Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

This thesis deals with the determination of biological activity of adiponectin, a recombinant protein using cell culture. First it was important to acquire the working skills for the cell culture of cell line 3T3-L1. An optimal concentration of inactivated fetal bovine serum in cell culture media was determined. A stimulation of the cell proliferation by HB-EGF, PDGF-BB and bFGF growth factors was observed at various concentration levels. Afterwards the biological activity of adiponectin was determined as an inhibition of growth stimulation with 5 ng/ml PDGF-BB. This biological activity assay for adiponectin was also conducted with lyophilized adiponectin and a growth factor bFGF (0.1 ng/ml). The lyophilization did not affect the biological activity of adiponectin.

Obesity and the metabolic complications associated with it are increasing globally. Sedentary lifestyles, poor diet and genetic predisposition contribute to obesity. In addition, environmental chemicals such as Bisphenol A (BPA) may play a significant role. Exposure to BPA has been correlated with an array of adverse health effects on the endocrine system and whole-body homeostasis. This has resulted in manufacturers replacing it with structural analogues such as Tetra Methyl Bisphenol F (TMBPF), Bisphenol F (BPF), Bisphenol AP (BPAP), and fluorine-9-bisphenol (BHPF). BPA is a suspected obesogen as it can induce adipogenesis in human and murine preadipocytes. The effects of the BPA analogues listed above on adipogenesis have yet to be evaluated. The aim of this project is to investigate their adipogenic effects. For this purpose, we used 3T3-L1 mouse embryonic fibroblasts. This cell model can be differentiated into mature adipocytes with appropriate inducers including 3-isobutyl-1-methylxanthine (IBMX), insulin and dexamethasone, a synthetic steroid. To assess the effects of BPA analogues, the cells were treated with varying concentrations of TMBPF, BPF, BHPF, BPA, or BPAP in place of dexamethasone. The expression levels of mature adipocyte markers were assessed at mRNA and protein levels to determine the adipogenic potential of the analogues. Lipid accumulation was evaluated by Nile Red staining. A time course was performed to assess the expression levels of known transcriptional regulators of adipogenesis. The results indicate that TMBPF, BPF and BPA increase 3T3-L1 adipogenesis. BHPF and BPAP did not affect adipogenesis in this model. BPF appears to be at least as good as BPA at inducing adipogenesis. TMBPF, on the other hand, can induce adipogenesis to a greater extent than the other chemicals, including BPA, as evidenced by increased expression of adipogenic markers and lipid accumulation. Finally, key transcription factors C/ebpδ and C/ebpα, part of the adipogenic transcriptional cascade, were up-regulated at III two and six hours post-treatment by TMBPF. BPA also up-regulated C/ebpδ at two hours post-treatment. Though the adipogenic effects have become apparent for some of these analogues, the mechanism by which they elicit their effects remains to be discovered. More research is required to deduce the mechanism of action and to provide consensus on what the effects of these replacement bisphenols actually are.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Os hormônios tireoidianos atuam sobre o metabolismo dos diversos tecidos do organismo e participam da regulação do consumo de glicose pelas células. Estudos já evidenciaram que o T3 atua, dependendo do tipo celular, aumentando a expressão de algumas isoformas dos transportadores de glicose (GLUTs) e a translocação do GLUT4 para a membrana plasmática, melhorando, também, a captação de glicose (CGlic) em poucos minutos. Os mecanismos envolvidos nessas ações do T3, contudo, ainda não estão bem esclarecidos. O objetivo do presente estudo foi investigar as possíveis vias de sinalização envolvidas na ação aguda do T3 sobre a CGlic em células adiposas 3T3-L1. Nossos dados demonstraram que o T3 promove aumento na CGlic, com pico aos 10 min, retornando ao nível do controle após 30 min de incubação das células com o hormônio. Sugerimos que essa ação depende, ao menos, de duas vias de sinalização. Uma delas envolve a ativação das proteínas Src, PI3K e Akt. A outra, aparentemente, é iniciada a partir da membrana plasmática via integrina aVb3 / Thyroid hormones act on the metabolism of many tissues and participate in the regulation of glucose consumption by cells. Studies from this and other laboratories have demonstrated in muscle and adipose cells that T3 increases, in a short period (minutes), the expression of some glucose transporter (GLUTs) isoforms and GLUT4 translocation to the plasma membrane leading to an improvement of glucose uptake. However, the mechanisms involved in these T3 actions are still not clear. The aim of this study was to investigate the possible signaling pathways involved in the acute T3 action on glucose uptake in 3T3-L1 adipocytes. Our results have shown that T3 increases glucose uptake with a peak at 10 min returning to the control level after 30 min of the cell incubation with the hormone. We suggest that this action depends on at least two parallel signaling pathways. One involves the activation of Src, PI3K and Akt proteins, while the other involves another mechanism triggered by T3, apparently, from the plasma membrane at aVb3 integrin.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-07-03T18:22:09Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Daniele Kosmo.pdf: 1666129 bytes, checksum: 21d35993408cf04cf3327fefb3c5a4a2 (MD5) / Made available in DSpace on 2018-07-03T18:22:09Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Daniele Kosmo.pdf: 1666129 bytes, checksum: 21d35993408cf04cf3327fefb3c5a4a2 (MD5) Previous issue date: 2017-07-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer de pulmão está entre os tipos mais comuns dentre todos os tumores malignos. Sendo a quimioterapia o principal tratamento preconizado, todavia além dos seus diferentes efeitos colaterais, verifica-se a resistência a múltiplos fármacos, referida como a perda da sensibilidade tumoral aos agentes quimioterápicos. Óleos essenciais (OE) podem contornar esta refratariedade dos tumores aos fármacos e se apresentam também como alternativa antitumoral, em especial pela possibilidade de serem administrados por via inalatória. O objetivo deste trabalho foi o de avaliar a citotoxicidade da fase vapor do OE de Jasminum officinale em células Calu-3 (adenocarcinoma de câncer de pulmão humano) e células 3T3 (linhagem padrão de fibroblasto murino) bem como investigar as alterações morfológicas e mecanismo de ação deste OE em Calu-3. Foram realizados ensaios de avaliação metabólica com redução de MTT e ensaio de marcação de proteína com SRB, onde verificou-se a redução da viabilidade celular de maneira dose-dependente. A IC50 estimada para células Calu-3 foi de 1495,57 μg/mL e para as células 3T3 foi de 3795,05 μg/mL. Para investigação das alterações morfológicas associados à citotoxicidade, foram avaliados (1) a morfologia celular com o emprego dos corantes alaranjado de acridina, brometo de etídio; (2) distribuição de células entre as fases do ciclo celular com emprego de iodeto de propídio (PI) por citometria de fluxo; e foi avaliado o potencial inibitório da fase vapor do OE de Jasminum officinale na atividade da glicoproteína P empregando o ensaio de acúmulo de Rodamina 123. Os resultados obtidos indicaram que a morte das células foi causada pelo processo de apoptose tardia, inicial e necrose, com pequena redução da porcentagem de células nas fases sub-G0 e G2/M do ciclo celular. A fase vapor do OE de Jasminum officinale apresenta efeito inibitório sobre a atividade da glicoproteína P em sinergia com a curcumina.Tais resultados sugerem a possibilidade da utilização do OE de Jasminum officinale, em sua fase vapor no tratamento do câncer de pulmão por via inalatória, porém ainda são necessários mais estudos para aprofundar o conhecimento sobre óleos essenciais, frente a células normais e tumorais de pulmão, incluindo os mecanismos envolvidos. / Lung cancer is among the most common types among all malignancies, with an increase of 2% per year in its incidence. Chemotherapy is the main treatment recommended, but in addition cause collateral effects and also resistance to multiple drugs can occur, that is, the loss of tumor sensitivity to chemotherapeutic agents. Essential oils (OE) may be an option for this refractoriness of tumors to drugs and also present as an antitumor alternative, especially for the possibility of being administered by inhalation. The objective of this work was to evaluate the cytotoxicity of the vapor phase of jasmine OE in Calu-3 cells (human lung cancer adenocarcinoma) and 3T3 cells (murine fibroblast control line model). The vapor phase of jasmine OE reduces cell viability in a dose-dependent manner, as demonstrated by the MTT and SRB reduction assays. The estimated IC 50 for Calu-3 cells was 1495.57 μg / mL and for 3T3 cells was 3795.05 μg / mL. The possible effects associated with cytotoxicity, (1) cellular morphology, (2) cell cycle phase distribution and (3) P-glycoprotein activity were evaluated. Results indicated that cell death was caused by the late apoptosis and necrosis, reduction in the percentage of cells in the sub-G0 and G2/M cell cycle phases. The vapor phase of the jasmine oil presented an inhibitory effect on the activity of the glycoprotein P in synergy with curcumin. These results suggest the possibility of the use of the OE of jasmine, in its vapor phase in the treatment lung cancer, by inhalation. Further studies are needed to knowledge about essential oils, especially the so-called alternative, against normal and tumor cells of the lung, included the mechanism involved.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Two Systems were designed. The first permits tunable on-demand softening of a hydrogel network. The second permits reversible on demand ligand exchange within a hydrogel network. Both means were shown to be cytocompatible and their uses demonstrated in cell culture of mesenchymal stem cells and 3T3 fibroblast cells.