Container Line Supply Chain (CLSC), which transports cargo in containers and accounts for approximately 95 percent of world trade, is a dominant way for world cargo transportation due to its high efficiency. However, the operation of a typical CLSC, which may involve as many as 25 different organizations spreading all over the world, is very complex, and at the same time, it is estimated that only 2 percent of imported containers are physically inspected in most countries. The complexity together with insufficient prevention measures makes CLSC vulnerable to many threats, such as cargo theft, smuggling, stowaway, terrorist activity, piracy, etc. Furthermore, as disruptions caused by a security incident in a certain point along a CLSC may also cause disruptions to other organizations involved in the same CLSC, the consequences of security incidents to a CLSC may be severe. Therefore, security analysis becomes essential to ensure smooth operation of CLSC, and more generally, to ensure smooth development of world economy. The literature review shows that research on CLSC security only began recently, especially after the terrorist attack on September 11th, 2001, and most of the research either focuses on developing policies, standards, regulations, etc. to improve CLSC security from a general view or focuses on discussing specific security issues in CLSC in a descriptive and subjective way. There is a lack of research on analytical security analysis to provide specific, feasible and practical assistance for people in governments, organizations and industries to improve CLSC security. Facing the situation mentioned above, this thesis intends to develop a set of analytical models for security analysis in CLSC to provide practical assistance to people in maintaining and improving CLSC security. In addition, through the development of the models, the thesis also intends to provide some methodologies for general risk/security analysis problems under complex and uncertain environment, and for some general complex decision problems under uncertainty. Specifically, the research conducted in the thesis is mainly aimed to answer the following two questions: how to assess security level of a CLSC in an analytical and rational way, and according to the security assessment result, how to develop balanced countermeasures to improve security level of a CLSC under the constraints of limited resources. For security assessment, factors influencing CLSC security as a whole are identified first and then organized into a general hierarchical model according to the relations among the factors. The general model is then refined for security assessment of a port storage area along a CLSC against cargo theft. Further, according to the characteristics of CLSC security analysis, the belief Rule base Inference Methodology using the Evidential Reasoning approach (RIMER) is selected as the tool to assess CLSC security due to its capabilities in accommodating and handling different forms of information with different kinds of uncertainty involved in both the measurement of factors identified and the measurement of relations among the factors. To build a basis of the application of RIMER, a new process is introduced to generate belief degrees in Belief Rule Bases (BRBs), with the aim of reducing bias and inconsistency in the process of the generation. Based on the results of CLSC security assessment, a novel resource allocation model for security improvement is also proposed within the framework of RIMER to optimally improve CLSC security under the constraints of available resources. In addition, it is reflected from the security assessment process that RIMER has its limitations in dealing with different information aggregation patterns identified in the proposed security assessment model, and in dealing with different kinds of incompleteness in CLSC security assessment. Correspondently, under the framework of RIMER, novel methods are proposed to accommodate and handle different information aggregation patterns, as well as different kinds of incompleteness. To validate the models proposed in the thesis, several case studies are conducted using data collected from different ports in both the UK and China. From a methodological point of view, the ideas, process and models proposed in the thesis regarding BRB generation, optimal resource allocation based on security assessment results, information aggregation pattern identification and handling, incomplete information handling can be applied not only for CLSC security analysis, but also for dealing with other risk and security analysis problems and more generally, some complex decision problems. From a practical point of view, the models proposed in the thesis can help people in governments, organizations, and industries related to CLSC develop best practices to ensure secure operation, assess security levels of organizations involved in a CLSC and security level of the whole CLSC, and allocate limited resources to improve security of organizations in CLSC. The potential beneficiaries of the research may include: governmental organizations, international/regional organizations, industrial organizations, classification societies, consulting companies, companies involved in a CLSC, companies with cargo to be shipped, individual researchers in relevant areas etc.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-22T15:16:36Z No. of bitstreams: 1 Tomas Andrade de Azevedo.pdf: 1309593 bytes, checksum: d63061f0e02d6c07d13728945ef882bd (MD5) / Made available in DSpace on 2017-02-22T15:16:36Z (GMT). No. of bitstreams: 1 Tomas Andrade de Azevedo.pdf: 1309593 bytes, checksum: d63061f0e02d6c07d13728945ef882bd (MD5) Previous issue date: 2012-06-06 / This study evaluated the wild fish aggregation associated to floating fish cages used for the culture of Cobia (Rachycentrum canadum) in Pernambuco, northeast Brazil. Aggregation took place gradually from first fish farm structure installation. Forty seven species recorded, belonging to 28 families. Haemulon aurolineatum was the first specie to colonize the structure and the one with the highest frequency of occurrence. Seven species who formed large schools represented the highest number of fishes associated to the structure: Haemulon aurolineatum, Haemulon squamipinn, Harengula clupeola, Sarda sarda, Sardinella brasiliensis, Selar crumenophthalmus, and Sphyraena picudilla. Seven species were observed feeding on uneaten pellets from cultured fish: Balistes capriscus, Calamus pennatul, Carangoides crysos, H.aurolineatum, Haemulon parra, H.squamipinna, and Lutjanus synagris. The diversity and number of individuals was higher in areas adjacent to the cages, while the lowest values were found around the anchoring structures, distant 100 m from the cages. The study concludes that the presence of marine fish farms contributes positively to the colonization of wild fish, suggesting that these places may serve as a small marine protected areas favoring fisheries in adjacent grounds. / Este estudo avaliou a colonização da ictiofauna associada às gaiolas de cultivo de Beijupirá (Rachycentrum canadum), no litoral de Pernambuco, região nordeste do Brasil. A colonização foi gradativa a partir da instalação das primeiras estruturas da fazenda de peixes. Foram documentadas 47 espécies pertencentes a 28 famílias. Haemulon aurolineatum foi a primeira espécie a colonizar a estrutura e a espécie com maior frequência de ocorrência. Sete espécies que formavam grandes cardumes representaram o maior número de indivíduos associados à estrutura: H.aurolineatum, Haemulon squamipinn, Harengula clupeola, Sarda sarda, Sardinella brasiliensis, Selar crumenophthalmus, Sphyraena picudilla. Sete espécies foram observadas se alimentando de ração não consumida pelos peixes cultivados: Balistes capriscus, Calamus pennatul, Carangoides crysos, H.aurolineatum, Haemulon parra, H.squamipinna, e Lutjanus synagris. A diversidade e o número de indivíduos foram mais elevadas nas áreas adjacentes às gaiolas, enquanto os menores valores foram encontrados nas estruturas de fundeio distantes 100 m das gaiolas. O estudo conclui que a presença de fazendas de piscicultura marinha contribui positivamente para a colonização de peixes selvagens, o que sugere a possibilidade de que esses locais possam servir como pequenas áreas de proteção ambiental marinha favorecendo a pesca em áreas adjacentes.

Made available in DSpace on 2014-06-11T19:31:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-04-25Bitstream added on 2014-06-13T19:01:15Z : No. of bitstreams: 1 aratani_rg_dr_jabo.pdf: 549089 bytes, checksum: f8fb7f3d8000539e1210f3fff3fbdc79 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tem crescido o interesse em se avaliar a qualidade do solo nos diferentes manejos dentro do sistema plantio direto, bem como a sua evolução, em função de um aumento de densidade do solo e de resistência mecânica à penetração e redução da macroporosidade devido às pressões provocadas pelas rodas das máquinas e pelo pisoteio animal. Nesse sentido, este trabalho teve por objetivo (i) analisar os efeitos de diferentes manejos na qualidade física e química de dois Latossolos Vermelhos do Estado de São Paulo, (ii) avaliar a qualidade física do solo por meio do índice S e (iii) verificar a influência da condição edafoclimática na discriminação dos diferentes manejos do solo. Para isso, foram avaliadas duas áreas cultivadas com soja na safra 2005/06, uma em Latossolo Vermelho Acriférrico típico (LVwf) de Guaíra (SP), onde foram avaliados: PD12 – SPD irrigado há 12 anos; PD5 – SPD há 5 anos; PD5I – SPD há 5 anos, com 2 anos de integração lavoura-pecuária; PC – preparo convencional e MN – mata natural, e outra em Latossolo Vermelho Eutroférrico típico (LVef) de Pedrinhas Paulista (SP), onde foram avaliados: PD15 – SPD há 15 anos; PD8 – SPD há 8 anos; PD8S – SPD há 8 anos, com sucessão de culturas; PC – preparo convencional e MN – mata natural. A qualidade física dos solos foi avaliada por meio da estabilidade de agregados, densidade, porosidade total, macroporosidade, microporosidade, resistência à penetração, densidade relativa, condutividade hidráulica e índice S, e a qualidade química por meio de análises de rotina para fins de fertilidade do solo, nas camadas de 0-10,0, 0,10-0,20 e 0,20-0,30 m. Para atingir o objetivo (iii), foi utilizada a estatística multivariada, com análises de agrupamento hierárquico, não hierárquico e de componentes principais. Nos dois solos, o cultivo promoveu redução na qualidade física... / It has grown the interest in evaluating the soil quality in the different management on no-till system, as well as its evolution, in function of an increase of soil bulk density and resistance to penetration and reduction of the macroporosity due to the pressures caused by machine wheels and the animal trampling. In this sense, the aim of this work were (i) to analyze the effect of different soil managements in the physical and chemical quality of two Oxisols from State of São Paulo, (ii) to evaluate the soil physical quality through index S and (iii) to verify the influence of soil type and climatic condition in the discrimination of the different soil managements. For this, two field areas in 2005/06 had been evaluated, one in an Anionic Acrustox (LVwf) from Guaíra, State of São Paulo, where they were evaluated: PD12 – 12 years old irrigated no-till system; PD5 – 5 years old no-till system; PD5I – 5 years old no-till system, with 2 years of croppasture rotation; PC – conventional till and MN – natural forest, and another one in an Eutrudox (LVef) from Pedrinhas Paulista (SP), where they were evaluated: PD15 – 15 years no-till system; PD8 – 8 years no-till system; PD8S – 8 years no-till system without crop rotation; PC – conventional till and MN – natural forest. The soil physical quality was evaluated through the stability of aggregates, soil bulk density, total porosity, macroporosity, microporosity, resistance to the penetration, relative density, hydraulic conductivity and index S, and the soil chemical quality through the data of routine analyses for the soil fertility test, in the layers of 0-0.10, 0.10-0.20 and 0.20-0.30 m. To reach the objective (iii), the multivaried statistics was used, with hierarchical clustering method, k-means method and principal components analysis. The results showed that in both soil types, the soil use by agriculture promoted... (Complete abstract click electronic access below)

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Operculina macrocarpa (L.) Urb. (âjalapa brasileiraâ) (Convolvulaceae) à uma espÃcie comum do Nordeste brasileiro, sendo popularmente utilizada pelas suas propriedades laxativa, purgativa e âdepurativaâ. A tintura do tubÃrculo de jalapa, matÃria-prima majoritÃria, ao lado da tintura de Convolvulus scammonia (TCS) compÃem o fitoterÃpico Aguardente Alemà(AAL) ou Tintura de Jalapa Composta, indicada como antitrombÃtico na medicina popular. O objetivo do presente trabalho foi investigar o potencial antiagregante plaquetÃrio e anticoagulante de O. macrocarpa em plasma humano, incluindo estudo de bioprospecÃÃo e determinaÃÃo do possÃvel mecanismo de aÃÃo. A avaliaÃÃo da atividade antiagregante plaquetÃria das drogas testes (tintura e fraÃÃes de O. macrocarpa, TCS e AAL) em plasma humano rico em plaquetas (PRP) foi mensurada por mÃtodo turbidimÃtrico, sendo a agregaÃÃo induzida por vÃrios agonistas como difosfato de adenosina (ADP), epinefrina (EPI), Ãcido araquidÃnico (AA), colÃgeno (COL) ou trombina (TROM). Ao contrÃrio da TCS e da AAL, tanto a tintura de O. macrocarpa (TOM/precipitado(P) e sobrenadante(S)) quanto uma das fraÃÃes orgÃnicas obtidas da planta (TOM/1-F4) mostraram atividade antiagregante plaquetÃria, onde a TOM/1-F4 (100 Âg/mL) apresentou efeito comparÃvel ao Ãcido acetilsalicÃlico (AAS). CaracterizaÃÃo fÃsico-quÃmica (CLAE e espectrofotometria) da TOM/PS (teor de resinas 1,38g%) e da TOM/1-F4 permitiu a identificaÃÃo de Ãcidos fenÃlicos (clorogÃnico, gÃlico e cafÃico), bem como a determinaÃÃo do teor de fenÃis totais da TOM (0,14g%). O efeito antiagregante plaquetÃrio da TOM/PS e da TOM/1F-4 parece resultar de vÃrias aÃÃes intracelulares. Contudo, a TOM/PS na presenÃa do AAS, L-ARG, ODQ, pentoxifilina (PTX) ou ticlopidina (TIC) teve seu efeito antiagregante plaquetÃrio modificado principalmente pela TIC, sugerindo um papel importante dos receptores purinÃrgicos na bioatividade da TOM/PS. A TOM/PS apresentou atividade antioxidante determinada atravÃs do teste do DPPH. AlÃm disso, a TOM/PS e a TOM/1F-4 nÃo mostraram aÃÃo anticoagulante em plasma humano, mas a TOM/PS aumentou o tempo de sangramento em camundongos. Foi observada uma toxicidade relativa da TOM/PS em neutrÃfilo humano. Dessa forma, o presente estudo comprovou, de maneira inÃdita, o potencial antiagregante plaquetÃrio da tintura e da fraÃÃo orgÃnica de jalapa, que estÃo pelo menos em parte relacionados à presenÃa de fenÃis, particularmente Ãcidos fenÃlicos, e resinas na planta. / Operculina macrocarpa (L.) Urb ("Brazilian jalapa") (Convolvulaceae) is a common species of the Brazilian Northeast. It is popularly used because of its laxative and purgative properties. The tincture of Operculina macrocarpa tubers (major constituent) and Convolvulus scammonia composes the raw materials of Aguardente AlemÃÂ, herbal medicine, referred to as antithrombotic in folk medicine. The objective of this study was to investigate the antiplatelet and anticoagulant potential for Operculina macrocarpa in human plasma, including the bioprospection study and determination of possible mechanism of action. The evaluation of antiplatelet activity of drugs (tincture and fractions of O. macrocarpa, tincture of C. scammonia and AAL) in human platelet-rich plasma (PRP) was measured by the turbidimetric method where the aggregation was induced by the addition of agonists (adenosine diphosphate (ADP), epinephrine (EPI), arachidonic acid (AA) collagen (COL) or thrombin (TROM). Unlike tincture of C. scammonia and AAL, both O. macrocarpa tincture (TOM/preciptade (P) and supernatant (S)) and one of the organic fractions obtained from O. macrocarpa (TOM/1-F4) showed antiplatelet activity in human plasma where TOM/1-F4 (100 Âg/mL) presented comparable effect to AAS. Physicochemical characterization (HPLC and spectrophotometer) of TOM/PS (content of resins 1,38g%) and TOM/1-F4 allowed the identification of phenolic acids (chlorogenic, galic and caffeic) as well as the determination of total phenols of TOM (0,14g%). The antiplatelet effect of TOM/PS and TOM/1-F4 seems to result from many intracellular actions. However, TOM/PS in presence of AAS, L-ARG, ODQ, pentoxifilin (PTX) or ticlopidine (TIC) had its antiplatelet effect modified mainly because of TIC, suggesting an important role of purinergic receptors in TOM/PS. TOM/PS presented antioxidant activity determined by DPPH test. Furthermore, TOM/PS and TOM/1-F4 did not showed anticoagulant action in human plasma but TOM/PS increased the bleeding time in mice. It was observed a relative toxicity of TOM/PS in human neutrophil. Therefore, this study demonstrated, as never before, the antiplatelet potencial of tincture and the organic fraction of "jalapa" and that at least related to the presence of phenols, particulary phenolic acids, and resins in plant.

INTRODUÇÃO: Os Inibidores de Bombas de Prótons (IBP´s) são comumente prescritos a pacientes em uso de dupla antiagregação plaquetária (DAP) com ácido acetilsalicílico (AAS) e clopidogrel. Entretanto, esta classe de medicamentos, especialmente o omeprazol, tem sido associada à redução da potência antiplaquetária do clopidogrel, levando em muitos casos ao uso de ranitidina como alternativa. MÉTODOS: Foram analisados pacientes com doença arterial coronária (DAC) estável em uso de AAS 100 mg uma vez ao dia. A agregabilidade plaquetária foi medida no momento basal e após uma semana de terapia com clopidogrel na dose de 75 mg uma vez ao dia. Após essa fase inicial, os participantes foram randomizados de modo duplo-cego e duplo-mascarado para omeprazol 20 mg duas vezes dia ou ranitidina 150 mg duas vezes ao dia, sendo os testes de agregação plaquetária novamente repetidos após uma semana. A agregabilidade foi avaliada com a utilização dos seguintes métodos: VerifyNow P2Y12® (Accumetrics - San Diego, CA, EUA, meta principal do estudo), utilizando-se Unidades de Reatividade ao P2Y12 (\"P2Y12 Reactivity Units\" - PRU) e Inibição Percentual da Agregabilidade (IPA) na descrição da agregabilidade; agregometria de sangue total (AST) por bioimpedância utilizando os reagentes ADP e colágeno, sendo a agregabilidade medida em Ohms; \"Platelet Function Analyser\" 100® (Siemens Healthcare Diagnostics®, Newark, Delaware, EUA) utilizando o cartucho de colágeno/ADP, com a agregabilidade avaliada pelo tempo de fechamento do orifício em segundos. Além disso, foi feita dosagem de tromboxano B2 (TXB2) sérico na última visita a fim de se avaliar o efeito do AAS. RESULTADOS: Oitenta e cinco pacientes foram incluídos na análise final, sendo 41 no grupo omeprazol e 44 no grupo ranitidina. Houve redução significativa da IPA após o acréscimo de omeprazol (de 26,3 ± 32,9% para 17,4 ± 33,1%; P = 0,025), enquanto o grupo ranitidina não demonstrou modificação significativa (de 32,6 ± 28,9% para 30,1 ± 31,3%; P = 0,310). Levando-se em conta o valor em PRU, houve um aumento numérico porém não significativo estatisticamente no grupo omeprazol (de 159,73 ± 83,06 para 173,54 ± 72,29; P = 0,116) enquanto no grupo ranitidina houve uma diferença muito pequena (de 153,61 ± 70,12 to 158,77 ± 76,37; P = 0,440). Em relação aos demais testes de agregabilidade e à dosagem de TXB2 sérico, não houve alterações significativas em qualquer um dos grupos. CONCLUSÃO: A ranitidina não influenciou o efeito antiplaquetário do clopidogrel, ao contrário do omeprazol, que reduziu a atividade antiplaquetária do medicamento. Esses achados podem ter um importante impacto na tomada de decisão quanto ao protetor gástrico a ser utilizado em pacientes submetidos a DAP com AAS e clopidogrel. / BACKGROUND: Proton-pump inhibitors (PPIs) are often prescribed to patients taking dual antiplatelet therapy (DAPT) with acetylsalicylic acid (ASA) and clopidogrel. However, this class of medication, especially omeprazole, has been associated with a reduction of clopidogrel efficacy, leading many to substitute omeprazole with ranitidine. METHODS: The present study analyzed patients with stable coronary artery disease (CAD) in use of ASA 100 mg daily. Platelet aggregability was measured at baseline and after one week of clopidogrel 75 mg daily. Then, the subjects were randomized, in a double-blinded, doubledummy fashion, to omeprazole 20 mg twice a day or ranitidine 150 mg twice a day. After one more week, aggregability tests were repeated. Platelet aggregability was evaluated by the following methods: VerifyNow P2Y12TM (Accumetrics - San Diego, California, USA, main endpoint of the study), with aggregability depicted as percent Inhibition of Platelet Aggregation (IPA) and as P2Y12 Reactivity Units (PRU); whole blood aggregometry by bioimpendance using ADP and collagen with aggregability measured in Ohms; and Platelet Function Analyser 100TM (Siemens Healthcare Diagnostics, Newark, Delaware, USA) using collagen/ADP cartridge with aggregability measured in time to closure in seconds. Besides that, serum thromboxane B2 dosage was done on the last visit to evaluate ASA effect. RESULTS: Eighty-five patients were included in final analysis (41 in the omeprazole group and 44 in the ranitidine group). IPA was significantly decreased after addition of omeprazole (from 26.3% ± 32.9 to 17.4% ± 33,1; P = 0.025), with no significant changes being observed in the ranitidine group (from 32.6% ± 28.9 to 30.1% ± 31.3; P = 0.310). When taking into account PRU values, there was a numerical, but statistically non-significant increase in the omeprazole group (from 159.73 ± 83.06 to 173.54 ± 72.29; P = 0.116), with a very slight difference in the ranitidine group (from 153.61 ± 70.12 to 158.77 ± 76.37; P = 0.44). There were no significant changes taking into account other aggregability tests and serum thromboxane B2 dosage. CONCLUSION: In patients with stable CAD, ranitidine did not influence clopidogrel antiplatelet activity, in contrast to omeprazole, which reduced antiplatelet drug effect. These findings may have a great impact in clinical decision making regarding gastrointestinal prophylaxis choice in patients taking DAPT with ASA and clopidogrel

A L- asparaginase é uma enzima aplicada no tratamento de Leucemia Linfoide Aguda, que atua na hidrólise da L- asparagina, privando a célula tumoral de um aminoácido essencial para o seu crescimento. A L- asparaginase, como outros biofármacos, deve ser estável, manter sua atividade específica e formar poucos agregados. A fim de manter a integridade do biofármaco, são utilizados adjuvantes nas formulações farmacêuticas, e dentre os mais importantes estão os osmólitos. Essas moléculas protegem a estrutura nativa da proteína, sendo capazes de interferir na formação de agregados e garantir a estabilidade proteica. O presente trabalho teve o objetivo de estudar o efeito dos osmólitos sacarose, sorbitol, arginina e glicina na atividade específica, estabilidade, cinética e caracterização de agregados na solução de L- asparaginase II de Erwinia chrysanthemi. Os resultados mostraram que a maioria dos osmólitos testados aumentou a atividade específica e a estabilidade da enzima, o que pode estar relacionado com o aumento da velocidade máxima e do kcat observados no ensaio cinético realizado com sacarose e sorbitol. Um perfil diferente de agregados foi encontrado para cada tipo de osmólito. A presença de sacarose ou sorbitol resultou na menor quantidade de agregados na faixa de, respectivamente, 100 a 200 e 200 a 300 nm em relação a enzima sem osmólito. Por outro lado, aumento no número total de agregados e presença de moléculas de alto peso molecular (300 a 500 nm) foram observados nas soluções enzimáticas contendo, respectivamente, glicina e arginina. Dessa forma, os resultados obtidos neste trabalho poderão auxiliar na produção e escolha da formulação de biofármacos, e, consequentemente, melhorar o tratamento medicamentoso de pacientes. / L L-Asparaginase is an enzyme applied in the treatment of Acute Lymphoblastic Leukemia, which acts on the hydrolysis of L- asparagine, depriving the tumor cell of an essential amino acid for its growth. L-asparaginase, as other biopharmaceuticals, must be stable, maintain its specific activity and form few aggregates. In order to maintain the integrity of the biopharmaceutical, adjuvants are used in the pharmaceutical formulations, and among the most importants adjuvants are the osmolytes. These molecules protect the native structure of the protein, being able of interfering in the formation of aggregates and guarantee protein stability. The present work had the objective of studying the effect of the osmolytes sucrose, sorbitol, arginine and glycine in the specific activity, stability, kinetic and aggregates characterization, in L- asparaginase II solution of Erwinia chrysanthemi. The results showed that the majority of the tested osmolytes increased the specific activity of the enzyme and its stability, which may be related to the augment of maximum velocity and kcat observed in the kinetic assay performed with sucrose and sorbitol. A different profile of aggregates was found for each type of osmolyte. The presence of sucrose or sorbitol resulted in the least amount of aggregates in the range of, respectively, 100-200 and 200-300nm in relation to the enzyme without osmolyte. On the other hand, increase in the total number of aggregates and the presence of high molecular weight molecules (300 to 500 nm) were observed in the enzymatic solutions containing, respectively, glycine and arginine. Thus, the results obtained in this work may help in the production and choice of the formulation of biopharmaceuticals and, consequently, improve the drug treatment of patients.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-22T16:51:30Z No. of bitstreams: 1 SamiraAbdala.pdf: 3051742 bytes, checksum: 816c5c802400bcf5a75deb6a99de0617 (MD5) / Made available in DSpace on 2017-05-22T16:51:30Z (GMT). No. of bitstreams: 1 SamiraAbdala.pdf: 3051742 bytes, checksum: 816c5c802400bcf5a75deb6a99de0617 (MD5) Previous issue date: 2017-04-07 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Platelets, the blood cells involved in maintaining hemostasis, play a key role in the development of acute ischemic coronary, cerebrovascular events and are critically involved in the thrombosis process. In response to vascular injury, changes in blood flow or chemical stimuli, platelets trigger three functional mechanisms: adhesion, activation and aggregation. After platelet capture, a rapid stabilization of adhesion is required for thrombus formation to occur. Platelet activation results from conformational changes dependent of the protein disulfide isomerase (PDI), so that it has recently been proposed as a molecular target in platelet antiaggregant activity. The use of plant species rich in phenolic compounds as a source of bioactive substances is a promising strategy for the development of new therapeutic alternatives for thromboembolic diseases. Previously, we have shown that Syzygium cumini (L.) Skeels leaf contains multiple polyphenols, which support its use for antiplatelet purposes. Therefore, this study sought to evaluate the effects of polyphenol-rich extract (PESc) from S. cumini leaf on platelet activation and aggregation, as well as on PDI reductase activity. Platelet-rich plasma from healthy volunteers (n=5) was incubated with PESc (10-1000 μg/mL), for 25 min, before activation with ADP, thrombin or PMA. To analyze PESc effect on integrin αIIbβ3 activation, flow citometry protocols were conducted in washed platelets pre-treated with PESc (10-1000μg/mL) and activated with thrombin before tagging with PAC–1 antibody. Finally, PESc (0.1-100 μg/mL) effects on PDI reductase activity were assessed in absence or presence of polyphenolic standards gallic acid, myricetin and quercetin. PESc dose-dependently inhibited platelet aggregation despite the agonist used, even though lower agonist concentration potentiated PESc inhibitory effects to a maximal 77% inhibition at 2.5 μM ADP. Similarly, PESc dose-dependently reduced the proportion of activated αIIbβ3 molecules per platelet up to one third of control at 1000 μg/mL. These effects correlated with the strong inhibitory action of PESc on PDI activity, an effect synergically augmented in presence of standards. Therefore, our data show that PESc reduces platelet aggregation and activation, probably through PDI inhibition, strengthening its prominent antiplatelet activity. / As plaquetas, células sanguíneas envolvidas na manutenção da hemostase, exercem uma função essencial no desenvolvimento de eventos isquêmicos agudos coronarianos, cerebrovasculares e estão criticamente envolvidas no processo de formação da trombose. Em resposta à lesão vascular, às alterações no fluxo sanguíneo ou a estímulos químicos, as plaquetas desencadeiam três mecanismos funcionais: adesão, ativação e agregação. Após a captura da plaqueta, uma rápida estabilização da adesão é necessária para que ocorra a formação do trombo. A ativação plaquetária resulta de alterações conformacionais dependentes da proteína dissulfeto isomerase (PDI), de tal modo, que recentemente foi proposto o seu uso como alvo molecular na atividade antiagregante plaquetária. O uso de espécies vegetais ricas em compostos fenólicos como fonte de substâncias bioativas apresenta-se como uma estratégia promissora para o desenvolvimento de novas alternativas terapêuticas das doenças tromboembólicas. Anteriormente, temos mostrado que as folhas de Syzygium cumini (L.) Skeels contém múltiplos polifenóis, que apoiam a sua utilização para fins antiplaquetários. Portanto, este estudo procurou avaliar os efeitos do extrato rico em polifenóis (ERP) da folha de S. cumini sobre a ativação e agregação plaquetária, bem como, sobre a atividade redutase da PDI. Para tanto, plasma rico em plaquetas de voluntários saudáveis foi incubado com ERP (10 - 1000μg/mL), durante 25 min, antes da ativação com ADP, trombina ou PMA. Para analisar o efeito de ERP sobre a ativação da integrina αIIbβ3, os protocolos de citometria de fluxo foram conduzidos em plaquetas lavadas pré-tratadas com ERP (10 -1000 μg/mL) e ativadas com trombina antes da marcação com anticorpo PAC-1. Finalmente, os efeitos de ERP (0,1-100 μg/mL) na atividade redutase da PDI foram avaliados na ausência ou presença de padrões polifenólicos de ácido gálico, miricetina e quercetina. ERP inibiu a agregação plaquetária dependente da dose apesar do agonista utilizado, embora uma menor concentração de agonistas potencializasse os efeitos inibitórios de ERP até uma inibição máxima de 77% a 2,5 μM de ADP. De modo semelhante, o ERP reduziu a dose proporcionalmente a proporção de moléculas de αIIbβ3 ativadas por plaquetas até um terço do controle a 1000μg/mL. Estes efeitos correlacionaram-se com a forte ação inibitória de ERP na atividade da PDI, um efeito sinergicamente aumentado na presença de padrões. Portanto, nossos dados mostram que o ERP reduz a agregação e ativação plaquetária, provavelmente através da inibição da PDI, fortalecendo sua proeminente atividade antiplaquetária.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T18:35:57Z No. of bitstreams: 1 HiranReisSousa.pdf: 2489901 bytes, checksum: 8a82807f0600af87559439a496bd0d2a (MD5) / Made available in DSpace on 2017-06-14T18:35:57Z (GMT). No. of bitstreams: 1 HiranReisSousa.pdf: 2489901 bytes, checksum: 8a82807f0600af87559439a496bd0d2a (MD5) Previous issue date: 2016-12-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Recent researches have emphasized the importance of redox mechanisms for platelet function modulation. The platelet surface contains a large variety of integrin receptors and other molecules presenting functional thiol groups in their structures, which are potential targets for redox regulation. Among these various thiol-containing proteins, integrin αIIbβ3 stands out for being the convergence path of platelet activation induced by various agonists. Activation of αIIbβ3 integrin is catalyzed by protein disulfide isomerase (PDI) through an essential conformational change leading to the exposure of fibrinogen-binding site. Thus, PDI has been shown to be an important target for the development of antiplatelet drugs. In recent years, many studies have described substances from plan (DE A. PAES et al., 2011), as well as synthetics that are capable of inhibiting PDI. In a previous study of our research group has shown that the synthetic peptide CxxC, which contains the redox motif of PDI in its original sequence CGHC, inhibited reductase activity of this enzyme, effect not observed with AxxA peptide, whose cysteines were replaced with alanine and Scr peptide, which contains the same aminoacids from CxxC peptide, but under random sequence. It has been also demonstrated that CxxC peptide was the only to reduce by 30% ADP-induced aggregation (5μM) in platelet rich plasma, an effect apparently mediated by the association of CxxC and PDI at platelet surface. Thus, in this work, we further assessed the effects of CxxC and its control peptides on platelet aggregation. Washed human platelets were incubated with CxxC peptide at concentrations of 3, 6 and 10 μM, resulting in a dose-dependent inhibition of maximum aggregation activated by thrombin (0.02 U/mL) at 25, 60 and 74%, respectively with IC50 of 6.13 ± 1.09 μM. The presence of control peptides did not produce any inhibitory effect. CxxC peptide also reduced the activation of αIIbβ3 integrin at platelet surface, but did not affect the expression of the markers CD 62-P and CD 63. Control peptides did not alter the expression of these markers. Analysis by mass spectrometry of the interaction of recombinant human PDI with the peptide showed that only CxxC peptide associated with the redox Cys400 of a’ motif of PDI, which has been considered essential for platelet aggregation. Together, these results demonstrate that CxxC peptide reduces platelet aggregation by association with PDI and can be further used as a model for the development of new antithrombotic drugs. / Investigações recentes têm enfatizado a importância de mecanismos redox na modulação da função plaquetária. A superfície da plaqueta contém grande variedade de integrinas e outras moléculas receptoras que possuem tióis funcionais em sua estrutura, os quais são alvos potenciais de regulação redox. Dentre estas várias proteínas tiólicas, a integrina αIIbβ3 destaca-se por ser a via de convergência da ativação plaquetária induzida por diversos agonistas. A ativação da integrina αIIbβ3 é catalisada pela proteína dissulfeto isomerase (PDI), essencial à mudança de conformação que leva à exposição do sitio de ligação ao fibrinogênio. Sendo assim, a PDI tem se mostrado como um alvo importante para o desenvolvimento de fármacos reguladores da agregação plaquetária. Nos últimos anos, diversos estudos têm descrito substâncias de origem vegetal, animal e sintéticas que são capazes de inibir a PDI. Em trabalho do nosso grupo de pesquisa (DE A. PAES et al., 2011), demonstrou que o peptídeo sintético CxxC, o qual contém o motivo redox da PDI na sua sequência original CGHC, inibiu a atividade redutase desta enzima; efeito não observado com os peptídeos AxxA, que possui as cisteínas substituídas por alanina e Scr, peptídeo controle contendo os mesmos aminoácidos do peptídeo CxxC, porém com sequência aleatória sem formação de ditiol. Demonstrou-se, também, que apenas o peptídeo CxxC reduziu em 30% a agregação induzida por ADP (5M) em plasma rico em plaquetas, efeito aparentemente mediado pela associação do CxxC com a PDI na superfície plaquetária. Sendo assim, neste trabalho continuamos a avaliação dos efeitos do peptídeo CxxC e seus controles sobre a agregação plaquetária. Para tanto, incubamos lavado de plaquetas humanas com o peptídeo CxxC nas concentrações de 3, 6 e 10 μM, resultando em inibição concentração-dependente da agregação ativada por trombina (0,02 U/mL) em 25, 60 e 74 %, respectivamente, com IC50 de 6,13 ± 1,09 μM. A presença dos peptídeos controle não produziu quaisquer efeitos inibitórios. O peptídeo CxxC reduziu a ativação da integrina αIIbβ3 na superfície da plaqueta, porém não impactou a expressão dos antígenos CD 62-P e CD 63. Os peptídeos controle não alteraram a expressão desses marcadores. A análise por espectrometria de massas da interação da PDI recombinante humana com os peptídeos, mostrou que apenas o peptídeo CxxC associa-se com a Cys400 do motivo redox a’ da hPDI, o qual tem sido considerado fundamental para a agregação plaquetária. Em conjunto, estes resultados demonstram que o peptídeo CxxC reduz a agregação plaquetária via associação com a PDI, podendo ser empregado como modelo para o desenvolvimento de fármacos novos antitrombogênicos.

Neste trabalho, aprofundamos o estudo sobre risco sistêmico via funções de agregação. Consideramos três carteiras diferentes como proxy para um sistema econômico, estas carteiras são consistidas por duas funções de agregação, baseadas em todos as ações do E.U.A, e um índice de mercado. As medidas de risco aplicadas são Value at Risk (VaR), Expected Shortfall (ES) and Expectile Value at Risk (EVaR), elas são previstas através do modelo GARCH clássico unido com nove funções de distribuição de probabilidade diferentes e mais por um método não paramétrico. As previsões são avaliadas por funções de perda e backtests de violação. Os resultados indicam que nossa abordagem pode gerar uma função de agregação adequada para processar o risco de um sistema previamente selecionado. / In this work, we deepen the study of systemic risk measurement via aggregation functions. We consider three different portfolios as a proxy for an economic system, these portfolios are consisted in two aggregation functions, based on all U.S. stocks and a market index. The risk measures applied are Value at Risk (VaR), Expected Shortfall (ES) and Expectile Value at Risk (EVaR), they are forecasted via the classical GARCH model along with nine distribution probability functions and also by a nonparametric approach. The forecasts are evaluated by loss functions and violation backtests. Results indicate that our approach can generate an adequate aggregation function to process the risk of a system previously selected.

Proteins play a major role in living systems and present a wide spectrum of functionalities. Many different types of proteins are involved into biological processes, such as the catalysis of biochemical reactions, cellular membrane transport, immune system response and DNA replication. However, some proteins and peptides might become harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative disorders including Alzheimer disease (AD). One of the causes of AD is the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The number of AD patients is increasing, but a cure has not been founded yet. Therefore, it is crucial to investigate the mechanisms underlying amyloid aggregation and screening for compounds able to prevent this irreversible process. Microfluidics permits characterising the physicochemical properties of proteins, investigate their aggregation and study their interactions with other molecules. Chemical kinetics allows studying the microscopic events occurring during protein self-assembly. The combination of these two techniques provides a powerful tool for the identification of compounds inhibiting the aggregation process. In this thesis by using microfluidics, chemical kinetics and other biophysical assays, I have investigated the proteins isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process. Firstly, I describe the development of a microfluidic platform allowing for the measurement of the protein pI, in a gradient-free manner. This approach overcomes a fundamental limitation of convectional techniques that is the achievement of a stable and well-controlled pH gradient. Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42) aggregation. The findings from this study show that nanobodies target monomeric species with high affinity whereas interactions with fibril surfaces are weak. Finally, I discuss the use of other compounds inhibiting specific nucleation stages. These include the chaperones clusterin and brichos, as well as soot and pure carbon nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.