Gold nanoparticle to antibody conjugates for diagnosis applications: molecular interactions and immunoassay development

Thesis for the Master degree in Structural and Functional Biochemistry

Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis

Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).

The relationship between knowledge acquisition on international assignments and career development: an exploratory study with portuguese repatriates

This paper explores the relationship between the expatriates’ knowledge acquisition (KA) and their career development after an international assignment (IA). The purpose of this paper is to examine the role of expatriates in KA and transfer within International Portuguese multinational corporations. Furthermore, with this empirical study we try to analyse how the knowledge that is acquired and transferred translates into a basis for career development after the IA. This phenomenon has a special relevance in the Portuguese context, because this country is known a growing process of globalization in recent years. Furthermore, (a) there are no empirical studies concerning knowledge transfer and career development of repatriates from Portuguese companies; (b) little is known about the repatriates’ contributions to their home company after IA. This paper is one of the first to focus specifically on the repatriates’ role in KA and transfer from the host company to their Portuguese home company. A qualitative research methodology is used, specifically through an exploratory case study approach, which examines how knowledge management (KM) acquisition or transferring during IA are important for the repatriates’ career development in the Portuguese home company. Data were collected through semi-structured interviews to 42 Portuguese international assignees and 18 organizational representatives from nine Portuguese companies. Preliminary results show that KA and transfer made by Portuguese expatriates contributes directly to their career development. Moreover, evidence reveals that not all repatriates were promoted after their IA; rather some repatriates were even demoted after their IA. Furthermore, the results obtained suggest that the type of knowledge which acquired or transferred plays a central role in the career development after repatriation. According to these results, the paper discusses the major theoretical and practical implications. Suggestions for future research are also presented.

Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

In vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants

Terrestrial plants have been demonstrated to be sources of antimalarial compounds. In Cuba, little is known about antimalarial potentials of plant species used as medicinals. For that reason, we evaluated the antimalarial activity of 14 plant species used in Cuba as antimalarial, antipyretic and/or antiparasitic. Hydroalcoholic extracts were prepared and tested in vitro for the antimalarial activity against Plasmodium falciparum Ghana strain and over human cell line MRC-5 to determine cytotoxicity. Parasite multiplication was determined microscopically by the direct count of Giemsa stained parasites. A colorimetric assay was used to quantify cytotoxicity. Nine extracts showed IC50 values lower than 100 µg/mL against P. falciparum, four extracts were classified as marginally active (SI < 4), one as partially active (Parthenium hysterophorus) exhibiting SI equal to 6.2 and two extracts as active (Bambusa vulgaris and Punica granatum), showing SI > 10. B. vulgaris showed the most potent and specific antiplasmodial action (IC50 = 4.7 µg/mL, SI = 28.9). Phytochemical characterization of active extracts confirmed the presence of triterpenoids in B. vulgaris and polar compounds with phenol free groups and fluorescent metabolites in both extracts as major phytocompounds, by thin layer chromatography. In conclusion, antimalarial use of B. vulgaris and P. hysterophorus was validated. B. vulgaris and P. granatum extracts were selected for follow-up because of their strong antimalarial activity.

Induction of phagocytic activity and nitric-oxide production in natural populations of Trypanosoma Cruzi I and II from the state of Paraná, Brazil

Twelve strains of Trypanosoma cruzi isolated from wild reservoirs, triatomines, and chronic chagasic patients in the state of Paraná, southern Brazil, and classified as T. cruzi I and II, were used to test the correlation between genetic and biological diversity. The Phagocytic Index (PI) and nitric-oxide (NO) production in vitro were used as biological parameters. The PI of the T. cruzi I and II strains did not differ significantly, nor did the PI of the T. cruzi strains isolated from humans, triatomines, or wild reservoirs. There was a statistical difference in the inhibition of NO production between T. cruzi I and II and between parasites isolated from humans and the strains isolated from triatomines and wild reservoirs, but there was no correlation between genetics and biology when the strains were analyzed independently of the lineages or hosts from which the strains were isolated. There were significant correlations for Randomly Amplified Polymorphic Deoxyribonucleic acid (RAPD) and biological parameters for T. cruzi I and II, and for humans or wild reservoirs when the lineages or hosts were considered individually.

Contributo das organizações do terceiro setor no desenvolvimento dos países de píngua pficial portuguesa

O empreendedorismo social é um campo que tem vindo a ganhar uma importância crescente nas sociedades atuais, sendo reconhecido como uma ferramenta útil na promoção do desenvolvimento sustentável. Como se trata de um campo emergente, apresenta-se ainda pouco explorado. Em Cabo Verde não existem ainda estudos sobre o tema, embora existam já algumas iniciativas de empreendedorismo social. A investigação foi desenvolvida de forma a responder ao objetivo de se conhecer qual o contributo que as Organizações Não Governamentais para o Desenvolvimento (ONGD) portuguesas têm dado para a sedimentação do empreendedorismo social em Cabo Verde. Assim, procurou-se apurar e analisar as ONGD que desenvolvem projetos em Cabo Verde, as áreas em que estas atuam para promoverem o desenvolvimento economico-social, os meios de financiamentos a que recorrem, as dificuldades encontradas no desenvolvimento das suas atividades, bem como compreender a razão que levou a que Cabo Verde fosse beneficiado com as ações dessas ONGD. Para conseguir atingir estas metas recorreu-se à metodologia qualitativa onde se fez uma análise exploratória e descritiva. A técnica utilizada para a recolha da informação primária foi a entrevista dirigida aos responsáveis de seis ONGD portuguesas que atuam em Cabo Verde, nomeadamente a Associação para a Cooperação Entre os Povos, a Associação de Defesa do Património de Mértola, Instituto Marquês de Valle Flôr, Meninos do Mundo, Terras Dentro e a Associação Raia Histórica. Os resultados deste estudo permitiram identificar que as ONGD portuguesas agem impulsionando o empreendedorismo social em Cabo Verde através da promoção do desenvolvimento integrado e sustentável, apoiado em parcerias estabelecidas com outras organizações locais caboverdeanas. Estes parceiros são atores chave que estão no terreno e possuem o conhecimento da realidade do país. Cabo Verde foi beneficiado pelos projetos por pertencer aos Países Africanos de Língua Oficial Portuguesa e/ou à Comunidade dos Países de Língua Portuguesa. Na obtenção de recursos, a maioria das ONGD portuguesas recorre a recursos em espécie e a voluntários portugueses, desempenhando estes o papel de formadores. A geração de valor social por parte destas entidades é feita muitas vezes de uma forma indireta, através da criação de valor económico que depois se repercute em valor social. Este valor social resulta da sua atuação em várias áreas como a saúde e segurança alimentar, desenvolvimento rural, meio ambiente, educação e formação profissional, emprego, economia alternativa ou microcrédito, pobreza e exclusão social, ambiente, habitação, promoção social e do turismo, capacitação e reforço institucional e coerência das políticas públicas para o desenvolvimento. Sendo Cabo Verde um arquipélago, as principais dificuldades encontradas pelas ONGD na sua atuação prende-se com a acessibilidade às ilhas devido à falta de transportes e meios de comunicação.

The use of indicators and evidence in governance and policy development of science, technology and innovation

This paper was first presented at the 2012 – EU SPRI Conference “Towards Transformative Governance? - Responses to mission-oriented innovation policy paradigms”, Fraunhofer ISI, June 2012, Karlsruhe

Territorial Strategic Planning as a support instrument for Regional and Local Development : a comparative analysis between Lisbon and Barcelona metropolitan areas - a first approach

Trabalho de Projecto apresentado para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Metropolização, Planeamento Estratégico e Sustentabilidade.

Adult B-Cell Acute Lymphoblastic Leukemia Cells Display Decreased PTEN Activity and Constitutive Hyperactivation of PI3K/Akt Pathway Despite High PTEN Protein Levels

Adult B-cell acute lymphoblastic leukemia remains a major therapeutic challenge, requiring a better characterization of the molecular determinants underlying disease progression and resistance to treatment. Here, using a phospho-flow cytometry approach we show that adult diagnostic B-cell acute lymphoblastic leukemia specimens display PI3K/Akt pathway hyperactivation, irrespective of their BCR-ABL status and despite paradoxically high basal expression of PTEN, the major negative regulator of the pathway. Protein kinase CK2 is known to phosphorylate PTEN thereby driving PTEN protein stabilization and concomitant PTEN functional inactivation. In agreement, we found that adult B-cell acute lymphoblastic leukemia samples show significantly higher CK2 kinase activity and lower PTEN lipid phosphatase activity than healthy controls. Moreover, the clinical-grade CK2 inhibitor CX-4945 (Silmitasertib) reversed PTEN levels in leukemia cells to those observed in healthy controls, and promoted leukemia cell death without significantly affecting normal bone marrow cells. Our studies indicate that CK2-mediated PTEN posttranslational inactivation, associated with PI3K/Akt pathway hyperactivation, are a common event in adult B-cell acute lymphoblastic leukemia and suggest that CK2 inhibition may constitute a valid, novel therapeutic tool in this malignancy.

Urban deforestation and urban development

This paper has developed a model of a single forest owner operating with perfect foresight in a dynamic open-city environment that allows for switching between alternative competing land uses (forest and urban use) at some point in the future. The model also incorporates external values of an even-aged standing forest in addition to the value of timber when it is harvested. Timber is exploited based on a multiple rotation model a la Faustmann with clear-cut harvesting. In contrast to previous models, our alternative land use to forest land is endogenous. Within this framework, we study the problem of the private owner as well as that of the social planner, when choosing the time to harvest, the time to convert land and the intensity of development. We also examine the extent to which the two-way linkage between urban development and forest management practices (timber production and provision of forest amenities) contributes to economic efficiency and improvements in non-market forest benefits. Finally, we consider policy options available to a regulator seeking to achieve improvements in efficiency including anti-sprawl policies (impact fees and density controls) and forest policies such a yield tax. Numerical simulations illustrate our analytical results.

Territorial Strategic Planning as a support instrument for Regional and Local Development: a comparative analysis between Lisbon and Barcelona Metropolitan Areas

MARQUES, B.P. (2011) "Territorial Strategic Planning as a support instrument for Regional and Local Development: a comparative analysis between Lisbon and Barcelona Metropolitan Areas", in Atas do 17.º Congresso da APDR, do 5.º Congresso de Gestão e Conservação da Natureza e do Congresso Internacional da APDR/AECR, Bragança e Zamora, pp. 1265-1272, ISBN 978-989-96353-2-6.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10% phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

Evaluation of larvicidal activity and brine shrimp toxicity of rhizome extracts of Zingiber zerumbet (L.) Smith

Introduction In this study, we used dichloromethane (DCM) and methanol (MeOH) extracts of the Zingiber zerumbet rhizome to evaluate brine shrimp lethality and larvicidal activity on Aedes aegypti and Anopheles nuneztovari mosquitoes. Methods Bioassays were performed by exposing third-instar larvae of each mosquito species to the DCM or MeOH extracts. Results Probit analysis with DCM and MeOH extracts demonstrated efficient larvicidal activity against A. aegypti and A. nuneztovari larvae. Conclusions The DCM and MeOH extracts showed higher activity against A. nuneztovari larvae than against A. aegypti larvae, suggesting that the extracts have species-specific activity.