Females are known to be more susceptible to alcoholic liver disease (ALD), but the precise mechanism behind this increased susceptibility is not well understood. The objective of this study was to identify the molecular mechanism behind the increased susceptibility of females to alcoholic steatohepatitis (ASH). Female rats in ASH model were found to have significantly higher neutrophilic infiltration in the liver as compared to the males. Osteopontin (OPN), a member of the SIBLING family of proteins was also found to be induced in females. Neutralizing OPN antibody experiments provided further evidence that OPN acts as a chemokine in attracting neutrophils into the liver making females more susceptible to ASH. Since neutrophil transmigration in the liver is mediated by intergins, the mechanism for OPN-mediated neutrophil infiltration was tested. Females in ASH had significantly higher expression of α4β1 and α9β1 integrins, and animals treated with neutralizing OPN antibody had significantly lower expression of these integrins, wherein hepatic neutrophilic infiltration was also decreased by 50% compared to the untreated group. Immunoprecipitation experiments suggested the binding of OPN to α4β1 and α9β1 integrins. OPN-mediated neutrophilic infiltration was further confirmed using Boyden chamber assays. Antibodies directed against α4, β1 integrins and SLAYGLR sequence was also found to significantly inhibit neutrophilic migration in vitro, suggesting that higher hepatic neutrophil chemokinesis in the female ASH appears to be mediated through both α4β1 and α9β1 integrin signaling. In addition, higher liver injury and higher expression of OPN in females were also found to be regulated by estrogen in a biphasic pattern; ovariectomy resulted in significantly increased liver injury compared to intact rats. Depending on dose, estradiol supplementation in the ovariectomized rats fed ethanol resulted in both a protective and damaging effect on liver. Besides OPN, several other oxidative stress related proteins like Ferritin H Chain, ER60, HSP60, Peroxiredoxin 6 were identified by proteomics approach. Females in ASH were found to have differentially regulated levels of these proteins as compared to their male counterparts, highlighting the potential novel mechanisms associated with higher liver injury noted in our female rat ASH model.

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Thesis (M. Sc.)--University of Toronto, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M. Sc.)--University of Toronto, 1996. / Includes bibliographical references.

No description available.

Thesis (Ph.D.) in Biological Sciences--University of Maine, 2004. / Includes vita. Includes bibliographical references (leaves 129-166).

No description available.

Orientador: Paulo Tambasco de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T15:44:04Z (GMT). No. of bitstreams: 1 Teixeira_LucasNovaes_D.pdf: 3520593 bytes, checksum: e42a5497740bc09fe80b9a6a035947df (MD5) Previous issue date: 2015 / Resumo: O carcinoma espinocelular (CEC) oral representa a neoplasia maligna mais prevalente das estruturas bucais, podendo invadir o tecido ósseo e promover sua reabsorção em até 56% dos casos. A expressão da proteína matricelular osteopontina (OPN) tem sido relacionada a uma maior agressividade de neoplasias malignas, incluindo o CEC oral. No tecido ósseo, a OPN representa a proteína mais abundante da matriz não colágena, concentrada nas interfaces ósseas, i.e. lâminas limitantes, linhas de cimentação e reversas, sendo essencial para adesão e funções de osteoblastos e osteoclastos. Apesar de no microambiente tumoral a OPN estar associada a um fenótipo neoplásico mais agressivo, ainda não está estabelecido o papel da OPN secretada por osteoblastos sobre células neoplásicas derivadas de CEC oral e o impacto sobre osteoclastos. O presente estudo teve como objetivos avaliar a expressão de OPN em sistemas de coculturas de células osteoblásticas e epiteliais neoplásicas malignas humanas e os efeitos da expressão de OPN secretada por osteoblastos sobre o fenótipo neoplásico in vitro. Adicionalmente, avaliou-se o efeito das coculturas sobre a atividade osteoclástica. Células epiteliais neoplásicas malignas derivadas de CEC oral (SCC9) foram plaqueadas sobre membranas de Transwell®, recobertas ou não por uma camada fina e uniforme de Matrigel, e cocultivadas com células osteoblásticas (SAOS-2) durante seu pico de expressão de OPN (10o dia de cultura). Células SCC9 expostas a culturas SAOS-2 silenciadas para OPN por RNA de interferência (RNAi) e células SCC9 cultivadas isoladamente foram usadas como controles. Após 24 h de cocultivo, células SCC9 foram avaliadas, quantitativamente, para adesão, proliferação, migração e invasão de Matrigel. A atividade de osteoclastos derivados de células monocíticas U-937 foi avaliada, quantitativamente, por meio dos ensaios de reabsorção de fosfato cálcio e de dosagem de citocinas em eluentes obtidos de células SCC9 e SAOS-2 após o cocultivo durante o pico de OPN ou com o seu silenciamento. A análise estatística foi realizada pelo teste não-paramétrico de Kruskal-Wallis (p < 0,05). Os resultados indicaram indução recíproca na expressão de OPN em SAOS-2 e SCC9 em cocultura. A OPN secretada por células SAOS-2 afetou o fenótipo de culturas SCC9, promovendo a adesão e a proliferação celulares e a invasão de Matrigel, a qual também estava aumentada, mas em menor intensidade, com o silenciamento para OPN. A migração celular não foi afetada. O cocultivo com SAOS-2, principalmente durante o pico de OPN, resultou na sobre-expressão das citocinas IL 6 e IL 8 pelas células SCC9, aumentando a capacidade de células osteoclásticas em reabsorver fosfato de cálcio. Conjuntamente, esses resultados sugerem que a OPN derivada de osteoblastos afeta as interações entre células epiteliais neoplásicas malignas, osteoblastos e osteoclastos, possivelmente contribuindo para a progressão de lesões ósseas do CEC oral / Abstract: The oral squamous cell carcinoma (OSCC) is the most prevalent malignant neoplasm of the oral structures. It may invade bone in up to 56% of the cases and promote osteoclast-mediated bone extracellular matrix (ECM) resorption. Expression of the matricellular protein osteopontin (OPN) in malignant neoplasms, including OSCC, has been positively correlated with aggressive tumor behavior. OPN is the most abundant non collagenous ECM protein in bone, where it preferentially accumulates at interfaces, including cement lines, laminae limitantes and reversal lines, being essential for the adhesion and function of osteoblasts and osteoclasts. Despite the importance attributed to OPN in the tumor microenvironment, indicative of more aggressive neoplastic phenotypes, the effects of osteoblast-derived OPN on OSCC cells and on OSCC-induced osteoclast activity are still not fully understood. The present in vitro study aimed to evaluate temporal expression of OPN in cocultures of human osteoblastic cells and malignant neoplastic epithelial cells and the effects of osteoblast-derived OPN on the neoplastic cell phenotype. Additionally, the effects of cocultures on osteoclastic activity were evaluated. Human OSCC-derived epithelial cells (SCC9 cell line) were plated on Transwell® membranes coated or not by a thin uniform layer of Matrigel and cocultured with human osteoblastic cells (SAOS-2 cell line) during its peak of OPN expression (day 10 of SAOS-2 culture). SCC9 cells exposed to OPN-silenced SAOS-2 cultures by means of interference RNA and SCC9 cells cultured alone were used as controls. At 24 h of coculture, SCC9 cells were quantitatively evaluated for cell adhesion, proliferation, migration and invasion of Matrigel. The impact of coculturing SCC9 and SAOS-2 cells either during the OPN peak expression or under the silencing of OPN was quantitatively evaluated in terms of calcium phosphate resorption by U-937-derived osteoclastic cells and expression of cytokines in the culture medium by ELISA assay. The statistical analyses were carried out using the non-parametric Kruskal-Wallis test (p < 0.05). The results showed a reciprocal induction of SAOS-2 and SCC9 cells in terms of OPN expression over the coculture interval. SAOS-2-secreted OPN altered the SCC9 cell phenotype, leading to enhanced cell adhesion and proliferation and higher Matrigel invasion, which was also enhanced, but to a lesser degree, by SAOS-2 cultures silenced for OPN. Cell migration was not affected. Cocultures with SAOS-2, mainly during the peak expression of OPN, resulted in overexpression of IL 6 and IL 8 by SCC9 cells, which corresponded with an enhanced resorptive capacity of osteoclastic cells. Taken together, the results suggest that osteoblast-derived OPN affects the interactions among malignant neoplastic epithelial cells, osteoblasts and osteoclasts, likely contributing to the progression of bone lesions in OSCC / Doutorado / Patologia / Doutor em Estomatopatologia

PEA3, ERM, and ER81 comprise a subfamily of ETS transcription factors that upregulate genes correlated with an increased metastastic potential of tumors. In mouse embryo fibroblast (MEF) cells, PEA3 is required for transformation by activated Ras or Neu, but the means by which PEA3 mediates Ras-transformation is not clear. Osteopontin (OPN) expression is induced upon B-ras-transformation and purified PEA3 can bind the OPN promoter by gel-shift analysis. In this study, OPN expressed higher transcript levels in the wildtype MEF 4 cell line than the PEA3 null MEF 1 cell line and was further characterized as a potential PEA3 target gene by Northern blot analyses and transient transfection studies. Northern blot analyses of 4 wildtype MEF (4, 100, 101, 104), 5 FEA3 null MEF (1, 115, B5, B10, B12), and 5 MEF 1 retransformant cell lines that stably reexpress PEA3 showed a good correlation between OPN and ERM transcript levels in 9/11 cell lines although at least 2 PEA3 subfamily members were coexpressed in 8/11 cell lines that expressed high OPN transcript levels. This suggested that the PEA3 subfamily additively regulated OPN and that ERM protein was more abundant than PEA3 and ER81 protein levels in the MEF cell lines. The relative PEA3 subfamily protein levels remain to be clarified. Transient transfection assays in the HEK 293-1 C cell line indicated that the OPN promoter was responsive to PEA3 and that the promoter region between -258 to -88 was required for maximal OPN promoter activity. There are 16 candidate core ETS binding sites in the -777 /+79 OPN promoter which could be responsible for PEA3 subfamily transactivation. The OPN promoter was more active in the MEF 4 cell line than the MEF 1 cell line, corresponding to their relative number of expressed PEA3 subfamily members. Ectopic expression of dominant negative PEA3 suppress ed OPN promoter activity in the MEF 4 cell line. Furthermore, ectopic expression of PEA3, ERM, or ER81 increased OPN promoter activity in the MEF 1 or COS-1 cell line. Thus OPN is transcriptionally regulated by the PEA3 subfamily and represents a target gene that can mediate the progression of tumor cells. / Thesis / Master of Science (MSc)

Die Arbeit befasst sich mit dem Anschwellen von Neuronen und Gliazellen der Netzhaut, was einen wichtigen pathogenetischen Faktor des Netzhautödems darstellt. Osteopontin ist ein neuroprotektiver Faktor, der durch GDNF-Stimulation (glial cell line-derived neurotrophic factor) aus Müllerzellen ausgeschüttet wird. Die durch Osteopontin vermittelte Inhibition der osmotischen Zellschwellung von Müllerzellen der Ratte in Anwesenheit von Bariumionen oder H2O2 wird beschrieben und es wird dargestellt, dass Osteopontin keinen Einfluss auf die osmotische Zellschwellung der Bipolarzellen hat. Der für Müllerzellen beschriebene Effekt war dosisabhängig mit einer mittleren effektiven Konzentration von ca. 0,6 ng/ml. Durch den Einsatz pharmakologischer Rezeptor- oder Enzymblocker bzw. Antikörper werden die Schritte der Osteopontinwirkung identifiziert. Osteopontin induziert die Freisetzung von VEGF, Glutamat, ATP und Adenosin aus Müllerzellen. Die Osteopontinwirkung wurde verhindert durch die Blockade von spannungsabhängigen Natriumkanälen, T-Typ Calciumkanälen, Kalium- und Chloridkanälen. Der Effekt ist außerdem abhängig von einem intrazellulären Calciumsignal, der Aktivierung der Phospholipase C und der Proteinkinase C und der vesikulären Exozytose von Glutamat. Die Arbeit kommt zu dem Schluss, dass der neuroprotektive Effekt von Osteopontin teilweise durch das Verhindern eines Anschwellens der Müllerzellen und durch die Induktion einer Freisetzung von VEGF und Adenosin vermittelt wird.