Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [106]-127).

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 129-157). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 129-157). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 18, 2007) Vita. Includes bibliographical references.

A leptospirose é uma zoonose de distribuição mundial causada por espiroquetas patogênicas do gênero Leptospira. Resultados experimentais demonstraram que a patogênese pode estar relacionada com a capacidade destas bactérias em aderir a proteínas da matriz extracelular, escapar da resposta imune do hospedeiro e de produzir toxinas. Este trabalho teve como objetivo identificar proteínas secretadas por Leptospira interrogans sorovar Pomona estirpe Fromm kennewicki (patogênica) e Leptospira biflexa sorovar Patoc estirpe Patoc I (saprófita), através de análise proteômica. As leptospiras foram cultivadas em meio EMJH suplementado com soro de coelho ou albumina bovina. Os sobrenadantes foram filtrados, dialisados e liofilizados para aplicação das tecnologias de análise proteômica utilizando gel bidimensional e análise em solução. A análise dos peptídeos obtidos, nos dois procedimentos, foi realizada utilizando-se LC/MS/MS. Foi possível a identificação de 159 proteínas diferentes nas amostras de L.interrogans, entre as quais 64 foram positivas em pelo menos uma das ferramentas usadas para a predição. Em L. biflexa, 104 proteínas diferentes foram identificadas, entre elas 43 proteínas foram positivas pela análise in silico. Entre as proteínas identificadas, estão aquelas que possuem peptídeo sinal sec ou tat dependentes. Em outras, a predição da localização celular é desconhecida ou podem ter múltiplos sítios de localização, e ainda, proteínas que não possuem peptídeo sinal e que podem ser secretadas por mecanismos não convencionais. Muitos destas são proteínas hipotéticas sem domínios conservados detectados. No que diz respeito à atividade proteolítica, foi identificada a presença de metaloproteases no secretoma de L.interrogans. Não houve detecção da presença significativa de proteases bacterianas em amostras de L. biflexa. A identificação e a caracterização funcional de proteínas secretadas poderão contribuir para a elucidação dos mecanismos patogênicos e no desenvolvimento de novas estratégias para o tratamento e prevenção de leptospirose. / Leptospirosis is a zoonosis of worldwide distribution caused by pathogenic spirochetes of the genus Leptospira. The mechanisms by which leptospires invade the host and cause the disease are not yet fully understood. Experimental results have shown that the pathogenesis may be related to the ability of these bacteria to bind to extracellular matrix proteins, to escape hosts immune responses and to produce toxins. This work aimed to identify secreted proteins by Leptospira interrogans serovar Pomona strain Fromm kennewicki (pathogenic) and Leptospira biflexa serovar strain Patoc Patoc I (saprophyte) through proteomic analysis. The leptospires were grown in EMJH supplemented with rabbit serum or BSA. Supernatants were filtered, dialyzed and lyophilized to proteomic technology, two-dimensional gel and non-gel. The analysis of the obtained peptides in two procedures was performed using LC/MS/LC. It was possible to identify 159 different proteins in the samples of L.interrogans; among them, 64 were positive proteins in at least one of the tools used for prediction. In L. biflexa, 104 different proteins were identified; among them, 43 positive proteins were positive by in silico analysis. Among the identified proteins are those that possess sec or tat dependent signal peptide. In others, the prediction of the cellular location is unknown or may have multiple sites of localization, and even proteins which have no signal peptide can be secreted by unconventional mechanisms. Many of these are hypothetical proteins with no detected putative conserved domains. The presence of metalloproteases has been identified in the L.interrogans´ secretome, using proteolytic assay. There was no significant detection of the presence of bacterial proteases in samples of L. biflexa. The identification and functional characterization of secreted proteins may contribute to the elucidation of pathogenic mechanisms and in the developing of new strategies for the treatment and prevention of leptospirosis.

No description available.

Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.

Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.

Introdução: O remodelamento da matriz extracelular no líquen escleroso (LS) caracteriza-se histologicamente por uma faixa hialinizada situada predominantemente na derme superficial, semelhante ao que ocorre na lipoidoproteinose (LPE), uma genodermatose rara, na qual ocorre deficiência na produção da proteína 1 da matriz extracelular (ECM-1). Recentemente, em casos de LS foram descobertos auto-anticorpos contra a ECM-1 e um novo caminho foi proposto para desvendar a sua etiopatologia. O LS também é freqüentemente comparado com a Esclerodermia, visto que alguns autores consideram que são espectros de uma mesma doença. Na Esclerodermia e na LPE há aumento do colágeno tipo V (COL V), mas pouco se sabe sobre este tipo de colágeno no LS. Assim, o objetivo do presente trabalho foi demonstrar a localização e a quantidade de COL V e ECM-1 nas vulvas de pacientes com LS. Materiais e métodos: foram estudadas 21 biópsias de pacientes com LS vulvar e 21 biópsias de vulvas normais. A morfometria foi realizada nas imagens geradas através da imunofluorescência marcada com anticorpos contra os colágenos I (COL I), III (COL III) e V, e também nas de imunoistoquímica para ECM-1. Ademais, utilizou-se microscopia confocal a laser para visualizar o COL V e a ECM-1 na mesma lâmina. Resultados: Peles do grupo controle mostraram fraca e homogênea distribuição das fibras de COL I, III e V na imunofluorescência. Em contraste, peles com LS exibiram desarranjo da arquitetura da derme superficial e difuso aumento da fluorescência das fibras de COL I, III e V na faixa hialina. A fração de área das fibras de COL I, III, e V foram significativamente maiores nas peles de pacientes com LS em relação ao controle (p<0,05). Peles controles mostraram co-localização da ECM-1 e do COL V na parede de pequenos vasos e ao longo da membrana basal. Entretanto, no LS houve perda de expressão da ECM-1 na parede dos vasos sanguíneos (p<0,001) e difuso aumento da fluorescência verde do COL V (p<0,001). Conclusão: Houve inversa relação entre o COL V e a proteína ECe constatado pela histomorfometria, imunofluorescência, imunoistoquímica e reconstrução tridimensional, sugerindo que estratégias terapêuticas para prevenir o aumento da síntese de COL V e a diminuição da ECM-1 poderão ser promissoras no prognóstico e tratamento do LS / Background: The extracellular matrix remodeling in lichen sclerosus (LS) is characterized in routine light microscopy by a hyalinized band predominantly located in the superficial dermis. The same pattern occurs in the lipoid proteinosis (LPE), a rare genodermatosis, in which the production deficiency of extracellular matrix protein 1 (ECM-1) is responsible for triggering the disease. Recently, in cases of LS, autoantibodies were discovered against ECM-1 and a new way to unravel their aetiopathology was proposed. LS is also frequently compared with Scleroderma, since some authors consider that they are spectra of a similar disease. In Scleroderma and LPE there is an increase of type V collagen (COL V), but little is known about this type of collagen in LS. Thus, the objective of the current study was to demonstrate the location and quantity of COL V and ECM-1 on the vulvas of patients with LS. Materials and methods: Twenty one biopsies from patients with vulvar LS matched with 21 biopsies from normal vulvas were included in this study. Immunofluorescence against type I (COL I), (COL III) and V collagen and immunohistochemistry for ECM-1 was performed and the slides were analysed by morphometry. Furthermore, laser confocal microscopy was used to visualize the COL V fibers and the ECM-1 protein on the same slide. Results: Skins of the control group showed low and homogeneous distribution of fibers COL I, III and V in immunofluorescence. In contrast, skins with LS exhibited disruption of the architecture of the superficial dermis and diffuse increase in fluorescence fibers COL I, III and V in the range hyaline. The area fraction of fibers COL I, III, and V were significantly higher in the skin of patients with LS compared to control (p <0.05). Skins controls showed colocalization of ECM-1 and COL V the wall of small vessels and along the basement membrane. However, the LS had loss of expression of ECM-1 the wall of blood vessels (p <0.001) increase the fluorescence and diffuse green COL V (p <0.001). Conclusion: There was an inverse relationship between COL V and ECM-1 protein in LS as demonstrated by histomorphometry, immunofluorescence, immunohistochemistry and three-dimensional reconstruction, suggesting that therapeutic strategies to prevent the increased synthesis COL V and ECM-1 protein in LS as demonstrated by histomorphometry, immunofluorescence, immunohistochemistry and three-dimensional reconstruction, suggesting that therapeutic strategies to prevent the increased synthesis COL V and decreased ECM-1 may be promising in the prognosis and treatment of the LS

O microrganismo gram-negativo Aggregatibacter actinomycetemcomitans, fortemente associado a quadros de periodontite agressiva, coloniza a cavidade oral, aderindo e invadindo as células epiteliais e participando da formação de biofilme nas superfícies do hospedeiro. A. actinomycetemcomitans expressa Aae, uma proteína de superfície, relacionada com a adesão às células epiteliais. Este estudo avaliou o papel de Aae na adesão a outros substratos tais como proteínas extracelulares colagenosas e não-colagenosas, hidroxiapatita recoberta por saliva (SHA) e na formação de biofilme. Um mutante nulo em aae foi construído e o fenótipo comparado com o da linhagem selvagem (VT1169). O mutante nulo exibiu significativa redução na adesão às células epiteliais e a SHA, na formação de biofilme, além de apresentar-se menos hidrofóbico que a linhagem parental. A capacidade de adesão ao colágeno V e a fibronectina foi fracamente afetada pela interrupção do gene aae. Entretanto, o mutante nulo em aae exibiu uma capacidade diminuída na adesão à laminina e colágenos I, III e IV. Estes dados sugerem que Aae desempenha um importante papel na colonização da cavidade oral, não somente promovendo sua adesão às células epiteliais, mas também a outros substratos. / The gram-negative organism Aggregatibacter actinomycetemcomitans, associated with aggressive periodontitis, colonizes the oral cavity by binding to and invading epithelial cells and by participating in biofilms formed in host surfaces. A. actinomycetemcomitans express Aae, a surface protein, implicated in the adhesion to epithelial cells. This study evaluated the role of Aae in adhesion to other substrates such as collagen and non-collagen extracellular matrix proteins and saliva coated hydroxyapatite (SHA) and in biofilm formation. A null mutant in aae was constructed and its behavior was compared with the wild type strain VT1169. The null mutant exhibited a decreased ability to bind to epithelial cells and to SHA, formed less biofilm and was less hydrophobic than the parental strain. The abilities to bind to collagen V and fibronectin were very poorly affected by aae interruption. However, the null aae mutant exhibited a decreased ability to adhere to laminin, collagen I, III and IV. These data suggest that Aae may play an important role in the colonization of the oral cavity by A. actinomycetemcomitans, not only by promoting its adhesion to epithelial cells, but by mediating adhesion to other substrates.