Made available in DSpace on 2014-10-09T12:32:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:10Z (GMT). No. of bitstreams: 1 17893.pdf: 2260443 bytes, checksum: b4dd83093861c3fa62cf99384a958491 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de São Paulo, São Paulo

Made available in DSpace on 2014-10-09T12:32:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:10Z (GMT). No. of bitstreams: 1 17893.pdf: 2260443 bytes, checksum: b4dd83093861c3fa62cf99384a958491 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de São Paulo, São Paulo

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Indiana University-Purdue University Indianapolis (IUPUI) / The proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry.

Osteocytes are widely regarded as mechanosensors, capable of detecting changes in the mechanical environment of the bone tissue and modifying cellular responses accordingly. Indeed, an intact osteocyte network is required for bone changes in response to unloading, and studies have shown that loading/unloading influences osteocyte expression of proteins that modulate bone turnover, such as sclerostin and receptor activator of nuclear factor kappa B ligand (RANKL). However, mechanisms underlying osteocyte mechanotransduction remain unclear. For instance, one of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular, or cytosolic, calcium (Ca2+cyt), but the mechanisms by which osteocytes generate or utilize Ca2+ signals to direct bone adaptation are largely unknown. In this thesis, I explored the mechanisms underlying the sustainment of Ca2+cyt oscillations in osteocytes as well as downstream consequences of these patterns. I discovered that Ca2+cyt oscillations are generated in osteocytes by Ca2+ release from the endoplasmic reticulum and that the predominant expression of T-Type voltage sensitive Ca2+ channels in these cells facilitates this behavior. I also explored the role of the actin cytoskeleton – another prominent feature in osteocytes – and found that actin dynamics are important for the generation of Ca2+cyt signals. Furthermore, I confirmed that Ca2+cyt transients subsequently activate actomyosin contractions in osteocytes by monitoring interactions of osteocytes exposed to Ca2+ agonists on micropillar substrates. With this information, I sought to relate Ca2+cyt signaling and actomyosin contractility in osteocytes to their roles as coordinators of bone adaptation. Ca2+-dependent contractions have been shown to facilitate the release of extracellular vesicles, small membrane-enclosed packages of proteins that cells use for communication, in other cell types. I found that mechanical stimulation increased the production and release of extracellular vesicles in osteocytes, and this was dependent on Ca2+ signaling. These extracellular vesicles contained key bone regulatory proteins and were small enough to plausibly transport through the lacunocanalicular system. Thus, I uncovered a novel mechanotransduction pathway by which osteocytes may coordinate tissue-level adaptation. As an extension of this work, I also characterized these behaviors in new osteocyte cell lines which may better reflect native cell physiology. The work in this thesis anchors Ca2+ signaling as a critical osteocyte response to mechanical loading and adds to the body of work exploring how and why these signals are generated. The results of these studies add new information to the still limited knowledge of this important bone cell and extend Ca2+ signaling research by connecting early mechanosensation events to subsequent protein responses to mechanical loading. Understanding the mechanisms behind the robust Ca2+cyt oscillations in osteocytes and how they relate to their roles as coordinators of bone adaptation may improve our ability to prevent or treat bone degeneration in diseases like osteoporosis where mechanosensitivity is impaired.

Orientador: Willian Fernando Zambuzzi / Resumo: Com o aumento progressivo da expectativa de vida da população, defeitos ósseos se tornou um problema de saúde pública. Por sua vez, o sistema esquelético abriga um conjunto de células que, de maneira hierárquica, sustentam a formação do osso ao longo da vida, sendo sua capacidade regenerativa comprometida durante o processo de envelhecimento. Com o aumento da expectativa de vida, a população idosa vem crescendo nos últimos anos e com ela o aumento eminente de fraturas ósseas. Sabe-se que o desenvolvimento e regeneração ósseos são eventos complexos e controlados por mecanismos parácrinos de sinalização intercelulares, destacando que a osteogênese está acoplada, principalmente, à angiogênese. Embora relatado, este mecanismo de comunicação entre células endoteliais e células osteoprogenitoras não está bem elucidado, sobretudo considerando o repertório de moléculas tróficas envolvidas. A fim de compreender melhor estes mecanismos, o objetivo deste trabalho foi desenvolver metodologias capazes de mimetizar o microambiente endotelial-ósseo, bem como desvendar eventos acoplados entre os diferentes tipos celulares envolvidos no processo, sobretudo gerando tecido endotelial in vitro, equivalente ao original. Para isso, fizemos uso de células humanas primárias, as quais foram submetidas a diferentes protocolos experimentais, onde a mesma densidade de células endoteliais arterial (HCAEC) e venosa (HUVEC) e de musculatura lisa (AoSMC) (cultivo misto) foi plaqueada em tubos cônicos sem tr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the progressive increase in the life expectancy of the population, bone defects became a public health problem. In turn, the skeletal system houses a set of cells that, in a hierarchical way, support the formation of the bone throughout life, and its regenerative capacity is compromised during the aging process. With the increase in life expectancy, the elderly population has been increasing in recent years and with it the eminent increase of bone fractures. It is known that bone development and regeneration are complex events and controlled by paracrine mechanisms of intercellular signaling, emphasizing that osteogenesis is mainly coupled with angiogenesis. Although reported, this mechanism of crosstalk between endothelial cells and osteoprogenitor cells is not well elucidated, especially considering the repertoire of involved trophic molecules. In order to better understand these mechanisms, the objective of this work was to develop methodologies capable of mimicking the endothelial-bony microenvironment, as well as unveiling the coupled events between the different cell types involved in the process, especially generating endothelial tissue in vitro, equivalent to the original one. For this, we used primary human cells, which were submitted to different experimental protocols, where the same density of arterial (HCAEC) and venous (HUVEC) endothelial cells and smooth muscle cell (AoSMC) (mixed culture) were plated in tubes (s), and after 72 hours the generated spheroid... (Complete abstract click electronic access below) / Mestre

Bibliography: leaves 86-103. Investigates the effect of zoledronic acid on myeloma cells and osteoblast-like cells to establish the molecular and cellular mechanisms responsible for the clinical effectiveness of bisphosphonates in the treatment of patients with myelomatosis. Concludes that zoledronic acid inhibits myelomatosis-induced osteolysis thorugh the mechanisms of myeloma cell death and proliferation and maturation of osteoblasts.

Addenda slip inserted in back. Includes bibliographical references (leaves 178-231). To further the understanding of the molecular mechanisms that contribute to the skeletal pathology of mucopolysaccharidosis type VI and to investigate the production of organic matrix by mucopolysaccharidosis VI osteoblasts

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

Addenda slip inserted in back. / Includes bibliographical references (leaves 178-231). / xiv, 234, [19] leaves, [56] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / To further the understanding of the molecular mechanisms that contribute to the skeletal pathology of mucopolysaccharidosis type VI and to investigate the production of organic matrix by mucopolysaccharidosis VI osteoblasts / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2001