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Orientador: Benedito Barravieira / Coorientador: Lucilene Delazani dos Santos / Banca: Carlos Antônio Caramori / Banca: Alexandre Leite Rodrigues de Oliveira / Resumo: INTRODUÇÃO: A cirrose é o estágio final da doença hepática. Causada principalmente pelas hepatites virais e ingestão de álcool, o único tratamento específico atual é o transplante O projeto Global Burden of Disease (GBD) publicou recentemente na BMC Medicine uma nova avaliação da mortalidade pela cirrose. Vários métodos e fontes de dados levaram a estimativa global de pouco mais de um milhão de mortes em 2010, cerca de 2% de todas as mortes. Nos últimos anos, as pesquisas biotecnológicas favoreceram a descoberta da terapia alternativa para cirrose usando células tronco. Estas geraram grande expectativa na regeneração tecidual e sua aplicação no tratamento. Um dos grandes desafios enfrentados foi sua reduzida fixação das células no fígado, com dispersão para todo organismo e sem a efetividade esperada. As metodologias ainda não encontraram uma maneira eficaz de impedir que estas células difundam para os outros órgãos diminuindo sua eficácia pretendida. OBJETIVO: Avaliar o selante de fibrina derivado da peçonha de serpente (SFDPS) como arcabouço para células tronco mesenquimais (CTMs) em fígados cirróticos. MÉTODOS: O modelo de cirrose experimental em ratos foi induzido pela tioacetamida (C2H5NS), aplicada duas vezes por semana por via intraperitoneal (i.v.) na dose de 200mg/kg. A coleta de sangue foi realizada uma vez por mês para as analises bioquímicas. O isolamento das células tronco foi realizado de acordo com Gasparotto et al., 2014. A marcação para citometria foi realizada em segunda passagem com os marcadores RT1 AW2 FITC, CLASS RT1 PE, CD11b, CD44, CD90, CD34, CD105, CD73, CD45, ICAM. A biomarcação da serinoprotease, um dos componentes do selante, foi realizada com o kit Alexa Fluor 488 Protein Labeling Kit (A30006v). A cirurgia foi realizada sob anestesia inalatória com isoflurano; os animais foram selecionados aleatoriamente em três grupos os quais receberam o tratamento especificado. Na... / Abstract: Introduction: Cirrhosis is the final stage of liver disease mainly caused by viral hepatitis, alcohol intake, and currently it can only be reversed by liver transplantation. In a new assessment of mortality of liver cirrhosis by design Global Burden of Disease (GBD) in the BMC Medicine, a variety of methods and data sources has led to an overall estimate of just over a million deaths in 2010, which was about 2% of all deaths. In recent years, research has intensified on biotech issues, favoring therapeutic discovery of stem cells to cirrhosis, which have generated great expectations in tissue regeneration of the liver and its application. However, a major problem faced was its low setting in cirrhotic liver, with spread to the whole body and without the expected effectiveness. The investigations have not yet found a way to effectively prevent stem cells spread to other organs reducing their effectiveness regeneration. AIM: Evaluate the fibrin glue derived from snake venom (SFDPS) as a scaffold for stem cells in cirrhotic livers. Methods: The model of experimental cirrhosis in rats was induced by Thioacetamide (C2H5NS), twice per week intraperitoneally (iv) at 200 mg / kg. The of blood collection was performed for biochemical analysis in B200 equipment. Isolation of stem cells was performed according Gasparotto et al. 2014. The markup for cytometry was performed on the second pass with markers RT1 AW2 FITC, CLASS RT1 PE, CD11b, CD44, CD90, CD34, CD105, CD73, CD45, ICAM. The biomarker of serine protease was performed with Alexa Fluor 488 Protein Labeling Kit (A30006v) kit. The surgery has been realized with inhalational anesthesia, the animals were separated into three groups where they received the treatment selected. Histology of 5μm sections were hematoxylin and eosin for staining (HE) and Picrosirius. Immunostaining was performed according to kit Click-it ® Imaging kits EdU. RESULTS: The development of cirrhosis was macroscopically ... / Mestre

Resumo: O Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP) foi criado em 1989 por um grupo de pesquisadores da Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP, com a finalidade de promover a pesquisa básica, aplicada e tecnológica das peçonhas ofídicas. Esse estudo teve como objetivo elaborar uma proposta para a implantação de um programa de manutenção em cativeiro mais eficiente, visando à sobrevida das serpentes e a independência de animais provenientes da natureza. Para tanto, foi necessária a construção de um sistema de cadastro de serpentes, a avaliação da infra estrutura e manejo utilizados, e a análise microbiológica de cada regime adotado. Foi observado que o Banco de Dados on line possibilita a organização dos dados e permite a rastreabilidade da peçonha. Ocativeiro intensivo apesar de proporcionar facilidade na vistoria dos animais necessita de modificações na infra estrutura e manejo. No cativeiro semi-extensivo, foi observado que as instalações facilitam o manejo dos animais, necessitando apenas de adequações nos aquecedores. No biotério de roedores são necessárias diversas mudanças estruturais, controle de temperatura, umidade, tratamento da ração, maravalha e água oferecida aos animais. Na análise microbiológica, não foi observada diferença na freqüência de bactérias entre as espécies, mas ocorreram diferenças entre os diferentes regimes de cativeiro utilizados, com destaque para o cativeiro intensivo. O isolamento de potencial patógenos nas amostras das serpentes estudadas apesar de não ser sinônimo de doença, foi considerado importante no processo de avaliação do tipo de manejo utilizado, considerando as numerosas condições estressantes proporcionadas pelo ambiente do cativeiro e o caráter oportunista desses agentes. Os resultados sugerem que o tipo de manejo adotado influencia na contaminação do ambiente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Center for the Study of Venoms and Venomous Animals (CEVAP) was founded in 1989 by a group of researchers from the Sao Paulo State University - UNESP, with the objective of promoting basic, applied, and technological research of snake venoms. The purpose of the present study was to develop a implementation program proposal for a more efficient captivity maintenance, aiming to extend the snakes' life expectancy and the independence of animals brought from the wild. Therefore, it w as necessary to build a snake registry system, an infrastructure and handling procedures evaluation, as well as a microbiological profile evaluation of each captivity environment adopted. It was observed that an on line data bank allow s arrangement of the data as well as the traceability of the venom. Although intensive captivity enables easy check of the animal, it demands modifications in the infrastructure and handling. In semiextensive captivity it was observed that the facilities meet all the structure and safety requirements in addition to making the animals' handling easier. In the rodents' the biotery, several structural changes are needed besides temperature and humidity control, feed treatment, saw dust and water. In the microbiological analysis, it was observed a difference in the frequency of bacteria betw een the species, but also between the distinct captivity environments used, w ith special remark to the intensive captivity. Even though the presence of pathogens does not mean illness, the isolation of potential pathogens in the snakes' samples analyzed was considered important in the evaluation process of the type of handling used, considering the numerous stressing conditions promoted by the captivity environment and the opportunistic character of these agents. Results suggest that the type of captivity adopted influences in the contamination of the environment... (Complete abstract click electronic access below) / Orientador: Benedito Barravieira / Coorientador: Rui Seabra Ferreira Junior / Banca: Augusto Abe / Banca: Anibal Melgarejo / Banca: Sandra de Moraes Gimenes Bosco / Banca: José Carlos Cogo / Doutor

Resumo: O veneno de Bothrops jararaca é constituído de misturas complexas de toxinas e entre os principais componentes estão as lectinas, metaloproteinases, serinoproteinases, desintegrinas, fosfolipases e os peptídeos. Esta composição pode variar de acordo com a idade, o sexo e a região da serpente. Este trabalho avaliou a variação individual do veneno de Bothrops jararaca da região de Botucatu - São Paulo, por meio da sua caracterização enzimática, bioquímica e farmacológica. Foram utilizados testes in vitro e ensaios biológicos. As atividades dos venenos foram comparadas ao Veneno Referência Nacional. A dosagem protéica apresentou variações entre os grupos de serpentes adultas (80% proteínas) e filhotes (40% proteínas). O perfil eletroforético apresentou perfis similares, geralmente com massas moleculares de 50 a 25 kDa, porém com variações intraespecíficas. O RP-HPLC revelou variações na concentração das proteínas. Na atividade coagulante não houve variação entre os grupos formados por adultos, no entanto, houve uma grande variação entre os grupos de filhotes e Veneno Referência Nacional, sendo estes mais coagulantes. A atividade hemorrágica mostrou maior atividade nos venenos de animais adultos, principalmente em machos recém chegados da natureza. Entretanto, os filhotes mantidos em regime de cativeiro apresentaram valores elevados, ficando muito próximos aos valores obtidos de machos adultos. Na atividade edematogênica, verificou-se um aumento do edema em todos os grupos de estudo não sendo possível avaliar se houve uma variação ontogenética entre os grupos estudados. Na dose letal média (DL50) houve uma grande variação de toxicidade em relação aos venenos, sendo as fêmeas cativas, três vezes mais tóxicas comparadas aos filhotes. Os resultados deste estudo ilustram a complexidade intra e interespecífica presentes em venenos de serpentes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bothrops jararaca venom comprises a complex mixture of toxins. Among its main components are: lectins, metalloproteinases, serine proteinases, disintegrins, phospholipases, and peptides. The venom composition can be present age, sex and region variation. This present study evaluated the individual variation of Bothrops jararaca venom in Botucatu region, São Paulo state, through enzymatic, biochemical and pharmacological characterizations of snake venoms which employed in vitro techniques and in vivo analysis. Venoms actions were compared with those of the National Venom Reference. Protein dosage varied between adult (80% proteins) and newborn (40% proteins). The electrophoretic profiles were similar, usually presenting molecular masses from 25 to 50 kDa; however, intraspecies variations were present. Due to the complexity of the technique, it was not possible to detect the individual chromatographic variations by means of RP-HPLC revealed individual protein variations on venom composition. There was no variation in the coagulant activity among adult groups; however, a great variation was observed between newborn groups and the National Venom Reference, the latter showing higher coagulant activity. Venoms from adult animals revealed greater hemorrhagic activity, mainly in newly captured males. Nevertheless, newborn kept in captivity also presented elevated hemorrhagic activity, close to that of adult males. Regarding the edematous activity, there was edema increase in all studied groups, therefore, it was not possible to evaluate if ontogenetic variations were present among studied animals. Concerning the median lethal dose (LD50), a great variation in venom toxicity was observed and captive females were three times more toxic when compared to newborn. Our findings showed the intraand interspecies complexity of snake venoms possibly represented by ontogenetic, sexual and environmental... (Complete abstract click electronic access below) / Orientador: Rui Seabra Ferreira Júnior / Coorientador: Andreimar Martins Soares / Banca: Daniel Carvalho Pimenta / Banca: Patrick Jack Spencer / Mestre

Orientador: Marcos Roberto de Mattos Fontes / Banca: Richard Garratt / Banca: Jonas Perales / Banca: Humberto D'Muniz / Banca: Marcia Gallaci / Resumo: O envenenamento por picadas de serpentes é um importante problema de saúde pública em muitos países tropicais e subtropicais. Aspectos científicos, médicos e sociais relacionados ao envenenamento por serpentes do gênero Bothrops são especialmente importantes no Brasil já que estes animais são responsáveis por aproximadamente 90% dos acidentes ofídicos relatados em nosso país. Um dos principais problemas relacionados ao acidente botrópico é o proeminente dano tecidual local cuja patogênese é complexa e envolve a ação combinada de uma variedade de componentes do veneno. As fosfolipases A2 (PLA2s) são os componentes mais abundantes no veneno e tem papel fundamental quando se trata do estabelecimento de lesão muscular. O presente trabalho descreve estudos estruturais com miotoxinas que adotam enovelamento de PLA2s (PLA2s homólogas) com o objetivo de melhor entender a relação estrutura - função destas toxinas. Para tanto, estudos estruturais por cristalografia de difração de raios X com Lys49-PLA2s de venenos botrópicos complexadas com os inibidores manganês e ácido rosmarínico (AR) foram realizados. A estrutura cristalográfica de uma miotoxina Asp49-PLA2 homóloga complexada com cálcio também foi elucidada. Adicionalmente, o arranjo quaternário de miotoxinas Asp49-PLA2s homólogas do gênero Bothrops foi revisado e características estruturais relevantes para a expressão da miotoxicidade destas proteínas foram apontadas. A estrutura cristalográfica do complexo formado entre uma Lys49-PLA2 e o inibidor AR evidencia que este inibidor interage com a proteína na entrada de uma dos canais hidrofóbicos do dímero protéico, estabelecendo ligações de hidrogênio com átomos dos resíduos Phe3, Lys7, Leu10, Gln11 e Gly15 do monômero com a qual interage. Já no... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Envenoming resulting from snakebites is an important public health problem in many tropical and subtropical countries. Scientific, medical and social aspects related to envenoming by snakes from Bothrops genus are especially important in Brazil because these animals are responsible for approximately 90 % of all ophidian accidents reported in our country. One of the main problems regarding bothropic accidents is a prominent local tissue damage whose pathogenesis is complex and involves the combined action of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms playing a central role in the muscle damage establishment. The present work reports structural studies with myotoxins that adopt a PLA2 fold (PLA2- like myotoxins) aiming a better understanding of their structure-function relationship. For this purpose, X-ray crystallographic studies of Lys49-PLA2s from bothropic snake venoms complexed to the inhibitors manganese and rosmarinic acid (RA) were performed. The crystallographic structure of the complex formed between an Asp49-PLA2-like myotoxin and calcium was also solved. Additionally, the quaternary assembly of Asp49-PLA2-like myotoxins from Bothrops genus was reviewed and relevant structural features for their myotoxicity expression were pointed out. The crystallographic structure of the complex formed between a Lys49-PLA2 and the inhibitor RA demonstrates that the inhibitor interacts with the protein in the entrance of one of its hydrophobic channel, establishing hydrogen bonds with atoms of the residues Phe3, Lys7, Leu10, Gln11 and Gly15 of the monomer with which it interacts. For the complex formed between a Lys49-PLA2 and the inhibitor Mn2+ the manganese ion is coordinated by atoms of the... (Complete abstract click electronic access below) / Doutor

Orientador: Raghuvir Krishnaswamy Arni / Banca: Patrick Jack Spencer / Banca: Adélia Cristina Oliveira Cintra / Banca: Jose Ramon Abrego / Banca: Fátima Pereira de Souza / Resumo: Um certo número de proteínas do veneno foram purificadas, caracterizadas e as suas estruturas foram determinadas para delinear as suas especialidades. (a) A LAAO de Bothrops jararacussu indicou uma maior especialidade para os resíduos hidrófobos, devido a uma substituição de Trp430por lle. O FAD ligado estava em uma conformação diferente do relatado em outros estudos. (b) O domínio rico em cisteína da metaloproteinase (MP III) de Bothrops moojeni pode adotar diferentes orientações relativas. (c) O local de distribuição de carga de serino proteinases é fundamental para determinar a especificidade da enzima. (d) A conformação tetramérica da Lys49 PLA2 de Bothrops brazili indica coexistência de dois estados diméricas. (e) A adição de uma segunda ponte dissulfeto estabiliza o loop catalítico e Trp 230 adota uma conformação diferente na ligação do substrato. Estes resultados contribuem para melhorar a nossa compreensão do modo de ação e função dessas proteínas / Abstract: A number of venom proteins were purified, characterized and their structures were determined to delineate their specificities. (a) The LAAO from Bothrops jararacussu indicated a higher specifity for hydrophobic residues due to a substitution of Trp430 by lle. The bound FAD was in a diferente conformation to that reported in other studies. (b) The cysteine rich domain of the metalloproteinase (MP III) from Bothrops moojeni can adopt different relative orientations. (c) The local charge distribution of serine proteinases is instrumental in determining enzyme specificity. (d) The tetrameric conformation of the Lys49 PLA2 from Bothrops brazili indicates coexistence of two dimeric states. (e) The addition of a second disulphide bridge stabilizes the catalytic loop and Trp 230 adopts a different conformation on substrate binding. These results contribute to improve our understanding of the mode of action and function of these proteins / Doutor

This paper analyzes a 2009 U.S. policy which provided short term federal subsidies for COBRA health insurance premiums. COBRA allows the recently unemployed to continue purchasing health insurance through their employment-based insurance plan for a short time period after they become unemployed. Early analysis found low take-up rates for COBRA insurance due to the exceedingly high cost of full health insurance premiums, especially for those who have just lost a steady employment income. A short term 65 percent federal subsidy for COBRA insurance was implemented as a part of the American Recovery and Reinvestment Act in 2009. Subsidy policy proposed to increase take-up rates of COBRA and to keep national insurance rates from dropping during a time of rising unemployment. This paper finds a 3.09 percentage point increase in insurance rates for the unemployed when subsidies became available in 2009. It also finds that the gains made in 2009 were lost by 2010, suggesting that subsidies may have provided temporary relief but did not represent a long-term solution for many of the unemployed. Demographic analysis within the unemployed population determines that educated, middle to upper income earning men saw the greatest increases in insurance rates during this time. My analysis affirms previous research finding that COBRA eligibility requirements do not allow the majority of the low income, uninsured to receive federal assistance for health insurance through this policy. I also provide a positive analysis for the impact of direct-purchasing federal subsidies on insurance rates.

Im Rahmen eines vom BMBF/BMWi geförderten WTZ-Vorhabens wurde dem Institute for Nuclear Research and Nuclear Energy (INRNE) in Sofia der Programmcode DYN3D sowie der gekoppelte Programmkomplex ATHLET-DYN3D verfügbar gemacht. Dabei stellt dieser Programmkomplex eine Anbindung des im FZR entwickelten 3D Kernmodells DYN3D an den thermohydraulischen Systemcode ATHLET der GRS dar. Zur Validierung dieser Programmcodes wurde eine Messdatensammlung zu einem Inbetriebnahmeexperiment am Block 6 des KKW Kozloduj (WWER-1000/320) erstellt. Die Ergebnisse der durchgeführten Validierungsrechnungen wurden mit den Messwerten aus der Datensammlung verglichen. In den Programmcode DYN3D wurde von den bulgarischen Experten ein vereinfachtes Modell zur Simulation von Querströmungen zwischen den Brennelementen integriert. Dieses Querströmungsmodell ermöglicht es, Prozesse mit asymmetrischen Randbedingungen realistischer zu betrachten. Eine erste Bewertung des implementierten Strömungsmodells erfolgt durch Vergleichsrechnungen zwischen dem modifizierten DYN3D Code und dem Unterkanalanalyse-Code COBRA-4I sowie auf der Grundlage von verfügbaren Messdaten aus dem KKW Kozloduj.

This thesis is concerned with lifecycle data support for the design of large made to order products. These products have so many complex functions to perforrn that no one designer will have all of the relevant skills such as in structural design Z): or electrical engineering to produce a comprehensive design. This therefore leads to the utilisation of a team of designers who will not only fulfil logically different design roles but often work at different physical locations. In such a design environment there may be several local models, represented in local Z-- databasesT. hese databases may or may not support versioning either of the data or of the schema which evolves as the product design grows. The interfaces to these databases ID will be varied as they are intended to suit the local needs of the design aIgDe nt. This thesis proposes a model for version control in a desig4nn environment which does not alter the designers existing view. Cý tý A system of distributed co-operatinZg:, aZgD ents is presented whose goal is to manatDg e change and orgCaIDni se version sets in an enrgDin eering environment. The agents are designed for full lifecycle support and inter-operation across heterogeneous networks. The agent communication is based on CORBA but an extra messaging layer is developed which utilises a language built in VDM-SL (Vienna Development Method - Specification Language). A version model is presented in two ways informally based on the assumptions on a general design process and formally in VDM-SL. ZP tP In order to demonstrate the effectiveness of the version model, two industrial case studies are presented. The first of these is a study of offshore process engineering. The second is a study of conceptual ship design.

Made available in DSpace on 2014-06-11T19:35:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-29Bitstream added on 2014-06-13T19:25:19Z : No. of bitstreams: 1 oliveira_l_dr_rcla_parcial.pdf: 195299 bytes, checksum: 4fe7fe890504ee214eb88018a7f1f55e (MD5) Bitstreams deleted on 2015-07-02T12:36:06Z: oliveira_l_dr_rcla_parcial.pdf,. Added 1 bitstream(s) on 2015-07-02T12:37:30Z : No. of bitstreams: 1 000726286.pdf: 2826703 bytes, checksum: 239ffdbf89f8a4502f199140708c65cf (MD5) / A origem e a evolução da função venenosa nas serpentes têm sido objeto de inúmeras discussões. De um modo geral, os estudos se restringem às glândulas de veneno ou de Duvernoy, ambas constituídas por células serosas. Pouca atenção é dispensada às demais glândulas orais e no papel biológico de suas secreções. Nos dipsadíneos goo-eaters, uma linhagem altamente especializada de serpentes comedoras de invertebrados, especialmente moluscos e anelídeos, as glândulas de Duvernoy são reduzidas ou ausentes, enquanto que as glândulas infralabiais são predominantemente seromucosas e associadas à musculatura adjacente. Além disso, uma série de modificações dentárias, particularmente nos dentes maxilares e dentários, são associadas à estas serpentes. Este conjunto de características glandulares, musculares e dentárias são frequentemente relacionados à dieta especializada. O presente trabalho teve o objetivo de realizar um estudo comparativo acerca da anatomia, histologia e histoquímica das principais glândulas cefálicas (infralabiais, supralabiais, Duvernoy e de Harder) e ultraestrutural dos dentes maxilares e dentários dos dipsadíneos gooeaters, utilizando para comparações, serpentes dipsadíneas que comem vertebrados. Além disso, foi realizado um estudo acerca do desenvolvimento embrionário das glândulas labiais (infra e supralabiais) de Sibynomorphus mikanii. Cabeças inteiras de espécies representativas dos diversos gêneros de dipsadíneos goo-eaters, bem como dos gêneros de dipsadíneos que comem vertebrados foram dissecadas e submetidas ao processamento histológico. Embriões de Sibynomorphus mikanii foram estudados através da morfologia externa e séries histológicas das cabeças, enquanto que os dentes maxilares e dentários foram estudados por microscopia eletrônica de... / The origin and evolution of the venomous function in snakes has been subject of recent debates. In a general way, studies on this subject are restricted to the venom and Duvernoy’s glands, both constituted by serous cells. Little attention has been given to the others oral glands, such as infralabial and supralabial, and the biological role of their secretions. In the goo-eater dipsadines, a highly specialized lineage of snakes feeding on invertebrates, specially mollusks and annelids, the Duvernoy’s glands are very reduced or absent, while the infralabial glands are predominantly constituted by seromucous cells and associated with adjacent muscles. In addition, several dental modifications, particularly in the maxillary and dentary teeth, are associated with these snakes. These muscular, glandular and dental features are frequently associated with highly specialized diet presented by the goo-eaters. This study aimed to perform a comparative study on the anatomy, histology and histochemisty of the major cephalic glands (infralabial, supralabial, Duvernoy and Harder), and the ultrastructure analysis on the maxillary and dentary teeth of the goo-eater dipsadines and the dipsadinae snakes feeding on vertebrates. Furthermore, a study on the embryological development of the labial glands (infralabial and supralabial) in Sibynomorphus mikanii was performed. Whole heads of representative species of several genera of goo-eater and vertebrate feeding dipsadines were dissected and submitted to histological procedures in paraffin. Embryos of Sibynomorphus mikanii in different stages of development were submitted to studies of external morphology and histological sections of the whole heads and the maxillary and dentary teeth were analyzed by scanning microscopy. In Dipsas, Sibynomorphus and Geophis, the... (Complete abstract click electronic access below)