CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Polyamines and ethylene have been cited as important regulators of plant growth and development, and may be involved in plant defense against several abiotic stresses, such as salinity. To withstand with salt harmful effects, plants respond through a coordinated set of physiological and molecular responses to improve their performance under salinity. In order to test the hypothesis that salt tolerance degree in maize genotypes is related to changes in polyamine metabolism associated with ethylene production, two experiments were performed. In the first one, BR5033 (salt-tolerant) and BR5011 (salt-sensitive) maize genotypes were subjected to 80 mM NaCl stress to identify the pattern of ethylene production in leaves and roots. Two peaks of ethylene production at 5.5 h (phase I) and 12.5 h (phase II) after onset the salinity treatment were registered in salt-sensitive leaves; whereas only the first peak of ethylene synthesis was detected in salt-tolerant leaves. Surprisingly, the biphasic ethylene production in roots was much less pronounced than in leaves. In the second experiment, we sought to investigate whether the phases I and II of ethylene production alter the polyamine metabolism in the leaves of maize genotypes. In salt-tolerant genotype, the phase I of ethylene synthesis was associated with signaling events, as evidenced by increased H2O2 levels, which were generated by putrescine (Put) catabolism. An early signaling (at 5.5 h) in the salt-tolerant genotype seemed to be effective to suppress the second peak of ethylene production, known as âstress ethyleneâ. Yet, in the salt-sensitive genotype, the decreased H2O2 concentration during the phase I was associated with a marked increase in ethylene production, which was resulted from upregulation of acid 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) activity and ZmACO5 gene expression. At the phase I, the total polyamine content was increased by salinity in the salt-tolerant, whereas it was decreased in the salt-sensitive one. In the salt-tolerant genotype, the increased total polyamine was sustained by high spermine (Spm) and spermidine (Spd) contents, while the decay in the salt-sensitive genotype was due to the reductions of Put and Spd forms. Otherwise, in the phase II, no significant changes in the total polyamines in salt-tolerant genotype (it was likely due to conversion of Put to Spm/Spd), and decreases in salt-sensitive genotype were registered. Under stress conditions, the salinity-induced improvement of Spd and Spm (free and soluble conjugated forms) in salt-tolerant genotype was bigger than in salt-sensitive one, thus suggesting a key role of polyamines in the maize salt stress acclimation processes. Finally, we investigated if the lack of ethylene production during phase II in salt-tolerant genotype was correlated to improved antioxidant capacity. Salt stress dramatically increased the superoxide levels, the electrolyte leakage and lipid peroxidation, it being more pronounced in both leaves and roots of salt-sensitive genotype. On the other hand, under salinity, salt-tolerant genotype displayed a better performance of enzymatic and non-enzymatic antioxidant system, evidenced by a higher ascorbate and glutathione content and upregulation of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase activity. In conclusion, our results suggest that the ethylene is intimately involved in salt stress acclimation through activation of intricate signaling pathways mediated by H2O2 that is originated from polyamine catabolism. An efficient signal network raises the polyamine content and antioxidant capacity and is responsible, at least in part, for greater tolerance to salinity of BR5033 maize genotype. / Poliaminas e etileno sÃo reguladores do crescimento e desenvolvimento vegetal, que tambÃm estÃo envolvidos nas respostas de defesa das plantas contra estresses abiÃticos, dentre eles a salinidade. Para lidar com o estresse salino, as plantas realizam ajustes fisiolÃgicos, bioquÃmicos e moleculares, que podem resultar em sua aclimataÃÃo diante dessa condiÃÃo adversa, tornando o indivÃduo mais tolerante ao estresse, em comparaÃÃo Ãqueles que nÃo se encontram aclimatados. Essa pesquisa foi desenvolvida para testar à hipÃtese de que o grau de tolerÃncia à salinidade entre genÃtipos de milho envolve alteraÃÃes no metabolismo das poliaminas associadas à produÃÃo de etileno. Para isso, foram realizados dois experimentos. No primeiro, plantas de milho dos genÃtipos BR5011 (sensÃvel) e BR5033 (tolerante) foram submetidas ao estresse salino (NaCl a 80 mM) para identificar o padrÃo de produÃÃo de etileno em folhas e raÃzes. Nas folhas do genÃtipo sensÃvel, a salinidade intensificou a produÃÃo de etileno apÃs 5,5 h (fase I) e 12,5 h (fase II) apÃs o inÃcio do estresse, enquanto no tolerante isso aconteceu somente com 5,5 h. Nas raÃzes, embora tenha sido observada a produÃÃo bifÃsica de etileno no genÃtipo sensÃvel, esse processo foi muito menos intenso que nas folhas. O segundo experimento teve como objetivo principal investigar se a produÃÃo de etileno pela salinidade nas fases I e II resultava em alteraÃÃes no metabolismo das poliaminas nas folhas dos genÃtipos de milho. No genÃtipo tolerante, a fase I de produÃÃo de etileno foi associada à eventos de sinalizaÃÃo, dado o aumento dos teores de H2O2, mediado pelo catabolismo da putrescina (Put). Essa sinalizaÃÃo pareceu ser eficiente para suprimir a produÃÃo do etileno em condiÃÃes de estresse (fase II ou âetileno do estresseâ) nesse genÃtipo. Jà no sensÃvel, a diminuiÃÃo dos teores de H2O2 na fase I foi acompanhada por um aumento acentuado na produÃÃo do etileno, decorrente de acrÃscimos na atividade da enzima oxidase do Ãcido 1-carboxÃlico-1-aminociclopropano (ACO) e na expressÃo de transcritos do gene ZmACO5 (principal membro expresso). Em geral, a salinidade aumentou os teores de poliaminas totais no genÃtipo tolerante, enquanto reduziu no sensÃvel. Na fase I, na condiÃÃo salina quando comparada com o controle, os teores de poliaminas totais foram aumentados no genÃtipo tolerante enquanto no sensÃvel esses teores foram reduzidos. No genÃtipo tolerante, o aumento nos teores de poliaminas totais foi sustentado principalmente pelo aumento nos teores de espermina (Spm) e espermidina (Spd), enquanto a diminuiÃÃo observada no genÃtipo sensÃvel foi devida, sobretudo, Ãs reduÃÃes nas formas de Put e Spd. Jà na fase II, no genÃtipo tolerante nÃo houve alteraÃÃes nos teores totais de poliaminas (provavelmente, devido a utilizaÃÃo de Put para a sÃntese de Spm e Spd), enquanto no sensÃvel esses teores foram reduzidos. Sob condiÃÃes de salinidade, o aumento nas formas livre e conjugada solÃvel de Spm e Spd foi mais pronunciado no genÃtipo tolerante do que no sensÃvel, sugerindo assim importante papel para essas duas poliaminas nos processos de aclimataÃÃo ao estresse salino em plantas de milho. Por fim, foi investigado se a ausÃncia de produÃÃo do etileno na fase II, causado pela salinidade no genÃtipo tolerante, foi relacionada com uma melhor capacidade antioxidante. O estresse salino aumentou drasticamente os teores do radical superÃxido, o vazamento de eletrÃlitos e a peroxidaÃÃo lipÃdica, sendo isso mais pronunciado nas folhas e raÃzes do genÃtipo sensÃvel. De modo geral, o genÃtipo tolerante teve melhor desempenho do sistema antioxidante enzimÃtico e nÃo enzimÃtico, sob condiÃÃes de estresse salino, evidenciado pelos maiores incrementos nos teores de ascorbato e glutationa e na atividade das enzimas dismutase do superÃxido, peroxidase do ascorbato e peroxidase do guaiacol. Em conclusÃo, os resultados aqui apresentados sugerem que o etileno està intimamente envolvido na aclimataÃÃo ao estresse salino, por meio da ativaÃÃo de vias de sinalizaÃÃo mediadas pelo H2O2 produzido a partir do catabolismo de poliaminas. AlÃm disso, sugere-se que essa sinalizaÃÃo induz o aumento nos teores de poliaminas e melhor capacidade antioxidante no genÃtipo BR5033, sendo isto, pelo menos em parte, responsÃvel por sua maior tolerÃncia ao estresse salino, quando comparado ao BR5011.

Protein PEGylation is the covalent bonding of polyethylene glycol (PEG) polymers to amino acid residues of the protein and it is one of the most promising techniques for improving the therapeutic effect of biopharmaceuticals and long-term stability of protein-based biosensors. This chemical modification brings advantages to biopharmaceuticals, such as an increased half-life, enhanced stability, and reduced immunogenicity. Moreover, in the analytical field, PEGylation improves the multiple properties of protein-based biosensors including biocompatibility, thermal and long-term stability, and solubility in organic solvents. However, the use of PEGylated conjugates in the analytical and therapeutic fields has not been widely explored. The limited industrial application of PEGylated bioconjugates can be attributed to the fact that the reaction and separation steps are currently a challenge. The correct selection of the PEGylation reaction design and the purification process are important challenges in the field of bioconjugation. In this sense, the design and optimization of site-specific PEGylation reactions and application of aqueous biphasic systems (ABS) as purification platforms for PEGylated conjugates are the two main objectives of this thesis. Regarding the purification step, the efficient fractionation (i) of the PEGylated conjugates from the native protein and (ii) of the PEGylated conjugates based on their degree of PEGylation was studied. Centrifugal partition chromatography (CPC) was applied as a continuous regime platform based on ABS technology to efficiently purify the PEGylated proteins. The two proteins under study are L-asparaginase, an important biopharmaceutical applied in the treatment of acute lymphoblastic leukemia and cytochrome c, a promising biosensor. The current work developed in this thesis demonstrates the great potential of ABS in the fractionation of PEGylated proteins, under batch and continuous regime. In addition, in situ recovery of the PEGylated products through one-pot bioconjugation and ABS purification was successfully demonstrated for both enzymes studied. Although further research on scale-up is still required, the results presented show the relevance of ABS platforms for the development of separation processes of PEGylated proteins. / A PEGuilação de proteínas é a ligação covalente de polímeros de polietilenoglicol (PEG) a resíduos de aminoácidos da proteína e é uma das técnicas mais promissoras para melhorar o efeito terapêutico dos biofármacos e a estabilidade a longo prazo de biossensores proteícos. Esta modificação química traz vantagens aos produtos biofarmacêuticos, como um aumento da meia-vida, maior estabilidade e imunogenicidade reduzida. Além disso, no campo analítico, a PEGuilação melhora as múltiplas propriedades dos biossensores baseados em proteínas, incluindo biocompatibilidade, estabilidade térmica e a longo prazo, e solubilidade em solventes orgânicos. No entanto, o uso de conjugados PEGuilados em campos analíticos e terapêuticos não tem sido amplamente explorado. A aplicação industrial limitada dos bioconjugados PEGuilados pode ser atribuída ao facto de as etapas de reacção e separação serem atualmente um desafio. A seleção correcta do design da reacção de PEGuilação e do processo de purificação são importantes desafios no campo da bioconjugação. Neste sentido, a concepção e otimização de reações de PEGuilação sítio-específicas e aplicação de sistemas aquosos bifásicos (ABS) como plataformas de purificação de conjugados PEGuilados são os dois principais objetivos desta tese. No que concerne à etapa de purificação foi estudado o eficiente fracionamento (i) dos conjugados PEGuilados, da proteína nativa e (ii) dos conjugados PEGuilados baseados no seu grau de PEGuilação. A cromatografia por partição centrífuga (CPC) foi aplicada como uma plataforma de regime contínuo baseada na tecnologia de ABS para purificar eficientemente as proteínas PEGuiladas. As duas proteínas em estudo são a L-asparaginase, importante biofármaco aplicado no tratamento da leucemia linfoblástica aguda e o citocromo c, um potencial biossensor. A partir dos trabalhos desenvolvidos, é possível confirmar o grande potencial dos ABS no fracionamento de proteínas PEGuiladas, em regime contínuo e descontínuo. Além disso, a recuperação in situ dos produtos PEGuilados através da integração em uma única etapa de bioconjugação e purificação por ABS foi comprovada com sucesso para ambas as enzimas estudadas. Embora ainda sejam necessários estudos adicionais sobre a viabilidade destes sistemas em larga escala, os resultados aqui apresentados demonstram a relevância dos ABS para o desenvolvimento de processos de separação de proteínas PEGuiladas.

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-22T19:45:32Z No. of bitstreams: 1 gustavomirandateixeira.pdf: 21289543 bytes, checksum: 065f79ef58fa71e8e9917d39cf048fe1 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:26:17Z (GMT) No. of bitstreams: 1 gustavomirandateixeira.pdf: 21289543 bytes, checksum: 065f79ef58fa71e8e9917d39cf048fe1 (MD5) / Made available in DSpace on 2017-08-07T19:26:17Z (GMT). No. of bitstreams: 1 gustavomirandateixeira.pdf: 21289543 bytes, checksum: 065f79ef58fa71e8e9917d39cf048fe1 (MD5) Previous issue date: 2011-07-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Simulação de Reservatórios é uma importante área da engenharia de reservatórios. Simuladores são baseados em modelos matemáticos que modelam o escoamento dos fluidos através do meio porosos. Modelos água-óleo incompressíveis são representados por sistemas de equações diferenciais parciais em duas variáveis: pressão e saturação dos fluidos. O problema consiste de um sistema de equações de difícil resolução e que exige o uso de métodos iterativos para aproximar a solução. Neste trabalho foi feita uma comparação entre métodos numéricos que aproximam a solução deste problema através uma abordagem acoplada, utilizando um método implícito e um esquema desacoplado. O sistema desacoplado utiliza um método implícito para obter a solução da equação da pressão, enquanto a solução da saturação é aproximada através dos métodos de Euler Explícito e BDF (Backward Differentiation Formulas). Para resolver as equações no espaço, os métodos de Kurganov-Tadmor e o Esquema Upwind foram utilizados. O Esquema Upwind é um método de primeira ordem, enquanto o Kurganov-Tadmor (KT) é um método central de segunda ordem. Testes foram realizados utilizando diferentes tipos de reservatórios e propriedades da rocha e do óleo. Os resultados foram comparados com soluções precisas para medir o erro numérico introduzido por cada método. Através dos resultados obtidos nesse trabalho foi possível comparar os métodos e observar que a solução através de um esquema desacoplado é mais vantajosa, principalmente ao utilizar o método BDF para resolver a equação da saturação no tempo. Comparando o esquema Upwind com o KT, verificamos que, como esperado, o KT é um método menos difusivo. Nosso trabalho sugere que o uso do método KT em associação com o BDF resulta em um método mais preciso. Por outro lado, o KT é computacionalmente mais custoso do que o Esquema Upwind. Contudo, para produzir resultados com erros numéricos semelhantes aos do KT, o método Upwind chega a ser mais de seis vezes mais lento. / Reservoir simulation is part of an important area of reservoir engineering. Reservoir simulators are based on mathematical models capable of predicting the fluid flow through porous medium. Water-oil incompressible models are represented by a system of differential partial equations in two variables: pressure and the fluid saturation. The system of equations of the problem is not simple to solve and it demands the use of iterative methods to get the approximate solution. In this work we compared a coupled implicit approach and a decoupled scheme. The decoupled system evolves in time using an implicit method to solve the pressure equation solution whereas the solution for the saturation equation is obtained using Explicit Euler and BDF (Backward Differentiation Formulas) methods. The solutions in space discretization use the Kurganov-Tadmor method and the Upwind Scheme. Upwind Scheme is a first-order finite volume based method whereas Kurganov-Tadmor (KT) is a second-order central scheme. Different kinds of reservoir types and different rock and oil properties were used to compare the results with more precise solutions in order to estimate the amount of numerical errors introduced by each method. These results allowed us to quantify the difference between the analyzed methods and to observe that the solution obtained from the decoupled method yields the best results, in particular when the BDF method is used to solve the saturation equation in time. The solution from the BDF method in association with the Upwind Scheme compared with the KT shows that, as expected, the KT method is less diffusive than the Upwind. Our work suggests that the use of the KT method in association with the BDF results in a more accurate method. On the other hand, the KT method is computationally more expensive compared to Upwind Scheme. Nevertheless, to produce numerical results with similar errors to KT, the Upwind Scheme can be more than five times slower.

The instantaneous mortality rate (M) is an important parameter in elasmobranch management and conservation, but is difficult to estimate directly. Thus, indirect estimates based on relatively easily obtained life history parameters are commonly used. Many indirect methods incorporate one or more parameters from the von Bertalanffy growth model (VBGM), which is often criticized for its inability to describe changes in growth associated with maturity. The Lester growth model (LGM) is a biphasic alternative to the VBGM that incorporates trade-offs between reproduction, growth, and survival, and may therefore more accurately estimate M. I used published data from 29 elasmobranch species to compare the performance of the LGM to four conventional growth models and nine conventional methods for indirectly estimating M. For three species (Heterodontus portusjacksoni, Rhizoprionodon taylori, and Carcharhinus limbatus), I obtained direct estimates of M to evaluate the accuracy of indirect M methods. According to AICc, the LGM was the best fitting model for 80.8% of datasets. Using one-sample t-tests, I found that five indirect M methods (two of which are dependent on the LGM) consistently generated estimates of M that were in close agreement with direct estimates. The most common methods in elasmobranch literature appear to be overestimating M by factors of 1.34 – 1.91. However, further research is needed to verify these results across a wider range of species. Overall, I recommend using the LGM to describe the lifetime growth of sharks, and estimating M by averaging across five indirect methods.

Legionella pneumophila, responsable de la maladie du légionnaire, est une bactérie aquatique parasitant les amibes, mais aussi les macrophages alvéolaires humains. Legionella alterne entre une forme infectieuse non réplicative et une forme réplicative intracellulaire, qui n'exprime pas les facteurs de virulence. Ce cycle de vie biphasique est gouverné par un système de régulation complexe permettant son adaptation dans différents hôtes. Le but de mon projet de thèse était d'étudier un des facteurs clés dans la régulation des ARNm, le régulateur post-transcriptionnel global Hfq. L'expression de Hfq est régulée au cours du cycle infectieux chez L. pneumophila: Hfq est peu exprimée en phase réplicative, mais fortement exprimée lors de la phase de transmission, ce qui suggère un rôle dans la transition entre ces deux phases. J'ai identifié un petit ARN (sRNA) que j'ai nommé Anti-hfq puisqu'il est transcrit dans l'orientation antisens à Hfq et chevauche sa région 5' non traduite (UTR). Mes recherches ont mis en évidence un mécanisme sophistiqué par lequel Anti-hfq régule l'expression de Hfq: Anti-hfq interagit avec l'ARNm du gène hfq par sa région complémentaire et ainsi contrôle la stabilité de la protéine. De plus, j'ai montré que la protéine Hfq auto-réprime sa propre traduction en facilitant l'interaction entre Anti-hfq et son propre ARNm. Finalement, Hfq régule son propre renouvellement par le recrutement de la RNase III. De plus, des tests de réplication intracellulaire ont montré que Hfq et Anti-hfq sont nécessaires pour la multiplication intracellulaire de L. pneumophila, ce qui a mis en évidence un rôle important de Hfq et Anti-hfq dans la virulence de cette bactérie. / Legionella pneumophila, the causative agent of the pneumonia-like Legionnaires’ disease, is commonly found in aquatic habitats worldwide where it multiplies within protozoa. To adapt between intra- and extracellular environments, L. pneumophila evolved a biphasic lifecycle wherein it alternates between an infectious and non replicative form and an intracellular form, which does not express the virulent phenotypes. This biphasic life cycle is governed by a complex regulatory network that comprises transcriptional and post-transcriptional regulatory elements, enabling the bacteria to adapt in diverse hosts. During my Ph.D., I focused my attention of the post-transcriptional regulator Hfq, a hexameric, RNA-binding protein and chaperon of small RNAs (sRNA). The expression of this fascinated protein is life cycle regulated: poorly expressed during the replicative phase of growth, whereas significantly upregulated upon entry into the transmissive phase of growth. Moreover, my research research lead to the identification of a sRNA transcribed antisense to the hfq gene overlapping its 5’UTR region. This antisense RNA, named Anti-hfq, was found to regulate hfq expression by base-pairing complementarity, describing a sophisticated mechanism of regulation. In addition, the Hfq protein controls its own translation by facilitating the interaction between Anti-hfq and its own mRNA. Thus, Hfq regulates its turnover, recruiting the endoribonuclease RNaseIII. Furthermore, infection assays revealed that Hfq and Anti-hfq are necessary for efficient replication of L. pneumophila in amoeba revealing an important role of both in bacterial virulence.

Orientador: Orlando Petrucci Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T07:48:01Z (GMT). No. of bitstreams: 1 Assumpcao_AntonioCarlos_D.pdf: 13951044 bytes, checksum: 4ebd9040bcd7ea540e6927bdc4258384 (MD5) Previous issue date: 2013 / Resumo: Introdução: O aumento do limiar de estimulação ventricular (LEV) tem sido observado após a administração de choque elétrico de cardioversão/desfibrilação transtorácico (CDT) para desfibrilação ventricular, contudo, poucos estudos têm avaliado este fenômeno no que diz respeito à energia e a forma de onda empregada para a CDT. Este estudo analisou o LEV após CDT de 360J, comparando-se os resultados após aplicação de onda de energia Mono e Bi. Método: Em suínos Landrace femininos foram implantados sistema de estimulação permanente de marcapasso, divididos em três grupos: sem indução de fibrilação ventricular (FV) e CDT com formato de onda monofásica (Mono) e bifásica (Bi) (Grupo I); indução FV, um minuto de observação sem intervenção, dois minutos de massagem cardíaca externa, e CDT Mono e Bi (Grupo II) e indução de FV, dois minutos de observação sem intervenção, quatro minutos de massagem cardíaca externa e CDT Mono e Bi (Grupo III). Após CDT, o LEV foi avaliado a cada minuto durante dez minutos. Resultados: Um total de 143 experimentos foram concluídos. No final do período de observação, os grupos I e II mostraram valores constantes LEV. O Grupo III mostrou aumento no LEV Mono e Bi com energia externa, sem diferença entre os formatos de ondas externas. O formato Mono foi associada a valores mais elevados de LEV quando o tempo de parada cardiorrespiratória (PCR) foi maior, o que não foi verificado na Bi. Conclusão: A CDT não tem impacto significativo sobre o LEV. Com onda Mono, observou-se aumento do LEV quando o período de PCR foi mais longo / Abstract: Introduction. Although an increase in the ventricular pacing threshold (VPT) has been observed after administration of transthoracic shock for ventricular defibrillation, few studies have evaluated the phenomenon in respect to the defibrillation waveform energy. Therefore, this study examined the VPT behavior after transthoracic shock with a monophasic or biphasic energy waveform. Method. Domestic Landrace female piglets implanted with a permanent pacemaker stimulation system were divided into 3 groups: no ventricular fibrillation (VF) induction and transthoracic shock with monophasic or biphasic energy (group I); VF induction, 1 minute of observation without intervention, 2 minutes of external cardiac massage, and transthoracic shock with monophasic or biphasic energy (group II); and VF induction, 2 minutes of observation without intervention, 4 minutes of external cardiac massage, and transthoracic shock with monophasic or biphasic energy (group III). After external shock, the VPT was evaluated every minute for 10 minutes. Results. A total of 143 experiments were performed. At the end of the observation period, groups I and II showed steady VPT values. Group III showed an increase in VPT with monophasic or biphasic external energy, with no difference between the external energy sources. The monophasic but not the biphasic waveform was associated with higher VPT values when the VF was longer. Conclusion. Defibrillation does not have a significant impact on pacing threshold, but a longer VF period is related to a higher VPT after defibrillation with monophasic waveform / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

Articular cartilage is a specialized connective soft tissue that resides onthe ends of long-bones, transfers the load smoothly between the bones in diarthrodialjoints by providing almost frictionless, wear resistant sliding surfacesduring joint articulation. Focal chondral or osteochondral defects in articularcartilage are common and show limited capacity for biological repair. Furthermore,changes in the bio-mechanical forces at the defect site may makethe tissue more susceptible to continued degeneration. Alternatively, the contouredfocal resurfacing metal implant can be used to treat such full thicknesscartilage defects. Physiological and biomechanical studies on animal modelswith metal implant have shown good clinical outcomes. However, the mechanicalbehavior of cartilage surrounding the implant is not clearly known withrespect to the joint function after treating such defects with metal implantsand also to improve the implant design. We developed a simple 3-dimensionalfinite element model by approximating one of the condyles of the sheep kneejoint. Parametric study was conducted in the simulations to verify differentprofiles for the implant, positioning of the implant with respect to cartilagesurface, defect size and to show the mechanical sealing effect due to the wedgeshape of the implant. We found the maximal deformations, contact pressuresand stresses which constitute the mechanical behavior of cartilages. We alsoconfirmed that using a metal implant to fill the full thickness chondral defectsis more beneficial than to leave the defect untreated from mechanical point ofview. The implant should be positioned slightly sunk into the cartilage basedon the defect size, in order to avoid damage to the opposing surface. The largerthe defect size, the closer the implant should be to the flush. We also simulatedthe time dependent behavior of the cartilages. In all the simulations, a staticaxial loading was considered. The wedge shape of the implant provided themechanical sealing of the cartilage surrounding the implant. The determineddeformations in the cartilages immediately surrounding the implant are instrumentalin predicting the sticking-up of the implant into the joint cavity whichmay damage opposing soft tissues. / <p>QC 20101125</p>

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