Abstract:We investigated the effects of photodynamic therapy (PDT) outcome when combining three laser systems that produce light in three different wavelengths (600, 630, and 660 nm). Cooperative as well as independent effects can be observed. We compared the results of the combined wavelengths of light with the effect of single laser for the excitation of the photosensitizer. In the current experiment, the used photosensitizer was Photogem R (1.5 mg/kg). Combining two wavelengths for PDT, their cumulative dose and different penetrability may change the overall effect of the fluence of light, which can be effective for increasing the depth of necrosis. This evaluation was performed by comparing the depth and specific aspect of necrosis obtained by using single and dual wavelengths for irradiation of healthy liver of male Wistar rats. We used 15 animals and divided them in five groups of three animals. First, Photogem R was administered; follow by measurement of the fluorescence spectrum of the liver before PDT to confirm the level of accumulation of photosensitizer in the tissue. After that, an area of 1 cm 2 of the liver was illuminated using different laser combinations. Qualitative analysis of the necrosis was carried out through histological and morphological study.
We present experimental evidence of the existence of cell variability in terms of threshold light dose for Hep G2 (liver cancer cells) cultured. Using a theoretical model to describe the effects caused by successive photodynamic therapy (PDT) sessions, and based on the consequences of a partial response we introduce the threshold dose distribution concept within a tumor. The experimental model consists in a stack of flasks, and simulates subsequent layers of a tissue exposed to PDT application. The result indicates that cells from the same culture could respond in different ways to similar PDT induceddamages. Moreover, the consequence is a partial killing of the cells submitted to PDT, and the death fraction decreased at each in vitro PDT session. To demonstrate the occurrence of cell population modification as a response to PDT, we constructed a simple theoretical model and assumed that the threshold dose distribution for a cell population of a tumor is represented by a modified Gaussian distribution.Microscopic image (100×) of a necrosis caused by PDT in a rat liver. The edge is the depth of necrosis
The slow darkening of grains is sought by bean breeders because the consumers consider that darker grains demand more time for cooking. The analysis currently used takes around 90 days to differentiate grain color among genotypes. The objective was to evaluate the color as a function of the value of L* (lightness) of carioca beans, by natural and accelerated methods to verify equivalence between methods, validation of the methodology and identification of genotypes tolerant to the darkening. The grain darkening was compared and evaluated by natural darkening method under shelf conditions, in days storage, and accelerated darkening method under ultraviolet light, in hours. The natural darkening time of 90 days was statistically equal to 24 hours of accelerated darkening, and the difference among the genotypes could be obtained in a shorter time, indicating a correspondence in the methods. The accelerated darkening method can be used to shorten the analysis time in the routine of breeding programs.
Estudo da distribuição de doses limiares em TFD para um modelo de cultura tridimensional de células obtido pelo método de levitação magnética São Carlos -SP 2014 LUIS GUSTAVO SABINO Dedico este trabalho aos meus pais e meus avós. AGRADECIMENTOSAgradeço a todas as pessoas que colaboraram para a realização deste trabalho. Para vocês, gostaria de citar um trecho de uma música que diz: "Sonho que se sonha só, é só um sonho que se sonha só, mas sonho que se sonha junto é realidade" (Raul Seixas). Um agradecimento especial aos meus pais Francisco Sabino e Nilce Teixeira Sabino pela dedicação e pelo apoio em todos os momentos da minha vida, sem vocês eu não chegaria até aqui. Agradeço aos meus irmãos, e toda a minha família pelo suporte e toda ajuda. Gostaria de agradecer a Ana Carina, minha companheira e amiga de todas as horas, das alegrias e das tristezas. Obrigado aos amigos do Laboratório de Biofotônica, Clóvão, Pri, Didi, Sebastião, Alessandra, Lili, Denis, Merê, Nat, Paulinha, Larissa, e tantos outros, vocês têm me ajudado muito nessa caminhada. Agradeço aos amigos do LAT (Blim Blim, Minduca, Jef, Guilherme), e ao pessoal da secretaria (Bel, Benê, Cris). Agradeço aos funcionários e professores do IFSC por toda a ajuda, especialmente ao Ítalo, a Renata, e a Neusa da biblioteca. Agradeço aos Professores Daniela e Tirapelli, e os alunos Fermino e Paulinho da FMRP-USP, pela ajuda nos experimentos de citometria. Um agradecimento especial à professora Cristina Kurachi e ao professor Vanderlei, por disponibilizar os recursos para que o trabalho fosse realizado. Um agradecimento especial aos professores Thomas Killian (Rice University) e Glauco Souza (Nano3D Biosciences), por todo o apoio e os ensinamentos. Obrigado também aos meus amigos de Houston, Jacob Gage, Sue Shiao, Jianbo Chan, e Shane Neeley, que tornaram minha estadia em Houston mais agradável. Gostaria de agradecer a participação, as lições e O amor é a única loucura de um sábio e a única sabedoria de um tolo. )). Developing methods for more realistic tissue emulation with in vitro cultures, such as three-dimensional (3D) cultures, have been encouraged aiming to avoid the above-mentioned dissimilarities. A 3D cell culture is preferable compared to monolayer cell cultures, because it provides cell-cell and cell-substrate interaction, and makes evaluating a culture and its volumetric dimension (which resembles the tumor morphology) possible. This study also includes the development of a 3D model, using the magnetic levitation method (MLM) and the magnetic printing method (MIM, from Portuguese método de impressão magnética) for PDT dosimetry. The aim is to define parameters for g (Dth), to investigate cell resistance to PDT, and to achieve a fast and consistent method for new PS in vitro tests. By comparing cultures from the different cell types studied, the ones obtained by MLM for more than 185 hours were found to present a denser cellular structure, which provided improved resistance to PDTinduced damage. Hep G2 cells showed a remarkable behavior by bein...
Ao bom Deus e aos meus pais, os responsáveis pela minha existência. Obrigado por iluminar meu caminho e me dar força na busca por meus sonhos e realizações. A toda a minha família, a base da minha vida, o meu chão, obrigado pelo apoio. Agradeço também a minha namorada Nayara, pela compreensão e apoio na fase final deste trabalho. Ao Professor Vanderlei S. Bagnato, exemplo de competência, perseverança e generosidade. Agradeço pelo suporte e a orientação durante o mestrado, e desejo que esteja presente nas próximas etapas da minha formação como pesquisador. À Professora Cristina Kurachi que está sempre presente e pronta para ajudar e ensinar o que é necessário. Sendo muito importante para meu crescimento pessoal e profissional.
We used three dimensional cell cultures (3D) based on the magnetic levitation method (MLM) to evaluate cytotoxicity of photodynamic therapy (PDT). First, we decorated Hep G2 and MDA-MB-321 cells with NanoShuttle by introducing it in the media and incubated overnight. Next day, we transferred the cells to a 6-well plate and placed a magnetic driver on the top of the plate to start levitation. We monitored the formation of the 3D cell culture by optical microscopy and after four days, we added the photosensitizer Photogem (PG) in the culture media in concentrations of 50, 25, 12.5, 6.25µg/ml. We incubated them for 24 hours, after that we washed the cultures with PBS and added fresh media. Samples were then illuminated for 600s using a 630nm LED-based device, generating light intensities of 30 mW/cm 2 in a total light fluence of 18 J/cm 2 . Following the illumination, we added fresh media, and 30 hours later, the 3D structures were broken using a pipettor and the cells seeded in 96 well plates, 10 5 cells per well, with a magnetic drive placed on the bottom of the plate to create cell culture dots. After 24 hours, we used a MTT assay to evaluate PDT cytotoxicity. The PDT effect, evaluated by the half maximal effective concentration (EC50), in MDA-MB-231 cells (EC50 =3.14 µg/ml) is more aggressive compared to the effect of PDT in Hep G2 cells (EC50 = 7.48 µg/ml). It suggests that the cell culture structure and its interaction facilitated the PG uptake and consequently elevated the Photodynamic effect for MDA-MB-231.
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