Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-09-28T12:04:15Z No. of bitstreams: 1 475275 - Texto Completo.pdf: 1472800 bytes, checksum: fe0676cb911e95563a60b4837d542763 (MD5) / Made available in DSpace on 2015-09-28T12:04:15Z (GMT). No. of bitstreams: 1 475275 - Texto Completo.pdf: 1472800 bytes, checksum: fe0676cb911e95563a60b4837d542763 (MD5) Previous issue date: 2015-08-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Low-intensity pulsed ultrasound (LIPUS) has been proposed as a high potential therapeutic technique for the treatment of metabolic bone diseases and fractures with delayed healing. However, recent investigations have shown controversial results, suggesting the need for more studies on the understanding of biological responses and the standardization of methods and parameters. For this reason, the present study aimed to evaluate the effect of ultrasound on the proliferation and mineralization of osteoblasts using in vitro bioassays. Pre-osteoblastic MC3T3-E1 cells were used and treated with therapeutic pulsed ultrasound, 20%, frequency of 1MHz and 0,2W/cm2 of intensity. In the study of cellular proliferation, intracellular calcium, TGF-?1, magnesium and osteopontin and osteocalcin mRNA levels, NF-?B1 and p38? were evaluated. In addition, nifedipine and rapamycin were used to investigate the proliferation pathways. Initially, the results showed an increase in proliferation of MC3T3-E1 and a decrease in calcium and magnesium content in supernatant with LIPUS exposure. In addition, LIPUS increased calcium deposition, activated NF-?B1 and mTOR complex via p38? and promoted a decrease in TGF-?1 synthesis, which is an inhibitor of cell growth. On cell mineralization assays, we evaluated mineral nodules deposition and expression of osteocalcin mRNA, collagen concentrations on culture supernatant, phosphate, calcium, TGF- ?1 and ALP on cell lysates. The results showed that US stimulates the mineralization of preosteoblasts 192h after treatment by stimulating osteocalcin mRNA expression, calcium and phosphate uptake amd consequent formation of HA. Later, in different experimental conditions, the results showed that LIPUS had the ability to stimulate pre-osteoblastic mineralization with decreased concentration of collagen, calcium and phosphate in the cell supernatant 192 hours after treatment. It also changed the alkaline phosphatase concentration, as well as the osteocalcin gene expression. / O ultrassom pulsado de baixa intensidade surge como recurso terap?utico de alto potencial para o tratamento de doen?as osteometabolicas e fraturas com atraso de consolida??o ?ssea. Entretanto, investiga??es recentes demonstram resultados controversos, sugerindo a necessidade de mais estudos acerca do entendimento das respostas biol?gicas e na padroniza??o dos par?metros das modalidades de tratamento. Sendo assim, o presente estudo teve o objetivo de avaliar o efeito do ultrassom na prolifera??o e mineraliza??o de pre-?steoblastos a partir de bioensaios in vitro. Foram utilizados pr?-osteoblastos da linhagem MC3T3-E1, sendo estas tratadas com ultrassom terap?utico no modo pulsado a 20%, com uma frequ?ncia de 1MHz e intensidade de 0,2 W/cm2. No estudo de prolifera??o celular, avaliamos c?lcio intracelular, o TGF-?1, magn?sio e os n?veis de mRNA de osteopontina e osteocalcina, NF-?B1 e p38?. Al?m disso, utilizamos nifedipina e a rapamicina para investigar rotas de prolifera??o. Inicialmente, os resultados mostraram que o ultrassom aumenta prolifera??o de MC3T3-E1, diminuindo o teor de c?lcio e magn?sio no sobrenadante. Al?m disso, aumenta a concentra??o de c?lcio intracelular, ativa NF-?B1 e o complexo mTOR via p38? e promove uma diminui??o na s?ntese de TGF-?1, que ? um inibidor do crescimento celular. Nos ensaios de mineraliza??o celular avaliamos inicialmente a deposi??o n?dulos de minerais na placa de cultura e a express?o g?nica da osteocalcina por PCR convencional, e, posterirormente a concentra??o no sobrenadante celular de col?geno, fosfato, c?lcio, TGF-? al?m de fosfatase alcalina no lizado de c?lulas. Os resultados mostraram que o ultrassom tem a capacidade de estimular a mineraliza??o pr?-osteoblastica em 192 horas ap?s o tratamento a partir do aumento da express?o osteocalcina, capta??o de c?lcio e fosfato e consequente forma??o de hidroxiapatita.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-05-17T12:16:55Z No. of bitstreams: 1 DIS_JULIANA_ROMEU_MARQUES_COMPLETO.pdf: 1432001 bytes, checksum: 2d6dc8303449f3f554bf97804d426795 (MD5) / Made available in DSpace on 2016-05-17T12:16:55Z (GMT). No. of bitstreams: 1 DIS_JULIANA_ROMEU_MARQUES_COMPLETO.pdf: 1432001 bytes, checksum: 2d6dc8303449f3f554bf97804d426795 (MD5) Previous issue date: 2016-02-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The liraglutide is a glucagon-like peptide 1 (GLP-1) based therapy and an alternative treatment to type 2 diabetes mellitus (T2DM). Apart from its hypogliycemic effect, the drug has been also associated with the prevention of cardiovascular diseases, regulation of blood lipid and anti-inflammatory action. Reports indicate, however, that liraglutide can increase the risk of thyroid cancer and cause pancreatitis. Besides, studies demonstrated that some analogues of GLP-1 have a deleterious bone effect, while others demonstrated to be benefic to the bone. The aim of this study was to evaluate the effect in vitro of liraglutide in mouse pre-osteoblastic cells, MC3T3-E1. The results demonstrated that liraglutide significantly decreased the number of viable cells without inducing apoptosis. The mechanism of this effect is associated to the reactive oxygen species production (ROS), which resulted in an increase of cell autophagy. The decreased number of viable cells indicates therefore that liraglutide may affect the bone formation. / A liraglutida ? uma terapia baseada no horm?nio GLP-1 utilizada como tratamento alternativo para a diabetes mellitus tipo 2 (DM2). O medicamento, al?m do efeito hipoglicemiante, tem sido associado ? preven??o de doen?as cardiovasculares, diminui??o dos lip?deos no sangue e ? a??o anti-inflamat?ria. Relatos indicam, por?m, que a liraglutida pode aumentar o risco de c?ncer de tireoide e provocar pancreatite. Al?m disso, estudos demonstram que alguns an?logos do GLP-1 t?m efeito ?sseo delet?rio, enquanto outros t?m efeito ben?fico. O objetivo deste estudo ? avaliar o efeito da liraglutida sobre o crescimento de c?lulas pr?-osteobl?sticas (MC3T3-E1). Os resultados t?m demonstrado que a liraglutida diminuiu significativamente o n?mero de c?lulas sem induzir a apoptose. O mecanismo deste efeito est? associado ? produ??o de esp?cies reativas de oxig?nio, que resulta em um aumento da autofagia das c?lulas, indicando, desta forma, que pode afetar a forma??o ?ssea.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-07-26T14:11:19Z No. of bitstreams: 1 TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf: 2184806 bytes, checksum: 196521807ad1dcdfa8bffc204202abe6 (MD5) / Made available in DSpace on 2016-07-26T14:11:19Z (GMT). No. of bitstreams: 1 TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf: 2184806 bytes, checksum: 196521807ad1dcdfa8bffc204202abe6 (MD5) Previous issue date: 2016-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Introduction: leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for exert an important role in cell signaling. However, most studies evaluating cellular responses mediated by Leu, works within a normal perspective in AA supply and little is known about the effects that supplementation can generate on the cell proliferation mechanisms. The effects of excess of this amino acid, have been extensively studied in many cell types, but there is an important limitation on the amount of information available in the scientific literature regarding their actions in bone cells. Objective: the aim of this study to determine the effects of leucine supplementation on proliferation of pre-osteoblasts of the MC3T3-E1 lineage. Methods: the MC3T3-E1 cells were kept in ?-MEM supplemented with 10% fetal bovine serum and 1% antibiotic. After initial determination of concentrations, the cells were treated during 48 hours, by the addition of 50 ?M Leu, which corresponds to 12,5% in addition of the amino acid to the culture medium. Untreated cells represented the control group. The evaluation of the viability and proliferation of cultured cells was performed with Trypan Blue dye (0.4%). To identify the mechanisms related to decreased cellular proliferation, assays were performed to verify cytotoxicity (LDH); apotosis (Annexin V); oxidative stress (TBARS and DCFH); inflammation (TGF-? 1 and CBA); autophagy (acridine orange and flow cytometry); senescence (DAPI and flow cytometry); and DNA damage (alkaline comet assay). Results and conclusions: Leu supplementation (50 ?M) decreases cell proliferation by 40% with causes not related to cell necrosis, apoptosis, oxidative stress, inflammation or autophagy. The Leu supplementation caused DNA damage, with consequent increase in senescence and decrease of proliferation of MC3T3-E1 cells. / Introdu??o: a leucina (Leu) ? um amino?cido (AA) essencial, de cadeia ramificada, presente na alimenta??o humana, principalmente no leite e seus derivados, que tem sido investigado por exercer um importante papel na sinaliza??o celular. Contudo, a maioria dos estudos que avalia as respostas celulares mediadas pela Leu, trabalha dentro de uma perspectiva de normalidade na oferta do AA e pouco se sabe sobre os efeitos que a suplementa??o pode gerar sobre os mecanismos de prolifera??o celular. Os efeitos do excesso deste amino?cido, t?m sido extensivamente estudado em diversos tipos de c?lulas, entretanto existe uma limita??o importante na quantidade de informa??es dispon?veis na literatura cient?fica em rela??o as suas a??es em c?lulas do tecido ?sseo. Objetivo: este estudo teve como objetivo analisar os efeitos da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1. M?todos: a cultura dos pr?-osteoblastos da linhagem MC3T3-E1, foi realizada com ?-MEM, suplementado com 10% de soro fetal bovino e 1% de antibi?tico. Ap?s determina??o de concentra??es, o tratamento foi feito com a adi??o de Leu, dilu?da ao meio de cultura nas concentra??es de 50 ?M, o que corresponde a um acr?scimo percentual de 12.5% a mais do amino?cido ao meio de cultura, por 48 horas. A viabilidade e a prolifera??o celular foram avaliadas pela t?cnica do Trypan Blue. Para a identifica??o dos mecanismos relacionados a inibi??o da prolifera??o celular, foram realizados ensaios que avaliaram a citotoxicidade (LDH); apoptose (Anexina V); estresse oxidativo (TBARS e DCFH); perfil inflamat?rio (TGF-? 1 e CBA); autofagia (laranja de acridina e citometria de fluxo); senesc?ncia (DAPI) e dano ao DNA (teste cometa). Resultados e conclus?es: a suplementa??o de Leu (50 ?M) inibe a prolifera??o celular em 40%, com causas n?o relacionadas a necrose celular, apoptose, estresse oxidativo, inflama??o ou autofagia. A suplementa??o de Leu provocou dano ao DNA, com consequente senesc?ncia e diminui??o da prolifera??o celular de pr?-osteoblastos da linhagem MC3T3-E1.