitorização e avaliação tem como primeiro passo o conhecimento da realidade no que se refere aos eventos adversos que afetam os utentes. Existem diversas metodologias de medição de eventos adversos. A revisão de processos clínicos, apesar de constituir o padrão de ouro, não permite, ao contrário da análise dos dados administrativos, avaliar de forma abrangente, os episódios de internamento. Esta metodologia a partir de dados recolhidos rotineiramente em inúmeros países, como Portugal, apresenta porém diversas limitações, para as quais têm sido instituídas soluções tais como a sinalização do momento de aquisição do diagnóstico, que pela recente instituição não foi utilizada neste trabalho. Num hospital do Sistema nacional de saúde em Portugal, pela análise dos dados administrativos, determinou-se nos episódios de internamento cirúrgico uma incidência de 2,5% de eventos adversos. Comprovou-se a relação de idade, sexo masculino e admissão urgente com a sua ocorrência. Os doentes que sofreram um evento adverso apresentaram uma probabilidade de óbito bastante superior (odds ratio 12,2) e apresentaram tempos de internamento médio prolongados em cerca de vinte dias. Estes dados não são contudo ajustados para o risco do doente e das intervenções a que são sujeitos. Se forem considerados os tempos de internamento das tabelas de GDH em Portugal, o prolongamento do internamento é de 8,4 dias. A avaliação dos custos adicionais, realizada pelos dias de internamento adicionais, está condicionada à questão metodológica atrás reportada, estimando-se implicações de 1,1% a 8,8% de dias de internamento, com custos de 1.000.000 a 8.600.000 Euros. Em Portugal a monitorização sistemática da ocorrência de eventos, e consequentemente das implicações para a saúde do doente e custos financeiros, não é ainda uma realidade. A implementação do código "presente na admissão" permitirá dar o passo seguinte na utilização dos dados administrativos na compreensão do fenómeno dos eventos adversos.

The brushstroke and materials of Amadeo de Souza-Cardoso combined in an authentication tool

Nowadays, authentication studies for paintings require a multidisciplinary approach, based on the contribution of visual features analysis but also on characterizations of materials and techniques. Moreover, it is important that the assessment of the authorship of a painting is supported by technical studies of a selected number of original artworks that cover the entire career of an artist. This dissertation is concerned about the work of modernist painter Amadeo de Souza-Cardoso. It is divided in three parts. In the first part, we propose a tool based on image processing that combines information obtained by brushstroke and materials analysis. The resulting tool provides qualitative and quantitative evaluation of the authorship of the paintings; the quantitative element is particularly relevant, as it could be crucial in solving authorship controversies, such as judicial disputes. The brushstroke analysis was performed by combining two algorithms for feature detection, namely Gabor filter and Scale Invariant Feature Transform. Thanks to this combination (and to the use of the Bag-of-Features model), the proposed method shows an accuracy higher than 90% in distinguishing between images of Amadeo’s paintings and images of artworks by other contemporary artists. For the molecular analysis, we implemented a semi-automatic system that uses hyperspectral imaging and elemental analysis. The system provides as output an image that depicts the mapping of the pigments present, together with the areas made using materials not coherent with Amadeo’s palette, if any. This visual output is a simple and effective way of assessing the results of the system. The tool proposed based on the combination of brushstroke and molecular information was tested in twelve paintings obtaining promising results. The second part of the thesis presents a systematic study of four selected paintings made by Amadeo in 1917. Although untitled, three of these paintings are commonly known as BRUT, Entrada and Coty; they are considered as his most successful and genuine works. The materials and techniques of these artworks have never been studied before. The paintings were studied with a multi-analytical approach using micro-Energy Dispersive X-ray Fluorescence spectroscopy, micro-Infrared and Raman Spectroscopy, micro-Spectrofluorimetry and Scanning Electron Microscopy. The characterization of Amadeo’s materials and techniques used on his last paintings, as well as the investigation of some of the conservation problems that affect these paintings, is essential to enrich the knowledge on this artist. Moreover, the study of the materials in the four paintings reveals commonalities between the paintings BRUT and Entrada. This observation is supported also by the analysis of the elements present in a photograph of a collage (conserved at the Art Library of the Calouste Gulbenkian Foundation), the only remaining evidence of a supposed maquete of these paintings. The final part of the thesis describes the application of the image processing tools developed in the first part of the thesis on a set of case studies; this experience demonstrates the potential of the tool to support painting analysis and authentication studies. The brushstroke analysis was used as additional analysis on the evaluation process of four paintings attributed to Amadeo, and the system based on hyperspectral analysis was applied on the painting dated 1917. The case studies therefore serve as a bridge between the first two parts of the dissertation.

Quality of essential drugs in tropical countries: evaluation of antimalarial drugs in the Brazilian Health System

INTRODUCTION: The emergence of drug resistance is one of the main problems concerning malaria treatment. The use of counterfeit and/or substandard antimalarial drugs can contribute to the development of parasite resistance. Thus, the aim of this study was to evaluate the quality of antimalarial drugs distributed in Brazil. METHODS: Samples containing chloroquine phosphate, mefloquine hydrochloride, primaquine phosphate, and quinine sulfate tablets were delivered to the Rio de Janeiro central storeroom (CENADI), state storerooms (SS), and Basic Health Units (BHUs) in the north region of Brazil - a total of 10 sample sets. After 5 months of storage, the samples were collected, and in vitro quality control analyses according to official and published methods were performed. RESULTS: Inadequate drug storage conditions were found in two SS and in all BHUs evaluated. There were no quality deviations found in the chloroquine samples. The quinine samples exhibited weight variation above the allowed limits. The primaquine samples were found to have packaging deficiency. The release of mefloquine in samples from some regions showed a statistically significant difference when compared with the CENADI samples. CONCLUSIONS: It is important to periodically evaluate the quality and storage conditions of essential drugs. The quality deviations found with the primaquine and quinine samples are not related to storage conditions and must be addressed urgently. The decreased mefloquine release from tablets is related to formulation problems or influenced by inadequate storage conditions, prompting further investigation. Even with the mentioned problems, the samples would probably not contribute to resistant parasite selection.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120); 5% (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

Work satisfaction and affective commitment of classical musicians: The European Union Youth Orchestra

This work project investigates career paths in the music field, by testing the application of general career and social theories for musicians. Using a sample from the European Union Youth Orchestra’ Alumni, the Boundaryless Career Theory, Intelligent Career Framework and Social Identity Theory were analysed through the impact on individual outcomes - musicians’ Overall work satisfaction and Affective commitment to the orchestra. Results suggest support for the three theories, and show their applicability for classical musicians’ careers.

Podem as nossas leis tornar-nos mais atrativos?

The concept of soft power offers the opportunity for the States, under the current power shifts, to thrive, in a competitive and globalised scene, shaping o t hers' preference in accordance with their goals. Portugal, though it i s a small country, has soft power skills, according with specialized rankings, due to i t s geography and climate, main economic activities, historical role, legal framework, culture and language. Therefore, we can and we should develop public policies to optimize our resources, converting them in planned outcomes. On the other hand, public entities engaged with foreign trade, investment and tour ism, aid f or development, promotion of culture and language should be structured in or-der to strengthen the performance of Portugal in this area. Being a member of the European Union or of the Community of Portuguese Speaking Countries is, at last, essential to expand our global presence. In this Master's work project, I decided to make a critical analysis of legislation related with public diplomacy i n Portugal, together wi th research about the approach of two other countries (United Kingdom and Finland) to the same topic, for the sake of improvement.

Contratos de prestação de serviços de comunicações electrónicas

The object of this dissertation is the analysis of the legal framework applicable to contracts for provision of electronic communications services, while trying to offer solutions to some of the issues regarding this matter. The main focus of this study will be the rules concerning service’s suspension, which have been recently amended. The technological development and the establishment of these services as information transmitters and work tools were noteworthy for its growing importance at the present time. These services include cable television, telephone (landline and mobile) and internet and they are regulated by Law nr 23/96, July 26th, along with other essential public services. Said law sets a group of principles and duties, such as good faith (article 3), continuity and quality of the service (article 7) and the duty to rightfully inform the user (article 4), in order to protect the users. For the analysis of legal framework applicable to these particular contracts it is also fundamental to mention Law nr 5/2004, February 10th, known as Electronic Communications Law. The provisions regarding the service’s suspension are currently prescribed in articles 52.º and 52.º-A of the law. Given the amendments introduced by Law nr 10/2013, January 28th, consumers are subjected to a regulation different from the one applicable to the other users, established in the new article 52.º-A. From our analysis, we have concluded that the main change from past provisions has to do with the automatic termination of the contract as consequence of the consumer’s failure to pay the price or to conclude a written payment arrangement after service’s suspension.

Ethical leadership and unethical pro-organizational behavior: A moderated mediation model of affective commitment and authenticity at work

Recently, unethical conduct in the workplace has been a focus of literature and media. Unethical pro-organizational behavior (UPB) refers to unethical conduct that employees engage in to benefit the organization. Given the complexity of UPB, there is an increasing need to understand how and under what conditions this attitude originates within organizations. Based on a sample of 167 employees and seven organizations, results support the moderated mediation model. An ethical leader increases employees’ organizational affective commitment which increases the likelihood to engage in UPB. However, the indirect relationship diminishes when employees feel authentic at work.

Characterization of an OPCPA laser amplifier based on the nonlinear crystal YCOB

The work described in this thesis was performed at the Laboratory for Intense Lasers (L2I) of Instituto Superior Técnico, University of Lisbon (IST-UL). Its main contribution consists in the feasibility study of the broadband dispersive stages for an optical parametric chirped pulse amplifier based on the nonlinear crystal yttrium calcium oxi-borate (YCOB). In particular, the main goal of this work consisted in the characterization and implementation of the several optical devices involved in pulse expansion and compression of the amplified pulses to durations of the order of a few optical cycles (20 fs). This type of laser systems find application in fields such as medicine, telecommunications and machining, which require high energy, ultrashort (sub-100 fs) pulses. The main challenges consisted in the preliminary study of the performance of the broadband amplifier, which is essential for successfully handling pulses with bandwidths exceeding 100 nm when amplified from the μJ to 20 mJ per pulse. In general, the control, manipulation and characterization of optical phenomena on the scale of a few tens of fs and powers that can reach the PW level are extremely difficult and challenging due to the complexity of the phenomena of radiation-matter interaction and their nonlinearities, observed at this time scale and power level. For this purpose the main dispersive components were characterized in detail, specifically addressing the demonstration of pulse expansion and compression. The tested bandwidths are narrower than the final ones, in order to confirm the parameters of these elements and predict the performance for the broadband pulses. The work performed led to additional tasks such as a detailed characterization of laser oscillator seeding the laser chain and the detection and cancelling of additional sources of dispersion.

Eco-epidemiologic study of emerging fungi related to the work of babaçu coconut breakers in the State of Maranhão, Brazil

Introduction: There are more than 300,000 extractors using the babaçu coconut as a source of income in the States of Maranhão, Pará, Tocantins and Piauí, and this activity is associated with fungal infections. The objective of this study was to examine the occurrence of emergent fungi in the conjunctiva, nails and surface and subcutaneous injuries of female coconut breakers in Esperantinópolis, Maranhão. Additionally, soil samples and palm structures were collected. Methods: The obtained samples were cultured in Petri dishes containing potato-dextrose-agar and chloramphenicol. The etiological agent was confirmed by a direct mycological exam and growth in culture. Results: In total, 150 domiciles were visited, and samples were collected from 80 patients. From the ground, the most frequently isolated fungus was Aspergillus niger (53. 8%). the most frequently detected fungus in babaçu coconut was Aspergillus niger (66.7%). Conjunctival fungal growth occurred in 76.3% of the women. The ocular fungal microbiota consisted of filamentous fungi (80.6%), and yeasts were present in 19.4% of cases. Onychomycosis was diagnosed in 44% (11/25) of the women. Conclusions: The identification of the genera Neosartorya, Rhizopus and Curvularia in onychomycoses shows that emergent filamentous fungi can be isolated. Aspergillus sp., Penicillium sp. and Scedosporium sp. were the predominant genera found in the babaçu coconut. From ocular conjunctiva, Candida spp. were the most prevalent species isolated, and Fusarium sp. was present only in one woman. The nearly permanent exposure of coconut breakers to the external environment and to the soil is most likely the reason for the existence of a mycotic flora and fungal infections, varying according to the individual's practices and occupation.