Dysregulation of endothelial cell barrier function is associated with a wide variety of human diseases ranging from tumor metastasis to inflammation. The barrier function of endothelial cells is maintained by cell adhesion molecules (CAMs). Immunoglobulin containing and proline-rich receptor-1(IGPR-1) was recently identified as a novel CAM involved in angiogenesis. However, the molecular mechanism of IGPR-1 function in endothelial cells remains largely unknown. The overarching goals of this study were: (A) to determine molecular mechanism by which IGPR-1 stimulates biological responses in cells and (B) to investigate regulation of phosphorylation of IGPR-1 at serine 220 (Ser220), and its role in IGPR-1 function. Our data demonstrate that IGPR-1 undergoes cis-dimerization, which leads to homophilic trans-dimerization of IGPR-1, which is required for its adhesive function. Moreover, we demonstrate that phosphorylation of Ser220 is regulated by trans-dimerization of IGPR-1 and that Glycogen Synthase Kinase 3 (GSK-3) is responsible for its phosphorylation as over-expression of kinase active increased and kinase inactive inhibited phosphorylation of Ser220, respectively. Taken together, the results demonstrate that the coordinated dimerization of IGPR-1 and its homophilic interaction regulates its adhesive function and serine phosphorylation. The adhesive function of IGPR-1 contributes to the barrier function of endothelial cells.

Orientador: Rosilene Fernandes da Rocha / Banca: Wilson Roberto Sendik / Banca: Mariane Spalding / Banca: Fernando Renó de Lima / Banca: Lafayette Nogueira Junior / Resumo: O propósito deste trabalho foi avaliar o efeito do plasma rico em plaquetas associados ou não ao enxerto ósseo autógeno no processo de reparação óssea em defeitos cirúrgicos confeccionados na tíbia de coelhos. Nesta pesquisa foram utilizados 25 coelhos adultos, nos quais foram realizados 2 defeitos em cada tíbia, divididos nos seguintes grupos de acordo com o tratamento: controle (C - defeito preenchido somente por coágulo sangüíneo), autógeno (A - defeito + enxerto), PRP (PRP = defeito + PRP) e autógeno + PRP (PRPA - defeito + enxerto + PRP). Todos os defeitos foram recobertos com uma barreira de PTFE e decorridos 15, 30 e 60 dias, 5 animais foram sacrificados por período, sendo as peças contendo os defeitos processadas para análises histológica e histomorfométrica. Outros 5 animais foram sacrificados aos 30 e 60 dias e submetidos à análise das propriedades biomecânicas e todos os espécimes foram submetidos ao exame radiográfico para análise da densidade óptica. Os resultados biomecânicos, radiográficos e histomorfométricos mostraram maior resistência, densidade óptica e maior formação óssea nos grupos A e PRPA quando comparados com os grupos C e PRP. Os resultados obtidos possibilitaram concluir que não houve uma melhora nos parâmetros radiográficos, mecânicos e na neoformação óssea quando o PRP foi usado isoladamente ou associado ao enxerto ósseo autógeno. / Abstract: The aim of this study was to evaluate the effect of platelet rich plasma associated or not to autogenous bone graft in bone repair process of tibia rabbits surgical defects. In this research 25 adult rabbits were used, 2 defects were accomplished in each tibia, divided into groups: control (C = bone defect only filled out by blood coagulum), autogenous (A = bone defect + autogenous graft), PRP (PRP = bone defect + PRP) and autogenous + PRP (PRPA = bone defect + autogenous graft + PRP). All of the defects were covered with a PTFE barrier and after 15, 30 and 60 days there were elapsed. Five animals were sacrificed by period, being the pieces containing the defects processed for histological and histomorphometric analysis. Other 5 animals were sacrificed to the 30 and 60 days and submitted to biomechanics analysis and all of the specimens were submitted to the radiographic examination of the optical density. The biomechanic, radiographic and histomorphometric results showed larger resistance, optical density and better bone formation in the groups A and PRPA when compared with the groups C and PRP. This study demonstrated that there was not an improvement in the radiographic, mechanics, and bone formation parameters when PRP was used separately or associated to the autogenous bone graft. / Doutor

Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics) / In recent years, the development and use of autologous platelet rich concentrates (PC's) has gained traction within the rapidly progressive, multidisciplinary field of regenerative medicine. A PC subtype, marketed as advanced platelet rich fibrin (A- PRF), is a recent advancement of the original PRF protocol and promoted as a "blood concentrate" containing platelets, leukocytes, circulating stem cells and endothelial cells. A-PRF in the form of membranes, plugs, or even shredded particulates are increasingly being used as surgical adjuncts in areas of previous infection or left exposed within the microbial rich oral environment. Although recent literature has noted the biologic benefits of this material within the context of wound healing and regeneration, the antimicrobial potential of APRF has remained unexplored. The aim of this investigation is to determine if A-PRF displays antimicrobial activity against microbes of the oral cavity with a null hypothesis that its activity is no different to a clot of unprocessed venous blood. Methodology: A-PRF and whole blood samples were obtained from consenting individuals and utilised to conduct an in-vitro agar disk diffusion investigation to determine their antimicrobial activity. Standardised samples of A-PRF, unprocessed clotted blood and 0.2% chlorhexidine gluconate (CHX) were tested against organisms cultured from fresh oral rinse samples and pure cultures of candida albicans, streptococcus mutans, staphylococcus aureus and enterococcus faecalis. The antimicrobial activity was assessed in accordance to the established principles of the agar disk diffusion method and measurement of inhibition zones. Results: A-PRF displayed antimicrobial activity against all of the individual organisms tested within this study following a 24 hour incubation period. However, no significant differences were noted between A-PRF and a natural clot of blood when tested against cultures of the oral rinse sample. Finally, the antimicrobial activity of A-PRF is significantly inferior to an equal volume of the CHX preparation. Conclusion: Although A-PRF displays antimicrobial activity; its strength, spectrum and biologic activity within a polymicrobial environment requires further investigation.

Insect, soil and vegetation sampling was carried out to determine processes driving insect and plant diversity in species rich grasslands in the White Carpathian Mountains Protected Landscape Area in the Czech Republic. At the same time, the occurence specialized insects was detected for Melampyrum nemorosum and Rhinanthus spp. (Orobanchaceae).

Title: Interactive crawling and data extraction Author: Bc. Petr Fejfar Author's e-mail address: pfejfar@gmail.com Department: Department of Distributed and Dependable Systems Supervisor: Mgr. Pavel Je ek, Ph.D., Department of Distributed and De- pendable Systems Abstract: The subject of this thesis is Web crawling and data extraction from Rich Internet Applications (RIA). The thesis starts with analysis of modern Web pages along with techniques used for crawling and data extraction. Based on this analysis, we designed a tool which crawls RIAs according to the instructions defined by the user via graphic interface. In contrast with other currently popular tools for RIAs, our solution is targeted at users with no programming experience, including business and analyst users. The designed solution itself is implemented in form of RIA, using the Web- Driver protocol to automate multiple browsers according to user-defined instructions. Our tool allows the user to inspect browser sessions by dis- playing pages that are being crawled simultaneously. This feature enables the user to troubleshoot the crawlers. The outcome of this thesis is a fully design and implemented tool enabling business user to extract data from the RIAs. This opens new opportunities for this type of user to collect data from Web pages for use...

xvii, 171 p. : ill. (some col.) / Crystals influence the migration of gas through magma. At low concentrations, they increase the bulk fluid properties, especially viscosity. At concentrations close to maximum packing, crystals form a rigid framework and magma cannot erupt. However, erupted pyroclasts with crystal contents close to the packing concentration are common at mafic volcanoes that exhibit Strombolian behavior. In this dissertation, I study the influence of solid particles on gas migration. I apply my results to Stromboli volcano, Italy, type locality of the normal Strombolian eruptive style, where gas moves through an essentially stagnant magma with crystallinity ∼50%. Specifically, I investigate the effect of crystals on flow regime, gas content (Chapter II), bubble concentration (number densities), bubble shapes, bubble sizes (Chapter III), and bubble rise velocities (gas flux) (Chapter IV). I find that gas-liquid flow regimes are not applicable at high particle concentrations and should be replaced by new, three-phase (gas-liquid-solid) regimes and that degassing efficiency increases with particle concentration (Chapter II). In Chapter III, I show that crystals modify bubble populations by trapping small bubbles and causing large bubbles to split into smaller ones and by modifying bubble shapes. In Chapter IV, I model Stromboli's crystal-rich magma as a network of capillary tubes and show that bubble rise velocities are significantly slower than free rise velocities in the absence of particles. In each chapter, I use analogue experiments to study the effect of different liquid and solid properties on gas migration in viscous liquids. I then apply my analogue results to magmatic conditions using simple parameterizations and/or numerical modeling or by comparing the results directly to observations made on crystal-rich volcanic rocks. Chapter V proposes a mechanism for Strombolian eruptions and gas migration through the crystalrich magma in which the effect of crystals is included. This model replaces the current twophase "slug" model, which cannot account for the high crystallinity observed at Stromboli. There are three appendices in this dissertation: a preliminary study of the influence of particles on gas expansion, image analysis methods, and the numerical code developed in Chapter IV. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Katharine Cashman, Chairperson; Alan Rempel, Member; Mark Reed, Member; Raghuveer Parthasarathy, Outside Member

In this dissertation I incorporate high dimensional data vectors in estimated Dynamic Stochastic General Equilibrium (DSGE) models, evaluating the labor market dynamics incorporated inside such data vectors, out-of-sample forecasting performance of many models estimated with such data vectors and analytically examining the reduction of macroeconomic volatility that can occur when such data vectors are used in the formation of expectations about the future. The second chapter investigates the extent to which modern DSGE models can produce labor market dynamics in response to a financial crisis that are consistent with the experience of the Great Recession. I estimate two New-Keynesian models, one with and one without financial frictions, in a data-rich environment. I find that negative financial shocks are associated with longer recoveries in real investment, capital-intensive sectors of the labor market and average unemployment duration. I also find the model with a financial accelerator is equipped with better tools to identify the dynamics associated with the Great Recession and its recovery in regard to many labor and financial metrics. The third chapter compares the out-of-sample forecasting performance of the two DSGE models of Chapter II when they are estimated both out of and in a data-rich environment. This chapter finds that many financial time series variance decomposition are significantly better explained using the structural set-up of the New-Keynesian model with financial frictions. DSGE models estimated with high dimensional data vectors significantly out forecast their regularly estimated counterpart in regard to output, investment and consumption growth. Lastly, the use of real-time optimal pool model weighting significantly out-forecasts traditional macroeconomic models as well as an equally weighted weighting scheme in terms of many macroeconomic variables. The fourth chapter examines the role forecasts derived by high dimensional data vectors can have on lowering macroeconomic volatility. Bounded rational agents are introduced into the Chapter II DSGE model with financial frictions and are given the option to use or ignore professionally generated forecasts from a dynamic factor model in their perceived forecasting model. In simulations, I find that professionally generated forecasts can significantly lower the volatility of many macroeconomic variables including inflation and hours worked.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

Orientadora: Profa. Dra. Christiane Bertachini Lombello / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Engenharia Biomédica, 2015. / A engenharia de tecidos visa cultivar celulas sobre substratos tridimensionais, para implantacao no organismo, restaurando desta forma partes danificadas. Os materiais utilizados como substratos devem apresentar caracteristicas propicias ao cultivo celular. Na cultura de celulas e necessario o estabelecimento de certas condicoes, como o modo de suplementacao do meio de cultura. Sendo assim, este estudo avaliou sistemas de cultura autologo (coagulo e soro do plasma rico em plaquetas) e xenogeno (gelatina e soro fetal bovino), como substratos e suplementos para a cultura de celulas. O plasma rico em plaquetas (PRP) apos ativacao da cascata de coagulacao apresenta uma estrutura tridimensional, o coagulo. Com a retracao do coagulo, ocorre a liberacao do soro do PRP. A gelatina e uma substancia com capacidade de gelificacao. Para manutencao de sua estrutura, a gelatina foi reticulada com glutaraldeido. A caracterizacao do substrato de gelatina e a liberacao de glutaraldeido foram avaliadas por espectroscopia. Foram realizadas avaliacoes do intumescimento e da degradacao do coagulo e da gelatina por 24 horas e cinco dias, respectivamente. A analise da ultraestrutura dos substratos foi realizada por microscopia eletronica de varredura (MEV). O diametro das fibras do coagulo e a rugosidade superficial da gelatina tambem foram examinados. Para as analises biologicas foram utilizadas celulas Vero. Nas culturas com o coagulo, o meio foi suplementado com 10% do soro do PRP e as culturas com gelatina tiveram o meio suplementado com 10% de SFB. A avaliacao da citotoxicidade foi realizada atraves do teste do extrato e de contato direto das celulas com os substratos. Nas analises do comportamento celular com os substratos, as celulas foram inoculadas sobre o coagulo e a gelatina. As culturas foram mantidas a 37¿C com 5% CO2. As avaliacoes por espectroscopia da gelatina nao identificaram alteracao da estrutura primaria e a presenca do agente reticulador. Os substratos do coagulo e da gelatina exibiram intumescimentos superiores a 200% e ambos demonstraram boa estabilidade, nao apresentando uma degradacao significativa no periodo de cinco dias. A ultraestrutura do coagulo e formada por uma rede irregular de fibras de diferentes diametros, com um valor medio de 0,210}0,097 ¿Êm. A gelatina apresentou uma estrutura densa e plana, exibindo uma micro-rugosidade de 0,11}0,02 ¿Êm. As avaliacoes da citotoxicidade confirmaram que os sistemas de cultura nao foram toxicos para as celulas Vero, exibindo valores de viabilidade celular superiores a 90%. Na analise da interacao celular com os substratos foi verificado que as celulas aderiram e se espalharam sobre os materiais. As celulas presentes na gelatina exibiram maior area superficial (866,32}390,53 ¿Êm) do que as celulas aderidas no coagulo (425,53}245,21 ¿Êm), indicando maior interacao com o substrato. Portanto, os resultados sugerem que ambos os sistemas de cultura sao promissores para utilizacao em engenharia de tecido. Todavia o sistema autologo apresenta a vantagem de ser proprio do organismo. / Tissue engineering aims to grow cells on three dimensional substrates for implantation in the body, restoring damaged parts. The materials used as substrates must have favorable characteristics to the cell culture. In cell culture, it is necessary to establish certain conditions, such as supplementation of the culture medium. Thus, this study evaluated autologous culture systems (clot and serum platelet-rich plasma) and xenogeneic (gelatin and fetal bovine serum) as substrates and supplements for cell culture. The platelet-rich plasma (PRP) upon activation of the coagulation cascade has a three dimensional structure, the fibrin clot. With clot retraction, the serum PRP is released. Gelatin is a substance with gelling capacity. To maintain its structure, gelatin was crosslinked with glutaraldehyde. Characterization of the gelatin substrate and the release glutaraldehyde were assessed by spectroscopy. For the characterization of the substrates were performed assessments of swelling and degradation for 24 hours and five days, respectively. The analysis of the ultrastructure of the substrates was performed by scanning electron microscopy (SEM). The diameter of the clot fiber and the surface roughness of gelatin were also examined. For biological testing, Vero cells were used. In cultures with the clot the medium was supplemented with 10% serum from PRP and gelatin medium were supplemented with 10% FBS. Assessment of cytotoxicity was performed using the test extract and direct contact of the cells with the substrates. In cellular behavior analysis with the substrates, the cells were inoculated onto the clot and gelatin. Cultures were maintained at 37°C with 5% CO2. The evaluations of gelatin by spectroscopy have not identified change in the primary structure and the presence of the crosslinking agent. The substrates of the clot and gelatin exhibited swellings higher than 200% and both demonstrated good stability, showing no significant degradation over a five day period. The ultrastructure of the clot is formed by an irregular network of fibers of different diameters, with an average value of 0.210±0.097 ìm. The gelatin presented a dense and planar structure, showing a micro-roughness of 0.11±0.02 ìm. The evaluation of cytotoxicity confirmed that culture systems were not toxic to Vero cells, exhibiting cell viability higher than 90%. In the analysis of cell interaction with the substrates, was found that the cells adhered and spread on the materials. Cells present in the gelatin exhibited a greater surface area (866.32±390.53 ìm) than the cells adhered in the clot (425.53±245.21 ìm), indicating a greater interaction with the substrate. Therefore, the results suggest that both culture systems are promising for use in tissue engineering. However autologous system has the advantage of being the body itself.

Parkinson's Disease (PD) is a late-onset neurodegenerative disease characterized by progressive motor dysfunction caused by a loss of dopaminergic neurons for which there is no known cure. Among the most common genetic causes of PD are mutations in the leucine-rich repeat kinase 2 gene (LRRK2), encoding a multi-domain protein with kinase activity. The LRRK2 G2019S mutation causes hyperactivity of the kinase domain and is the most frequent LRRK2 mutation in patients with familial PD, though its role in PD pathology remains unclear. Preliminary data from the lab of our collaborator, Dr. David Park, demonstrated through a genetic screen in Drosophila melanogaster that the deletion of rbp9 encoding an RNA-binding protein prevented pathology induced by PD-relevant mutations in the LRRK2 kinase domain. The neuronal homolog of RBP9 in humans is HuD, a member of the Hu family of RNA-binding proteins that regulates the expression of many transcripts involved in neuronal development, plasticity, and survival. In addition, HuD has been shown to modify the age-at-onset or risk of developing PD. Here, we studied the effect of LRRK2 on the post-transcriptional regulation of mRNAs bound by HuD in the context of PD. Our findings showed that HuD is a substrate for LRRK2 phosphorylation in vitro, and that LRRK2 G2019S hyperphosphorylates HuD. We demonstrated that LRRK2 kinase activity is required for the binding of several transcripts by HuD that encode PD-relevant proteins such as α-synuclein and neuronal survival factor BDNF. Our findings in human neuroblastoma cells indicated that LRRK2 regulates the protein levels of HuD mRNA targets α-synuclein and BDNF in a mechanism that can by modified by HuD. Finally, we showed that the combination of HuD knockout with LRRK2 G2019S expression in mice rescues aberrant expression of HuD targets in mice with only the LRRK2 G2019S mutation or the knockout of HuD alone. Together, our findings demonstrate that LRRK2 affects the post-transcriptional regulation of HuD-bound mRNAs, and suggest the use of HuD as a potential therapeutic target in patients with PD caused by the LRRK2 G2019S mutation.