Revascularização é uma técnica utilizada em dentes jovens, que apresentem rizogênese incompleta e danos irreversíveis ao tecido pulpar, necessitando de tratamento endodôntico, para formar novo tecido em lugar da polpa perdida. Clinicamente os resultados mostram a continuidade da rizogênese e a devolução da vitalidade dental. Porém, pouco se sabe sobre o novo tecido formado e não está estabelecido se este é capaz de desempenhar todas as funções da polpa dentária. Para melhorar as características do tecido formado pela técnica da revascularização, podemos utilizar ferramentas de engenharia tecidual, como célulastronco, fatores de crescimento e arcabouços de sustentação celular (scaffolds). As célulastronco (CTs) já estão presentes quando o sangue invade o canal radicular, e utilizar essa reserva de CTs que o hospedeiro possui, procedimento conhecido como homing, é uma vantagem em comparação com outras técnicas que injetam CTs obtidas por cultivo em laboratório. Entretanto os aspectos físicos do coágulo sanguíneo formado no interior do canal radicular podem ser melhorados com a adição de hidrogel de quitosana, que interage quimicamente com o sangue e forma um scaffold híbrido mais estável. Então, o objetivo deste estudo foi testar a hipótese de que o scaffold híbrido, composto por hidrogel de quitosana e sangue, ofereceria maior estabilidade física estrutural, bem como condições favoráveis à adesão e proliferação de células-tronco da papila apical humana (SCAPs; do inglês, Stem Cell from Apical Papila). Para isso, investigamos in vitro se a incorporação do sangue ao hidrogel de quitosana gera um scaffold mais estável, se este é favorável à adesão e proliferação de células-tronco da papila apical e se a fotobiomodulação potencializa essas características celulares. Para isso, SCAPs foram isoladas e caracterizadas por citometria de fluxo, tempo de dobra populacional, e contagem de unidades formadoras de colônias fibroblásticas (CFU-F; do inglês, Colony Forming Units - Fibroblastic). Ensaios de incorporação sanguínea, dissolução e embebição foram realizados para determinar o comportamento dos scaffolds híbridos. A adesão celular foi observada pela coloração PHK26® (do inglês, Red Fluorescent Cell Linker) e por microscopias eletrônicas de varredura (MEV); e a proliferação foi investigada pelo ensaio de alamarBlue®. Adicionalmente, a sobrevivência das SCAPs após a degradação do scaffold híbrido foi avaliada pela coloração Live/Dead®. A população celular estudada apresentou características de células tronco. O scaffold híbrido, constituído de densa rede alveolar com poros interconectados, embebeu e dissolveu rapidamente. De acordo com o PKH26® e alamarBlue® SCAPs aderiram e proliferaram no scaffold híbrido. SCAPs fotobiomoduladas exibiram maior taxa de proliferação e o ensaio Live/Dead® mostrou células vivas após 12 dias de cultivo. Concluiu-se que o scaffold híbrido apresenta biocompatibilidade e condições favoráveis de sobrevivência para SCAPs, que são potencializadas pela fotobiomodulação. / Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-07-17T22:01:31Z No. of bitstreams: 1 Tatiane Garcia Stancker.pdf: 1297386 bytes, checksum: b71324f95aa95befc8016ee8adc416fd (MD5) / Made available in DSpace on 2018-07-17T22:01:31Z (GMT). No. of bitstreams: 1 Tatiane Garcia Stancker.pdf: 1297386 bytes, checksum: b71324f95aa95befc8016ee8adc416fd (MD5) Previous issue date: 2017-10-30 / Osteoarthritis (OA) is a chronic degenerative process characterized by progressive deterioration of the cartilagereaching subchondral bone. Current interventions have little or no effect on the repair of articular cartilage. Therefore, the therapy throughmesenchymal stem cells (MSC’s) has been a target of interest due to its multilineage potential, becoming an important tool in the attempt of tissue repair. The photobiomodulation (PBMT) has been shown to be favorable in the proliferation of various cell types, including MSC’s. The aim of this study is to determine whether PBMT can increase the retention of adipocyte-derived stem cells (ADSC's) injected into the knee of the rat submitted to the experimental model of osteoarthritis and to analyze the chondroprotective effects of these therapies. ADSC's were collected from 3 male Fischer-344 rats and validated by flow cytometry. The sample was composed of 50 Fischer-344 rats and distributed into five groups: Control group (healthy animals), OA group (animals with OA), OA PBMT group (OA animals treated with PBMT therapy), OA ADSCs group (OA treated with ADSC's injection), and OA ADSC's PBMT group (animals with OA treated with ADSC's injection and PBMT). OA was induced by intra-articular injection of 4% papain solution. OA ADSC's and OA ADSC's PBMT animals received intra-articular injection of ADSC's (1x10⁶) diluted in 100μ of culture medium. For the groups that received PBMT (parameters used: wavelength of 808nm, power of 50mW, energy of 2J, density of energy 71.2J/cm² and spot size 0,028cm²) the applications were performed in 4 points of the articular line of the knee on consecutive days. The euthanasia was performed on the 3rd and 7th days after the treatments,which the articular cartilage was extracted for the quantification of the gene expression by RT/ PCR of SRY, IL-1β, IL-6, TNF-α, IL- MMP-1, MMP's 1 and 2, TIMP's 1 and 2 and collagen type II.For the statistical analysis, One way ANOVA with Tukey's post-hoc test was used andall data are expressed as mean±standard deviation with P<0,05. The results showed that the PBMT and ADSC’s therapies decreased the expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and MMP’s 1 and 2 when compared to the OA group. In addition, they increased the gene expression of TIMP's 1 and 2, IL-10 and Collagen type II. And the effects are more effective when therapies are associated. PBMT stimulated the retention of these cells in the intra-articular space. We conclude that the intra-articular ADSC’s injection associated with PBMT has chondroprotective action, modulating the inflammatory process and decreasing the cytokines and MMP's. / A osteoartrite (OA) é um processo degenerativo crônico caracterizado pela degradação progressiva da cartilagem chegando a atingir o osso subcondral. As intervenções atuais para a OAexercem pouco ou quase nenhum efeito sobre a reparação da cartilagem articular.Sendo assim, a terapia através de células-tronco mesenquimais (CTM’s) têm sido alvo de interesse devido a seu potencial de multilinhagem, tornando-se uma importante ferramenta na tentativa de reparação tecidual. A fotobiomodulação (PBMT) tem demonstrado favorecer a proliferação de vários tipos celulares, inclusive as CTM’s.O objetivo deste estudo é determinar se a PBMT pode aumentar a retenção de células-tronco derivadas de adipócitos (ADSC’s) injetadas em joelho de ratos submetidos à modelo experimental de osteoartrite e analisar os efeitos condroprotetores destas terapias. As ADSC’s foram coletadas de 3 ratos Fischer-344 machos e validadas através da citometria de fluxo. A amostra foi composta de 50 ratas Fischer-344 e distribuídas em cinco grupos: grupo Controle (animais saudáveis), grupo OA (animais comOA), grupo OA PBMT (animais com OA tratados com terapia porPBMT), grupo OA ADSC’s (animais com OA tratados com injeção de ADSC’s), e grupo OA ADSC’s PBMT (animais com OA tratados com injeção de ADSC’s e PBMT). A OA foi induzida através da injeção intra-articular de solução de papaína a 4%. Os animais dos grupos OA ADSC’s e OA ADSC’s PBMT receberam injeção intra-articular de ADSC’s (1x10⁶)diluídas em 100μde meio de cultura. Para os grupos que receberam PBMT (parâmetros utilizados:comprimento de onda de 808nm; potência de 50mW; energia de 2J; densidade de energia 71,2J/cm² e área do feixe 0,028cm²) as aplicações foram realizadas em 4 pontos da linha articular do joelho em dias consecutivos. A eutanásia foi realizada no 3° e 7° dias após os tratamentos onde foram extraídas as cartilagens articulares para a quantificação da expressão gênica através de RT/PCR deSRY, IL-1β, IL-6, TNF-α, IL-10, MMP’s 1 e 2, TIMP’s 1 e 2 e colágeno tipo II. Para a análise estatística foi utilizado ANOVA One way com post hoc test o teste de Tukey e os dados foram expressos através da média±desvio padrão com valor de P<0,05. Os resultados mostraram que as terapias através de PBMT e ADSC’s diminuíram a expressão de citocinas pró inflamatórias (IL-1β, IL-6 e TNF-α) e das MMP’s 1 e 2 quando comparados ao grupo OA. Além disso, aumentaram a expressão gênica das TIMP’s 1 e 2, IL-10 e do Colágeno tipo II. E quando as terapias são associadas os efeitos são mais eficazes. A PBMT também mostrou estimular a retenção destas células no espaço intra-articular. Concluímos que a injeção intra-articular de ADSC’s associada a PBMT tem ação condroprotetora, modulando o processo inflamatório, diminuindo as citocinas e MMP’s.

The photobiomodulation therapy (PBMT) has many demonstrated applications in the health area including anti-inflammatory and wound healing effects. The main objective of this work is to verify if the PBMT causes measurable changes in the mechanical properties of cells, specifically in red blood cells, epithelial cells and fibroblasts. In addition, to contribute to the knowledge of the action mechanisms of the PBMT, this study intends to support applications of the PBMT during invasive procedures, such as the direct photo-treatment of the blood in surgical procedures with cardiopulmonary bypass, regarding security of the cellular integrity. For this analysis, three experimental techniques were used: optical magnetic twisting cytometry (OMTC), defocusing microscopy and confocal laser-scanning microscopy. Human bronchial epithelial cells were evaluated with OMTC. The epithelial cell culture was either photo-treated or not, with red laser (lambda=660 nm), and fixed power and time (power density of 153 mW/cm2, time 300 s). It was not possible to observe significant differences between photo-treated and control epithelial cells, for the hysteresivity (ratio between the cell loss and elastic shear moduli). The defocusing microscopy, similar to a phase contrast microscopy, was used to study human red blood cells from fresh blood. The red blood cells were either photo-treated or not, with red laser (lambda=660 nm), and different powers and times (power densities from 0 to 510 mW/cm2, times from 0 to 180 s). Some morphological and mechanical characteristics of individual red blood cells were evaluated, such as volume, radial profile of cell thickness, lateral and vertical membrane fluctuations, for the photo-treated and control red blood cells. It was not possible to detect differences between the two groups, for any of the parameters analyzed. For both techniques, the absence of detectable differences might be due to several factors, such as the non-action of the PBMT, with the parameters used, in the epithelial cells and red blood cells or to the small sensitivity of each technique. Confocal laser-scanning microscopy was used to evaluate the actin filaments of mouse fibroblasts. The fibroblast cell culture was either photo-treated or not, with red (lambda=625 nm) or infrared (lambda=808 nm) light and fixed power and time (power density from 113 to 158 mW/cm2, time 300 s). The nucleus and cell areas increased slightly when comparing photo-treated and control cells. On the other hand, the total actin, total actin density and the number of filaments decreased. These changes were detected for a short time after treatment, however, after 24 h they are not anymore detectable. The total branch length does not seem to suffer any modifications. In summary, with the data acquired with the three techniques, it was found that the PBMT, in the red range, with the parameters used, could not cause noticeable changes in red blood cells and epithelial cells, in vitro. On the other hand, the PBMT in the red and near-infrared range, with the power and times used, cause changes in actin filaments of fibroblasts, in vitro, in particular the decrease of the total actin density. / A terapia por fotobiomodulação tem muitas aplicações na área de Saúde devido a sua ação anti-inflamatória e de reparação tecidual. O objetivo geral desse trabalho é verificar se a terapia por fotobiomodulação provoca mudanças nas propriedades mecânicas de células, em particular em hemácias, células epiteliais e fibroblastos. Além de contribuir com o conhecimento dos mecanismos de ação da terapia por fotobiomodulação, este estudo pretende subsidiar aplicações da terapia por fotobiomodulação durante procedimentos mais invasivos, como a iluminação direta do sangue em procedimentos cirúrgicos com circulação extracorpórea, sob o ponto de vista da segurança quanto à integridade celular. Para essa análise foram utilizadas três técnicas experimentais: citometria óptica magnética de oscilação (OMTC), microscopia de desfocalização e microscopia confocal. Com a técnica de OMTC foram avaliadas células epiteliais brônquicas humanas em cultura, foto-tratadas com laser vermelho (lambda=660 nm), com potência e tempo fixos (densidade de potência de 153 mW/cm2, tempo 300 s). Não foi possível constatar diferenças significativas entre as células epiteliais foto-tratadas e as células controle, para a histerisividade (razão entre os módulos viscoso e elástico das células). Com a técnica de microscopia de desfocalização, semelhante a uma microscopia de contraste de fase, foram estudadas hemácias humanas de sangue recém coletado. As hemácias foram tratadas com laser vermelho (lambda=660 nm), com potências e tempos variados (densidade de potência de 0 a 510 mW/cm2, tempo de 0 a 180 s). Foram avaliadas algumas características morfológicas e mecânicas das hemácias individualmente, como o volume, perfil radial de espessura, flutuações lateral e vertical da membrana, tanto para hemácias foto-tratadas quanto para hemácias controle. Não foi possível detectar diferenças entre as hemácias foto-tratadas e controle para nenhum dos parâmetros avaliados. Para ambas as técnicas, a falta de mudanças observáveis poderia ser devida a diversos fatores, como a não ação da terapia por fotobiomodulação nas células epiteliais e nas hemácias, com os parâmetros aqui empregados, ou à falta de sensibilidade de cada uma das técnicas usadas. A microscopia confocal foi utilizada para avaliar os filamentos de actina de fibroblastos de camundongo em cultura, os quais foram foto-tratados com luz vermelha (lambda=625 nm) ou infravermelha (lambda=808 nm) e potência e tempo fixos (densidade de potência de 113 a 158 mW/cm2, tempo 300 s). Foi possível constatar ligeiro aumento nas áreas nuclear e celular das células foto-tratadas em relação aos fibroblastos controle. Também foi possível verificar a diminuição da quantidade total de actina, densidade de actina e do número de filamentos de actina nos fibroblastos foto-tratados. Essas mudanças são detectadas para tempos curtos após o tratamento, sendo que depois de 24 h elas desaparecem. O tamanho total dos filamentos parece não sofrer alterações. A partir dos dados coletados com as três técnicas, foi possível constatar que a terapia por fotobiomodulação, com os parâmetros utilizados, não consegue provocar mudanças perceptíveis em hemácias e em células epiteliais, in vitro. Porém, causa mudanças nos filamentos de actina de fibroblastos, in vitro, em particular a diminuição da densidade de actina total.

The thesis presents a rational path for the optimization of the selection of optical treatment parameters in photobiomodulation of human skin fibroblasts. The project begins with an extensive analysis of 90 bibliographic reports in photobiomodulation published between 1985 and 2015, and revealed major inconsistencies in optical parameters selected for clinical applications. Seeking greater clarity for optimal parameter choice, a systematic approach to disentangle the multiple factors underpinning the response of human dermal fibroblasts in vitro to visible and near-infra red (NIR) light was employed. Light-based devices were constructed to specifically and systematically screen the optical parameter window (i.e. wavelength, irradiance and dose) observed in literature. Additionally, critical culture and treatment conditions that have dramatic impact on the outcome of specific light treatment of these human skin dermal cells were identified. In particular, environmental oxygen concentration, cell confluency and serum concentration were all found to have a great effect on the response of dermal fibroblasts to light. In parallel, the induction of reactive oxygen species (ROS) by short visible wavelengths on two dermal fibroblast sub-populations or lineage, reticular and papillary, was monitored by live-cell imaging. The ROS species were found to be created in or close to mitochondria. Lastly, gene expression studies revealed a strong impact of short visible wavelengths, as compared to long and NIR wavelengths on both subpopulations of human dermal fibroblasts. In particular, blue light (450 nm) specifically down-regulated proliferation, metabolism and protein synthesis molecular pathways. At the protein level, 450-nm light inhibited the production of procollagen I in human reticular and papillary fibroblasts in a dose-dependent manner. Gene expression results were in agreement i.e., the same light parameter down-regulated collagen fiber genes, integrins and up-regulated collagenase MMP1. This thesis concludes with a chapter presenting a characterization of the accuracy of a potential translation tool for the prediction of optical photon density inside human skin. / Marie Skłodowska-Curie Actions.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-06-05T19:10:49Z No. of bitstreams: 1 TeseEJA.pdf: 2933059 bytes, checksum: 5355b23e3d854a8e6867a11ec2458cde (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-06-05T20:04:57Z (GMT) No. of bitstreams: 1 TeseEJA.pdf: 2933059 bytes, checksum: 5355b23e3d854a8e6867a11ec2458cde (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-06-05T20:05:07Z (GMT) No. of bitstreams: 1 TeseEJA.pdf: 2933059 bytes, checksum: 5355b23e3d854a8e6867a11ec2458cde (MD5) / Made available in DSpace on 2017-06-05T20:10:17Z (GMT). No. of bitstreams: 1 TeseEJA.pdf: 2933059 bytes, checksum: 5355b23e3d854a8e6867a11ec2458cde (MD5) Previous issue date: 2017-02-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The effects of photobiomodulation by low level laser therapy (LLLT) on muscle tissue are well known. However, it is not found in the literature studies about the application of LLLT in the pelvic musculature under experimental disuse. The present thesis focused its analysis on three aspects: point out the anesthetic option which interferes the least in the metabolism of glucose, considering that muscle tissue is one of the main sites involved in glycemic regulation (study I), thus the results from this study could support the development of an non invasive pelvic immobilization model in rats (study II) and evaluate its effectiveness, through the chemo-metabolic response of the involved musculature before the muscular atrophy. Then we sought to evaluate if there is reversal of this muscle conditions (study III) by applying LLLT during and after the pelvic immobilization period. The three studies were performed with 80 male rats divided into different experimental groups. For the statistical evaluation, in study I the data were submitted to the Shapiro-Wilk normality test, followed by the ANOVA and Tukey test. In studies II and III the data were submitted to the Kolmogorov-Smirnov normality test followed by ANOVA and Tukey's test (a critical level of p <0.05 was stablished). In study we used 32 Wistar rats (n = 4), aged between 3 and 4 months, weighing 250±50g, distributed in eight groups, of which 4 groups were submitted to the glucose tolerance test (GTT) and another 4 submitted to the insulin tolerance test (ITT). We compared four anesthetics: Thiopental sodium (T, 40 mg/kg), Zolazepan (Z, 50 mg/kg), Ketamine-Xylazine (K, 35 mg/kg and 4 mg/kg) and Tribromoethanol (TRI, 250mg/kg). We concluded that Thiopental and Tribromoethanol anesthetics are the safest because they do not interfere in the glycemic control dynamics. In the study II, we distributed 18 rats were in three groups (n = 6): control (C), pelvic immobilization for 7 days (PI) and remobilized for 7 days after 7 days of device use (RP7). We found a difference (p <0.05) between groups: pelvic muscle glycogen reserves were reduced by 68% in the PI group and 50% in the RP7 group. The group PI had a delay of 10% in the glucose decay constant (KITT). Considering the total protein/DNA ratio groups PI and RP7 presented a reduction of 28% and 24% in the gluteus maximus and 32% and 16% in the iliopsoas, respectively. In the evaluation of the serum concentration of interleukin 6, the group PI had an increase of 27.3% and RP7 19.2% compared to the C. Interleukin 10 increased 30.4% and 14.9% in the IP and RP7 groups, respectively. Tumor necrosis factor (TNF-α) presented a discrete but significant increase of 5% only in the PI group compared to group C. Therefore, the experimental model proved to be effective in mimicking the deleterious effects of disuse, and this effects were not recovered after removing the immobilization device. Based on this findings, study III used 30 rats distributed in five groups (n=6), being; Control (C), Pelvic Immobilization (PI), Immobilized 7 days and remobilized for 7 days (IR), Laser during pelvic immobilization (LDPI) and Laser after pelvic immobilization (LAPI). Thus, the LLLT (Ga-Al-As, continuous λ = 830 nm, P = 30 mW, beam area = 0.116cm², T = 14s, fluency of 3,62J/cm², E = 0.42J; power density = 0.26mW/cm²) was applied on 8 points for 7 consecutive days. This study showed that, considering pelvic immobilization in the experimental condition, LLLT can minimize the catabolism by promoting an increase of glycogen reserves in the involved muscles, improving the total protein/DNA ratio, reducing pro-inflamatory activity (IL-6 and TNF- α) and increasing the anti-inflammatory activity (IL-10), especially when the LLLT was applied during the period of immobilization, providing that skeletal muscles maintain better metabolic and structural conditions even under disuse conditions. Finally, this work was successful in elucidating thiopental and tribromoethanol anesthetics as the ones that least interfere with glucose metabolism, so that this favored the development of an experimental model of disuse with marked muscular catabolism, which can be attenuated by means of photobiomodulation (830 nm). / Os efeitos da fotobiomodulação por terapia laser de baixa intensidade (LLLT) no tecido muscular são bem conhecidos. No entanto, não se encontra na literatura estudos que tenham verificado a aplicação de LLLT na musculatura pélvica sob desuso em caráter experimental. A presente tese concentrou suas análises em três vertentes: Apontar a opção anestésica que menos interfere no metabolismo da glicose, tendo em vista que o tecido muscular é um dos principais sítios envolvidos na regulação glicêmica (estudo I), de modo que os resultados provenientes desse estudo pudessem favorecer o desenvolvimento de um modelo experimental aplicado na imobilização da cintura pélvica de ratos (estudo II) e avaliar sua efetividade, por meio da resposta quimiometabólica, da musculatura envolvida e diante do quadro de atrofia muscular, buscou-se então avaliar se há reversão (estudo III) por meio da aplicação da LLLT durante e após o período de imobilização pélvica. Os três estudos foram realizados com 80 ratos machos da linhagem Wistar (Rathus novergicus var, albinus, Rodentia, Mamalia), com idade de 3 a 4 meses e massa corporal 250±50 g, distribuídos em diferentes grupos experimentais. Para a avaliação estatística, no estudo I os dados foram submetidos ao teste de normalidade de Shapiro-Wilk, seguido da aplicação da análise de variância ANOVA e teste de Tukey. Nos estudos II e III os dados foram submetidos ao teste de normalidade Kolmogorov-Smirnov seguido de ANOVA e teste de Tukey. Em todos os cálculos foi fixado um nível crítico de p<0,05. O estudo I foi realizado com 32 ratos Wistar, idade entre 3 a 4 meses, massa corporal 250±50g, que foram distribuídos em oito grupos (n=4), dos quais 4 grupos foram submetidos ao teste de tolerância a glicose (GTT) e outros 4 submetidos ao teste de tolerância a insulina (ITT). Os anestésicos utilizados foram: Tiopental sódico (T, 40 mg/kg peso,ip), Zolazepan (Z, 50 mg/kg peso, ip), Ketamina-Xilasina (KX, 35 mg/kg peso e 4 mg/kg peso, ip) e Tribromoetanol (TRI, 250mg/kg peso, ip). Concluiu-se que os anestésicos tiopental e tribromoetanol são os mais seguros por não interferirem nas dinâmicas de controle glicêmico. No estudo II foram utilizados 18 ratos distribuídos em três grupos (n=6): controle (C), imobilização pélvica por 7 dias (IP) e remobilizado por 7 dias após 7 dias de uso do dispositivo (RP7). Constatou-se que houve diferença (p<0,05) entre os grupos, onde as reservas glicogênicas da musculatura pélvica foram reduzidas em média 68% no grupo IP e 50% no grupo (RP7). A constante de decaimento (KITT) revelou retardo de 10% no grupo IP. A razão proteína total/DNA mostrou que houve redução de 28% e 24% no glúteo máximo e 32% e 16% no iliopsoas dos grupos IP e RP7, respectivamente. Na avaliação da concentração sérica de interleucina 6 houve elevação de 27,3% no grupo IP e 19,2% no grupo RP7 comparados ao C. A interleucina 10 expressou elevação de 30,4% e 14,9% nos grupos IP e RP7, respectivamente. O fator de necrose tumoral (TNF-α) apresentou discreta, porém significativa elevação de 5% somente no grupo IP comparado ao grupo C. Portanto, o modelo desenvolvido mostrou-se eficaz em mimetizar os efeitos deletérios do desuso, efeitos esses que não foram reestabelecidos após a remoção do modelo. Com base nos achados supracitados, o estudo III utilizou 30 ratos distribuídos em cinco grupos (n=6), sendo; Controle (C), Imobilização pélvica (IP), Imobilizado 7 dias e remobilizado por 7 dias (IR), Laser durante imobilização pélvica (LDIP) e Laser após imobilização pélvica (LAIP). Assim, a LLLT (Ga-Al-As, contínuo; λ = 830 nm; P= 30 mW; área do feixe = 0,116cm2, T= 14 s; fluência de 3,62J/cm; E= 0,42J; Densidade de potência de 0,25 mW/cm2) foi aplicada em 8 pontos por 7 dias consecutivos. Esse estudo revelou que frente a imobilização pélvica na condição experimental, a LLLT pode minimizar o catabolismo ao favorecer a elevação nas reservas glicogênicas da musculatura pélvica, melhorar a relação proteína total/DNA, reduzir a (IL-6 e TNF-α) atividade pró-inflamatória e aumentar (IL-10) a atividade anti-inflamatória, principalmente quando a LLLT foi aplicada durante o período de imobilização, propiciando que o músculo esquelético mantenha melhores condições metabólicas e estruturais mesmo sendo submetido ao desuso. Por fim, esse trabalho teve êxito em elucidar os anestésicos tiopental e tribromoetanol como os que menos interferem no metabolismo da glicose, de modo que isso favoreceu o desenvolvimento de um modelo experimental de desuso com acentuado catabolismo muscular, que pode ser atenuado por meio da fotobiomodulação (830 nm).

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-06-15T14:59:20Z No. of bitstreams: 2 Dissertação - Jessyca Luana Melo Costa Santos - 2018.pdf: 988766 bytes, checksum: e26f66c4c01ec695f377458e0e9daf89 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-15T15:00:58Z (GMT) No. of bitstreams: 2 Dissertação - Jessyca Luana Melo Costa Santos - 2018.pdf: 988766 bytes, checksum: e26f66c4c01ec695f377458e0e9daf89 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-15T15:00:59Z (GMT). No. of bitstreams: 2 Dissertação - Jessyca Luana Melo Costa Santos - 2018.pdf: 988766 bytes, checksum: e26f66c4c01ec695f377458e0e9daf89 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-05-03 / The light is used for treatment from the earliest days of civilization, considered as one of the oldest therapeutic modalities. Today, there are modern devices such as LEDs and lasers, which generate low-intensity light capable of interacting with biological tissues, triggering cellular responses and increased cellular metabolism necessary for the tissue repair process. The objective of this study was to evaluate the viability and activity of fibroblasts after irradiation with red LED 630nm in different potencies and fixed time. For the experiment, mouse fibroblast, L929 strain in RPMI medium supplemented with 10% fetal bovine serum, L-glutamine, Hepes, 2Mercaptoethanol, streptomycin and penicillin were cultured. After adequate confluence of the cells, the irradiation with fixed time of ten seconds was initiated, the power being varied according to each group: G50, power of 50mW; G75, 75 mW and G100, 100 mW and Control group (GC), without irradiation. The procedure was performed in triplicate. The cells were submitted to the MTT cytotoxicity test, evaluation of nitric oxide production (NO) and evaluation of collagen synthesis. Values were checked for normal distribution and, after failing to meet the assumptions (Kolmogorov-Smirnov, P <0.05), were analyzed by non-parametric Kruskal-Wallis and Mann-Whitney tests at a significance level of 5%. In the evaluation of cytotoxicity there was no significant difference between the control group, G50, G75 and G100. The results of nitrite production by fibroblasts show that there was a significant difference (P <0.05) between the control group and the experimental groups (G50, G75 and G100). Regarding the production of collagen, there was a significant difference (p ˂ 0.05) between the control group and the irradiated groups, but not between (G50, 752 and G100). In this study it was concluded that the irradiation of the L929 fibroblast culture by red led 630nm, at 50mW, 75mW and 100mW potentials for ten seconds, does not cause cell death, stimulates the production of nitric oxide and collagen. / Resumo: A luz vem é usada para tratamento desde os primórdios da civilização, considerada como uma das mais antigas modalidades terapêuticas. Hoje, existem modernos aparelhos como os LEDs e os lasers, que geram luz de baixa intensidade capazes de interagir com os tecidos biológicos desencadeando respostas celulares e aumento do metabolismo celular, necessárias para processo de reparação tecidual. O objetivo deste estudo foi avaliar a viabilidade e atividade de fibroblastos após a irradiação com LED vermelho 630nm em diferentes potências e tempo fixo. Para o experimento foi feito o cultivo de fibroblastos de camundongo, linhagem L929 no meio RPMI suplementado com 10% de soro fetal bovino, L-glutamina, Hepes, 2Mercaptoetanol, estreptomicina e penicilina. Após a confluência adequada das células, iniciou-se a irradiação com tempo fixo de dez segundos, variando-se a potência de acordo com cada grupo: G50, potência de 50mW; G75, 75 mW e G100, 100mW e Grupo controle (GC), sem irradiação. O procedimento foi realizado em triplicata. A células foram submetidas ao teste de viabilidade celular MTT, à avaliação da produção de Óxido Nítrico (NO) e à avaliação da síntese de colágeno. Os valores foram verificados e analisados utilizando ANOVA ao nível de significância de 5%. Na avaliação da citotoxicidade não houve diferença significativa entre o grupo controle, G50, 75 e G100. Os resultados da produção de nitrito pelos fibroblastos demonstram que houve diferença significativa (P < 0,05) entre o grupo controle, e os grupos experimentais (G50, 752 e G100). Em relação à produção de colágeno, houve diferença significativa (p ˂ 0,05) entre o grupo controle e os grupos irradiados, mas não entre o (G50, G75 e G100). Neste trabalho concluiu-se que a irradiação da cultura de fibroblastos L929 por led vermelho 630nm, nas potências de 50mW, 75mW e 100mW durante dez segundos, não ocasiona morte celular, estimula a produção de óxido nítrico e colágeno.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-08-16T13:19:25Z No. of bitstreams: 2 Dissertação - Ítalo Dany Cavalcante Galo - 2018.pdf: 2927565 bytes, checksum: 078e47430d5f7162d86e682c8a01e08b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-17T11:19:44Z (GMT) No. of bitstreams: 2 Dissertação - Ítalo Dany Cavalcante Galo - 2018.pdf: 2927565 bytes, checksum: 078e47430d5f7162d86e682c8a01e08b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-17T11:19:44Z (GMT). No. of bitstreams: 2 Dissertação - Ítalo Dany Cavalcante Galo - 2018.pdf: 2927565 bytes, checksum: 078e47430d5f7162d86e682c8a01e08b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-07-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Bacterial infections increase significantly the rate of morbidity and mortality in the hospital setting, which is substantially aggravated by the presence of bacterial resistance. For this reason, research that brings therapeutic alternatives that are efficient, cheap and safe in the fight against different infectious agents are necessary. One potential therapy is antimicrobial phototherapy, especially represented by blue light, which shows promise but still requires study and adjustments as to the best way of application. Thus, the present study aimed to verify the antibacterial action of blue light using different experimental protocols in order to clarify contradictions reported in the literature on the subject and to determine a reproducible protocol for the study of bacterial resistance. In vitro cultures of S. aureus, E. coli, P. aeruginosa, K. pneumoniae, S. epidermidis, E. faecalis and C. albicans were performed specifically for each test of light irradiation by blue ( 470 nm) or red (660 nm) light LED emitters. Different variations of light application were performed under two conditions: after inoculation and before inoculation in agar. In addition, preliminary testing was performed to verify the possible development of bacterial resistance to blue light. All of the in vitro assays evidenced important bacterial growth inhibition, showing that the possible inherent biases found in the literature can be eliminated with the adoption of specific protocol measures and that, in fact, the blue light inhibits the growth of these infectious agents. It has been demonstrated that, in all cases, the use high application times (and hence doses) of blue light is required to have better inhibition. The resistance test performed suggests higher sensitivity of S. aureus in relation to P. aeruginosa and E. coli, showing evidence that E. coli tends to become more resistant to successive applications of this photoemission. The present study evidences that the blue light has antibacterial action in all the in vitro protocols tested, being indicated the use of light application with the infectious agent in liquid medium rich in nutrients as a way of optimizing subsequent tests of greater complexity. The test of resistance to the blue light adopted evidences more tendency to resistance by the bacterium Escherichia coli in relation to other species tested. / Infecções bacterianas aumentam significativamente a morbidade e mortalidade no âmbito hospitalar, quadro que se agrava substancialmente na presença da resistência bacteriana. Por esta razão, pesquisas que tragam alternativas terapêuticas que sejam eficientes, baratas e seguras no combate a diferentes agentes infecciosos são necessárias. Uma terapia em potencial é a fototerapia antimicrobiana, representada especialmente pela luz azul, a qual se mostra promissora, mas, ainda demanda de estudo e ajustes quanto a melhor maneira de aplicação. Assim, o presente estudo teve como objetivo verificar a ação antibacteriana da luz azul utilizando diferentes protocolos experimentais a fim de esclarecer contradições reportadas na literatura sobre o tema e determinar um protocolo reprodutível para estudo de resistência bacteriana. Foram realizadas culturas in vitro de S. aureus, E. coli, P. aeruginosa, K. pneumoniae, S. epidermidis, E. faecalis e C. albicans de forma específica para cada teste de irradiação luminosa obtida por meio de emissores LED de luz azul (470 nm) ou vermelha (660 nm). Foram realizadas diferentes variações de aplicação da luz em duas condições: após semeadura e anterior à semeadura em ágar. Além disso, foi feito teste preliminar para verificação de possível desenvolvimento de resistência bacteriana à luz azul. Todas as análises in vitro adotadas evidenciam importante inibição bacteriana, mostrando que os possíveis viesses inerentes a trabalhos encontrados na literatura podem ser eliminados com a adoção de medidas protocolares específicas e que, de fato, a luz azul inibe o crescimento destes agentes infecciosos. Foi demonstrado que, em todos os casos, é necessário o uso de elevados tempos de aplicação (e, consequentemente, doses) de luz azul para se ter melhor inibição. O teste de resistência realizado sugere maior sensibilidade da S. aureus em relação a P. aeruginosa e E. coli, e mostra evidências de que a E. coli tende a se tornar mais resistente à aplicações sucessivas desta fotoemissão. O presente estudo evidencia que a luz azul possui ação antibacteriana em todos os protocolos in vitro testados, sendo indicado o uso de aplicação da luz com o agente infeccioso em meio líquido rico em nutrientes como forma de otimizar testes de maior complexidade subsequentes. O teste de resistência à luz azul evidencia maior tendência à resistência por parte da bactéria Escherichia coli em relação a outras espécies testadas.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-07-17T21:50:50Z No. of bitstreams: 1 Solange Almeida dos Santos.pdf: 759875 bytes, checksum: f16dbd72a00c1e89bdc4543f27aacd3f (MD5) / Made available in DSpace on 2018-07-17T21:50:50Z (GMT). No. of bitstreams: 1 Solange Almeida dos Santos.pdf: 759875 bytes, checksum: f16dbd72a00c1e89bdc4543f27aacd3f (MD5) Previous issue date: 2016-12-19 / Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by chronic and systemic inflammation, which leads to destruction of the cartilage and bone, and affects tissues in multiple joints. Oxidative stress has been implicated in involvement in various disease conditions, such as rheumatoid arthritis (RA). In vivo experimental studies using photobiomodulation therapy (FBM) have shown positive effects in reducing lipid peroxidation and increasing antioxidant activity. The regular practice of physical exercise has also been reported as a beneficial treatment capable of reducing oxidative damage. The aim of this research was to analyze the effect of photobiomodulation therapy at 2 joules and 4 joules doses associated with physical exercise on oxidative stress in an experimental model of rheumatoid arthritis in protein expression: superoxide dismutase (SOD); Glutathione Peroxidase (GPX) and Catalase (CAT) on thiobarbituric acid reactive substances (TBARS). Twenty-four male Wistar rats divided into 4 groups were submitted to an AR model (CIA). First immunization were performed at the base of the tail on the days 0 and 07, and after 28 days the third dose was administered intra-articular in both knees of the animals. After the last induction, FBM therapy was started immediately, transcutaneously at two points: medial and lateral, with a total of 15 applications. Treadmill exercise started the day after the last induction and lasted 5 weeks. As results we obtained the decrease of the lipid peroxidation and the increase of antioxidant activities of SOD, GPX and CAT with physical exercise associated to FBM in doses of 2 joules and 4 joules. Conclusion: Physical exercise associated with FBM therapy decreases lipid peroxidation and increases antioxidant activity. / A artrite reumatóide (AR) é uma doença inflamatória crônica redicivante caracterizada por uma inflamação crônica e sistêmica. O estresse oxidativo tem sido referido no envolvimento em várias condições de doenças, como artrite reumatóide (AR). Estudos experimentais in vivo, utilizando a terapia de fotobiomodulação têm demonstrado efeitos positivos na diminuição da peroxidação lipídica, e no aumento das atividades antioxidantes. A prática regular de exercício físico também vem sendo relatada como um tratamento benéfico capaz de diminuir os danos oxidativos. Sendo assim, esta pesquisa tem por objetivo analisar os efeitos da terapia de fotobiomodualçao nas doses 2 joules e 4 joules associado ao exercício físico sobre marcadores de estresse oxidativo em modelo experimental de artrite reumatóide. Foram analisadas expressão proteica: superóxido dismutase (SOD); e Glutationa Peroxidase (GPX) e Catalase (CAT), sobre as substâncias reativas ao ácido tiobarbitúrico (TBARS). 24 ratos machos wistar divididos em 4 grupos foram submetidos a um modelo de AR (CIA), 1ª imunização realizada na base da cauda nos dias 0, 07, e após 28 dias foi administrada 3ª dose intra-articular em ambos joelhos dos animais. Após última indução a terapia de fotobiomodulação foi iniciada imediatamente, por via transcutânea em dois pontos: medial e lateral, as aplicações seguintes aconteceram em dias alternados, totalizando 15 aplicações. O exercício na esteira começou no dia subsequente a última indução e teve duração de 5 semanas. Como resultados obtivemos a diminuição da peroxidação lipídica e aumento das atividades antioxidantes da SOD, GPX e CAT com exercício físico associado a terapia de fptpbiomodulação nas doses de 2 joules e 4 joules. Conclusão: O exercício físico associado a terapia de fotobiomodulação diminui peroxidação lipídica e aumenta atividades antioxidantes.

Yes / Background and Objective: Though devices for hair growth based on low levels of light have shown encouraging results, further improvements of their efficacy is impeded by a lack of knowledge on the exact molecular targets that mediate physiological response in skin and hair follicle. The aim of this study was to investigate the expression of selected light-sensitive receptors in the human hair follicle and to study the impact of UV-free blue light on hair growth ex vivo. Material and Methods: The expression of Opsin receptors in human skin and hair follicles has been characterised using RT-qPCR and immunofluorescence approaches. The functional significance of Opsin 3 was assessed by silencing its expression in the hair follicle cells followed by a transcriptomic profiling. Proprietary LED-based devices emitting two discrete visible wavelengths were used to access the effects of selected optical parameters on hair growth ex vivo and outer root sheath cells in vitro. Results: The expression of OPN2 (Rhodopsin) and OPN3 (Panopsin, Encephalopsin) was detected in the distinct compartments of skin and anagen hair follicle. Treatment with 3.2 J/cm2 of blue light with 453 nm central wavelength significantly prolonged anagen phase in hair follicles ex vivo that was correlated with sustained proliferation in the light-treated samples. In contrast, hair follicle treatment with 3.2 J/cm2 of 689 nm light (red light) did not significantly affect hair growth ex vivo. Silencing of OPN3 in the hair follicle outer root sheath cells resulted in the altered expression of genes involved in the control of proliferation and apoptosis, and abrogated stimulatory effects of blue light (3.2 J/cm2; 453 nm) on proliferation in the outer root sheath cells. Conclusions: We provide the first evidence that 1) OPN2 and OPN3 are expressed in human hair follicle, and 2) 453 nm blue light at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. / This study was supported by the European Marie-Curie Actions Programme, Grant agreement no.: 607886

Yes / Background and Objective Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light‐based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV‐blue light can activate Opsin 1‐SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. Materials and Methods Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro‐dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT‐PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch‐wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. Results Opsin receptors (OPN1‐SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. Conclusions Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light‐based therapies for cutaneous wound healing. / European Commission 7th Framework Programme for Research and Technical Development - Marie Curie Innovative Training Networks (ITN), Grant agreement no.: 607886