Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 101-119).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 131-157).

Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.

No description available.

No description available.

Metastatic cancers are problematic because they spread throughout the body. A crucial step in cancer metastasis is the separation of the cancer cells from their surrounding normal cells. This occurs due to suppression or destruction of cell adhesion molecules such as E-cadherin, occludin, and various claudins. The Snail and Slug transcription factors play a direct role in suppressing these cell adhesion molecules through their SNAG repression domain. We explored the possibility of developing an ELISA diagnostics capable of detecting soluble E-cadherin, occludin, and claudin fragments in the serum of cancer patients. Using several bioinformatics tools, unique extracellular antigenic sequences were identified on claudins-1, 4, 16, occludin, and E-cadherin. These sequences were cloned as GST fusion proteins, expressed, and purified in large quantities to raise antibodies. In parallel, expression profiling of metastatic cancer cell lines was carried out to derive a correlation between Snail-Slug expression and suppression of cell adhesion molecules. / by Michael Irvine. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

O presente estudo teve como objetivo caracterizar a expressão da família SIBLING (Small Integrin-Binding Ligand, N-linked Glycoproteins) após tratamento regenerativo de furca com enxerto de tecido reparativo de alvéolos dentários. Para isto, os 2os e 3os pré-molares superiores foram extraídos em quatro cães s.r.d. Cinco dias após as extrações, defeitos padronizados de furca classe II foram criados nos 2os, 3os e 4os pré-molares inferiores, bilateralmente. Estes defeitos foram tratados imediatamente com raspagem, alisamento e polimento corono-radicular (RAPCR) e retalho deslocado coronariamente (RDC) (Grupo Controle) ou com RAPCR + RDC + enxerto de tecido reparativo de alvéolos dentários (Grupo Teste) em um experimento de boca-dividida. Após um período de 6 semanas de reparação, os animais foram sacrificados e foi realizada análise imuno-histoquímica para avaliar a localização dos membros da família de proteínas SIBLING, composta pelas seguintes proteínas nãocolágenas da matriz extracelular: osteopontina (OPN), sialoproteína óssea (BSP), proteína da matriz dentinária 1 (DMP1), sialofosfoproteína da dentina (DSPP) e fosfoglicoproteína da matriz extracelular (MEPE). Não foram encontradas diferenças na expressão da família SIBLING entre os grupos teste e controle. Todas as proteínas foram expressas no novo osso, novo cemento e novo ligamento periodontal, em ambos os grupos. Os osteoclastos demonstraram imunolocalização intracelular intensa somente para a OPN. Cementócitos e o novo ligamento periodontal demonstraram, particularmente, marcação intensa para a MEPE. Houve uma diferença evidente entre o padrão de marcação entre o lado tratado (vestibular) e o não-tratado (lingual) de todos os espécimes, com presença de maior marcação do lado vestibular, para todos os anticorpos testados. Podemos concluir que não houve diferenças no padrão de expressão da família SIBLING após o uso do enxerto de tecido reparativo de alvéolos dentários. A família de proteínas SIBLING é expressa durante o processo de reparação de defeitos de furca, indicando possíveis papéis e funções para as proteínas OPN, BSP, DMP1, DSP e MEPE como moléculas alvo em terapias de regeneração periodontal. / The present study aimed in characterizing the expression of the SIBLING (Small Integrin- Binding Ligand, N-linked Glycoproteins) family in a regenerative treatment of furcation defects with a reparative tissue graft obtained from extraction sockets. The second and third upper premolars were extracted in four mixed breed dogs. Five days later, standardized class II furcation defects were created in the second, third and fourth mandibular premolars, bilaterally. The defects were immediately treated with either debridement and root planning (DRP) combined with a coronally positioned flap (CPF) (Control Group), or with DRP+CPF + a reparative tissue graft derived from the second and third premolar extraction sockets (Experimental Group) in a split-mouth design. After 6 weeks period of healing, the animals were sacrificed and immunohistochemistry was carried out to assess the localization of members of the SIBLING family of noncollagenous extracellular matrix proteins, namely osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). No differences in the SIBLING family of proteins expression were noted between the control and experimental group. All proteins were expressed in new bone, new cementum and new periodontal ligament in both groups. Osteoclasts exhibited intense intracellular localization only for OPN. Cementocytes and the newly formed periodontal ligament demonstrated particularly intense staining for MEPE. There was an evident difference between the staining pattern between the treated (buccal side) and non-treated (lingual) side of the specimens, with a more intense staining pattern in the buccal side, for all the tested antibodies. In conclusion, there were no differences in the pattern of SIBLING expression following the use of a reparative tissue graft obtained from extraction sockets. The SIBLING family of proteins is expressed during the healing process of furcation defects indicating possible roles and functions of OPN, BSP, DMP1, DSPP and MEPE as target molecules in periodontal regeneration therapies.

Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP. / Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3. / Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina. / by Asmaa Bilal Hamze. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

A aplicação de ozônio pode ser usada como uma alternativa no tratamento de inúmeras patologias. O objetivo é interferir positivamente na reparação tecidual e na anti-sepsia de injúrias, uma vez que o ozônio é um potente agente antimicrobiano e possui a capacidade de estimular a circulação sanguínea e a resposta imunomodulatória. Tais características justificam o interesse da sua aplicação na Medicina e na Odontologia. Quando em contato com fluidos orgânicos, o ozônio age como um oxidante gerando a formação de moléculas reativas do oxigênio e produtos de lipídeos oxidantes que influenciam em eventos bioquímicos do metabolismo celular. A proposição deste estudo foi avaliar a aplicação do ozônio diluído em água no processo de reparação óssea através de análise morfológica e imunoistoquimica em modelo animal. Nossos resultados mostraram que a irrigação da ferida cirúrgica com ozônio diluído em água ocasionou atraso na reparação e que este era mais exuberante no início do processo. / The ozone therapy can be used as an alternative for the treatment of numerous diseases. The goal is to positively affect tissue repair and antisepsis as ozone is a potent antimicrobial agent and has the ability to stimulate blood circulation and immunomodulatory response. These characteristics justify the interest of its application in Medicine and Dentistry. When in contact with body fluids, ozone acts as an oxidant generating reactive oxygen molecules and oxidative products of lipids influencing biochemical events of cellular metabolism. Therefore, the purpose of this study was to evaluate, in an animal model, the effect of ozone application during the bone wound healing process. These analyses were performed by the study of bone cells morphology and immunohistochemical expression of bone matrix proteins. Our results showed that the use of ozone diluted in water for irrigation caused a delay in early stages of bone repair. This delay was more prominent in the first phase of the bone wound healing.