Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-06-04T12:50:56Z No. of bitstreams: 2 Dissertação - Giuliana Thaíse Araújo da Silva - 2018.pdf: 1677560 bytes, checksum: fe8891f1722a8a10846220a3a8f2a5e9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-04T13:54:50Z (GMT) No. of bitstreams: 2 Dissertação - Giuliana Thaíse Araújo da Silva - 2018.pdf: 1677560 bytes, checksum: fe8891f1722a8a10846220a3a8f2a5e9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-04T13:54:50Z (GMT). No. of bitstreams: 2 Dissertação - Giuliana Thaíse Araújo da Silva - 2018.pdf: 1677560 bytes, checksum: fe8891f1722a8a10846220a3a8f2a5e9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-05-03 / The Light Emitting Diode (LED) is a semiconductor device that emits light source and can be used as a tissue modeler by means of the transformation of received photons. Photobiomodulation therapy (PBMT) is a specific term for the therapeutic application of light and acts as therapy, analgesia and anti-inflammatory. The objective of this work was to verify the effectiveness of LED at fixed time in the stimulation of fibroblasts. For this purpose, the L929 cell line submitted to LED irradiation (630 nm) was used, in triplicate, at the potency of 50, 75 and 100 mW, for five seconds and compared with non-irradiated control group. Next, cell viability, proliferation, nitric oxide production and collagen synthesis were observed. The results revealed that there was no cytotoxicity after 24, 48 and 72 hours of irradiation; there was an increase in the production of nitrite between the group of 72 hours and the other experimental groups and increase in the synthesis of collagen, directly proportional to the potencies used. Thus the irradiation allowed the fibroblastic stimulation with increased activity in the healing process. / O Diodo Emissor de Luz (Light Emissor Diode - LED) é um dispositivo semicondutor que emite fonte de luz e pode ser usado como modelador tecidual por meio da transformação de fótons recebidos. A fotobiomodulação (Photobiomodulation therapy - PBMT) é um termo específico para aplicação terapêutica da luz e atua como analgesia e anti-inflamatório. O objetivo deste trabalho foi de verificar a efetividade do LED em tempo fixo na estimulação de fibroblastos. Para tanto, utilizou-se a linhagem celular L929 submetida à irradiação LED (630 nm), em triplicata, nas potências de 50, 75 e 100 mW, por cinco segundos e comparou-se com grupo controle não irradiado. Em seguida, observou-se a viabilidade celular, proliferação, produção de óxido nítrico e síntese de colágeno. Os resultados revelaram que não houve citotoxicidade após 24, 48 e 72 horas de irradiação; houve aumento na produção de nitrito entre o grupo de 72 horas e os outros grupos experimentais e aumento na síntese de colágeno, diretamente proporcionais às potências utilizadas. Assim a irradiação possibilitou a estimulação fibroblástica com aumento de sua atividade no processo de cicatrização.

A polpa dental é formada de tecido conjuntivo frouxo sendo constituída por diversas células, dentre as quais os fibroblastos são as mais numerosas. Ao serem submetidas a agressões diversas, estas células respondem com a liberação de substâncias, tais como citocinas e quimiocinas, que participam de maneira ativa no processo inflamatório. Assim sendo, este trabalho teve como proposição: 1. avaliar a capacidade de fibroblastos de polpa dental humana em cultura em produzirem as quimiocinas MIP-l\'alfa\' /CCL3 e SDF-1/CXCL12; 2. avaliar a produção destas quimiocinas pelos fibroblastos quando estimulados por Enterococcus faecalis morto por calor com relação à quantidade de bactérias por célula e 3. avaliar a liberação destas quimiocinas com relação ao tempo de estímulo. Para o estabelecimento das culturas, foi coletada a polpa de terceiro molar hígido de um paciente saudável. O tecido foi extraído, armazenado e picotado em meio de cultura para fibroblastos (DMEM), os quais foram utilizados a partir da quarta passagem. Após adesão das células a placas de 24 poços, o meio de cultura contendo Enterococcus .faecalis morto por calor numa concentração correspondente a 1, 10 e 100 bactérias por fibroblasto foi adicionado aos poços. Após 1, 6 e 24 horas, o sobrenadante das células foi coletado para a análise por ELISA. A análise estatística foi realizada aplicando-se o teste Kruskal-Wallis com nível de significância de 5%. A produção de MIP-l\'alfa\' /CCL3 e SDF-l/CXCL12 pelas células pôde ser detectada por ELISA. Os fibroblastos pulpares se mostraram capazes de produzir SDF-1 constitutivamente sendo que o estímulo bacteriano levou a uma diminuição estatisticamente significativa desta produção. A produção de MIP-l\'alfa\' também foi detectada tanto de maneira constitutiva como em resposta ao desafio microbiano. Enquanto a concentração intermediária de bactéria por fibroblasto (10:1) mostrou uma produção semelhante ao grupo controle, as concentrações de 1 e 100 bactérias por fibroblasto induziram aumento maior na primeira hora de estímulo. Essas diferenças, entretanto, não foram estatisticamente significativas. A capacidade dos fibroblastos secretarem quimiocinas, como MIP-l\'alfa\' e SDF-1, reforça a importância dessas células dentro do contexto de imunidade e inflamação pulpar, principalmente por serem as células mais numerosas deste microambiente. / Dental pulp is a connective tissue structure constituted by many different cell types. Among them, the fibroblasts are the most frequent ones. When challenged by different aggressive agents, these cells are able to release some substances like cytokines and chemokines, which are essential to trigger the inflammatory process. The aims of this study were: 1. to evaluate the ability of fibroblasts to produce the chemokines MIP-l\'alfa\'/CCL3) and SDF-1/CXCL12; 2. to evaluate the expression of these chemokines by fibroblasts when challenged by heat killed Enterococcus. faecalis in gradual concentrations and 3. to evaluate the production of these chemokines in a time course manner. The dental pulp from non-carious third molar was collected from a healthy patient. Explants were made and stocked in culture medium (DMEM) for fibroblasts growth. The cells were used since passage four. In a 24-well plate and after reaching confluence, culture medium alone or containing heat killed E. faecalis at proportion 1:1, 10:1 and 100:1 bacteria:fibroblast, were added to the fibroblasts. After 1, 6 and 24 hours, the supernatants were collected for analysis. The protein detection of MIP-l\'alfa\'/CCL3 and SDF-1/CXCL12 was performed by ELISA. For statistical analysis, data were assessed by Kruskal-Wallis followed by Miller post-test. Significance levels of 5% were adopted. Production of both chemokines was detected by ELISA. Pulp fibroblasts were able to produce SDF-1 constitutively. This production decreased with the increase in the number of heat killed E. faecalis increased (p < 0.05). Production of MIP-l\'alfa\' was detected in unchallenged and challenged cells. The median bacterial concentration (10:1) presented a profile production similar to that of unstimulated cells. Bacterial concentrations of 1 and 100 microrganisms/cell showed a highly enhanced production of MIP-l\'alfa\' at the first hour of stimulum; however, these data were not statistically significant (p > 0.05). Fibroblasts ability to produce chemokines, like MIP-l\'alfa\' and SDF-1, confirms their importance at immune and inflammatory events in dental pulp, specially being fibroblasts the most abundant cells at this microenvironment .

Made available in DSpace on 2014-10-09T12:50:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:54Z (GMT). No. of bitstreams: 1 10826.pdf: 3720317 bytes, checksum: 26d9f64b9d44684f557ae44f37e86002 (MD5) / Dissertação (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Intituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo

Made available in DSpace on 2014-10-09T12:50:14Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:45Z (GMT). No. of bitstreams: 1 10825.pdf: 4609998 bytes, checksum: abaefc3f2b2d697034a947707dec7944 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia - USP

The ability of mammalian cells to locomote is important in a variety of normal and pathological processes. Previous work has suggested that low doses of x-irradiation may perturb cell motility, a finding that may have important consequences in embryogenesis, cancer metastasis, and immune response. This thesis has sought to study in more detail the effect of radiation on mammalian cell motility. Work performed in other laboratories used the colloidal gold assay and time lapse cinemicroscopy to study x-irradiation induced changes to 3T3 fibroblast motility in tissue culture. These studies were repeated here, with qualitative results similar to those reported earlier. However, these methods were not amenable to a detailed quantitative analysis. For this, spatial and temporal information on the motility and dynamic morphology of a large number of cells is required. Such a task would be impossible to perform manually, thus an automated microscope system was developed that used a computer-driven precision stage and a solid state optical sensor to track individual cells in tissue culture. Information on motility and morphology was concurrently extracted from many cells. As part of the thesis, several techniques were developed to analyze and display these data, and to correlate motility and morphology observations. These techniques were directed at preserving the actual process of 3T3 cell motility, and parameters were measured to quantify the short term persistence of cell movement (on a time scale of 0.5 to 2 hours), and the long term persistence of cells in maintaining certain characteristic behaviour (on a time scale of 3 to 12 hours). The response of 3T3 fibroblasts to x-irradiation was characterized by a number of parameters. The population average cell speed was measured following treatment, and a dose response and time response was determined in the range of 1.5 Gy. Other motility parameters indicate that the normal process of cell motility, evidenced by a series of motile segments, was disrupted by x-rays. This was thought to reflect perturbation to the control mechanisms of cell motility. The morphology of 3T3 cells stained with Coomassie blue was examined in an effort to correlate the observed motility changes with changes in the fixed cell morphology. This stain is a general structural protein stain with higher affinity toward microfilaments. High doses of x-rays were required to produce significant perturbation to cell morphology, and in the dose regime of interest, the morphology of irradiated cells was not identifiably different from control. Of note is that it was the well spread, quiescent cells that seemed least perturbed by large doses of irradiation. In summary, x-rays apparently disrupt the normal process of cell motility. Several lines of evidence suggest that actively migrating cells are the most perturbed by irradiation. This work has developed techniques to quantify cell motility in a meaningful way, and to characterize the x-ray induced perturbations. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

M.Tech. (Biomedical Technology) / Collagen Type I (Col- I) as well as collagen types III and V, form most of the connective tissues, smooth muscle cells and, endothelial cells in wound healing (Stuart and Leaper, 2008). Col-I is also the main extracellular matrix (ECM) protein (Ricard-Blum and Ruggiero, 2005). Low intensity laser irradiation (LILI) is a non-invasive, photobiomodulatory therapy. Huang et al., (2009a) have shown LILI to be involved in Col-I production both in vitro and in vivo. Enhanced collagen production in human skin fibroblasts is common shortly after irradiation (Illsley et al., 2000). However, its synthesis in wounded fibroblasts has not been well established in an in vitro model. Healing is impaired in chronic diabetic wounds which exhibit reduced proliferation rate and collagen synthesis (Beldon, 2010; Falanga, 2005). Studies have shown that LILI using a wavelength of 632.8 nm was not the only wavelength biostimulated in cultured cells: biological responses were also generated from various wavelengths within the visible to Near Infrared (NIR) spectral region (Hawkins and Abrahamse, 2005; Karu and Kolyakov, 2005). This study aimed to establish if LILI influenced collagen production and related cellular responses at a wavelength of 660 or 830 nm, with a fluence of 5 J/cm2 in an in vitro normal and wounded fibroblasts model. The study also evaluated the expression profiling of genes related to the ECM and adhesion. This study was performed on isolated human skin fibroblasts collected from a consenting adult undergoing abdominoplasty. Cells were routinely cultured according to standard techniques (Houreld and Abrahamse, 2010; Hawkins and Abrahamse, 2007a; Hawkins and Abrahamse, 2006a; Hawkins and Abrahamse, 2005).

Aims Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). Methods and resultsCFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 25 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 824 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β1 integrin receptor blockade by Tac β1 expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. Conclusion These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

Myocardial injury leads to scar formation and pathological fibrosis that has a significant impact on the development and progression of cardiac disease. Increasing evidence suggests alteration in the chromatin landscape of cells can exacerbate the extracellular matrix deposition and enhance disease progression. Chromatin alterations and fibrosis mediate several cardiac cellular changes, including scar formation, DNA damage, collagen deposition, and increased TGFB expression which are all disease-driving mechanisms during heart failure. Targeting epigenetic dependent fibrosis pathways is thus a promising strategy for the prevention and treatment after myocardial injury. The polycomb complex protein Bmi1, an epigenetic regulator, is associated with numerous biological functions including mediating DNA damage, cellular fate, and proliferation. However, there is currently a lack of understanding on how Bmi1 mediated epigenetic modifications affect adult heart function after injury. It was previously determined that Bmi1 modulates the epigenetic landscape of cardiac stem cells that mediates various molecular processes during a stress condition. In the present study, using a Bmi1 global and fibroblast specific knockout model, cardiac function was assessed through echocardiography using adult mice following cardiac injury. The loss of Bmi1 caused a significant decrease in heart function after injury, which was associated with increased fibrosis and DNA damage. Specifically, we found that the adult cardiac fibroblasts, isolated from the Bmi1 knockout model, had increased expression of pro-fibrotic genes including TGFB, aSMA, and Collagen1a1. Through multiomic sequencing, we found significant changes in the pathological fibrotic signaling pathways of TGFB, specifically with SMAD3 chromatin accessibility with the loss of Bmi1 epigenetic regulation. Concluding, Bmi1 epigenetic regulation mediates repair during pathological challenge by regulating adult cardiac fibroblasts and pathological fibrosis after cardiac injury. / Biomedical Sciences

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