Neste trabalho tentamos traçar quais seriam os efeitos funcionais e de expressão gênica de mutações patogênicas no gene TCOF1 em células não embrionárias. A partir do estabelecimento de culturas celulares oriundas de periósteo facial de quatro pacientes portadores da síndrome de Treacher Collins (STC), obtivemos populações celulares com mais de 95% de marcação positiva para antígenos que caracterizam células de origem mesenquimal. Demonstramos que as células-tronco mesenquimais e fibroblastos provenientes de pacientes com STC apresentaram redução da expressão de TCOF1 de aproximadamente 31% quando comparados aos controles. Tal redução se mostrou compatível com a diminuição da expressão dos transcritos portadores de mutação patogênica observada no seqüenciamento do cDNA dos pacientes. Portanto, estes resultados sugerem que há degradação do transcrito mutado, que muito possivelmente deve ser regulada pelo mecanismo de non-sense mediated mRNA decay (NMD) e corroboram o modelo de haploinsuficiência da Treacle. Como o pico de expressão do gene Tcof1 é detectado durante o estágio embrionário de formação das células da crista neural e embriões de camundongos Tcof1+/- apresentam redução da proliferação das células neuroepiteliais, testamos se haveria alteração da capacidade proliferativa de células mesenquimais adultas com mutações no TCOF1. A análise das taxas de crescimento das culturas celulares provenientes de pacientes com STC se mostrou semelhante aos controles normais, indicando que menores níveis de transcritos do gene não interferem na capacidade proliferativa celular. Além disso, avaliamos o potencial de diferenciação óssea in vitro, mostrando que menores níveis de TCOF1 também não parecem influenciar a capacidade de diferenciação celular. Apesar de ainda não termos identificado um fenótipo celular, a deficiência de TCOF1 em células não embrionárias resulta na alteração da expressão gênica, já que a análise da expressão genômica das culturas celulares identificou uma série de genes diferencialmente expressos. Ademais, genes de parada do ciclo celular e apoptose com expressão aumentada em células embrionárias de camundongo Tcof1+/- não apresentaram expressão aumentada nas células humanas com mutações patogênicas no TCOF1, sugerindo que a via da p53 não está ativa nestas células não embrionárias. Dentre os genes diferencialmente expressos encontrados, o aumento dos transcritos de DAXX nas células-tronco mesenquimais com mutações patogênicas no TCOF1 poderia modular as funções da p53 nessas células, constituindo um bom alvo de investigações futuras para a elucidação da função do TCOF1 em células não embrionárias. Os resultados obtidos sugerem que deficiência de TCOF1 em células mesenquimais leva à ativação de outras vias de sinalização, cujo efeito funcional é ainda desconhecido. Quais seriam as funções e em quais vias celulares o TCOF1 agiria, são questões a serem elucidadas a respeito do papel do gene na fase adulta. / In this work we tried to outline the effects of functional and gene expression of pathogenic mutations in the gene TCOF1 in non-embryonic cells. From the establishment of facial periosteum derived cell culture of four patients with Treacher Collins syndrome (TCS), we obtained cell populations that are more than 95% positive for mesenchymal origin characteristic antigens. We demonstrated that mesenchymal stem cells and fibroblasts from patients with TCS showed reduction of approximately 31% in the TCOF1 expression when compared to controls. This reduction was consistent with the decrease in the expression of transcripts carrying the pathogenic mutations observed when sequencing the cDNA of the patients. Therefore, these results suggest that degradation of the mutated transcript occurs and it may possibly be governed by the mechanism of non-sense mediated mRNA decay (NMD), thus corroborating the Treacle haploinsufficiency model. As the peak of TCOF1 gene expression is detected during the embryonic stage of the formation of neural crest cells and Tcof1+/- mice embryos had shown reduction of neuroepithelium cells proliferation rate, we tested if there is a change in proliferative capacity of adult mesenchymal cells with mutations in TCOF1. The analysis of the growth rate of TCS patients cells was similar to normal controls, indicating that lower levels of transcripts of the gene does not interfere with cellular proliferative capacity. Furthermore, we evaluated the bone differentiation potential of these cells in vitro, showing that lower levels of TCOF1 also seem does not influence cell differentiation ability. Although a cellular phenotype has not yet been identified, deficiency of TCOF1 in non-embryonic cells results in gene expression change, since genomic analysis of the expression of cell cultures identified a number of differentially expressed genes. Furthermore, arresting cell cycle and apoptosis related genes with increased expression in embryonic cells of Tcof1+/- mice showed no increased expression in human cells with pathogenic mutations in TCOF1 suggesting that the p53 pathway is not active in these non-embryonic cells. Among the differentially expressed genes found, the increase of DAXX transcripts in mesenchymal stem cells with pathogenic mutations in TCOF1 could modulate the function of p53 in these cells, providing a good target for future investigations to elucidate the function of TCOF1 in non-embryonic cells. The results suggest that deficiency of TCOF1 in mesenchymal cells leads to activation of other signaling pathways, whose functional effects are still unknown. The functions and cellular pathways in which the TCOF1 act, are issues yet to be elucidated concerning the role of the gene during adulthood.

O conhecimento do potencial citotóxico, das reações histológicas e propriedades clínicas é imprescindível para a escolha do material na terapia pulpar de dentes decíduos. O estudo teve como objetivo avaliar o efeito de fármacos desta terapia quanto à citotoxicidade e distribuição in vitro de proteínas da matriz extracelular e do citoesqueleto de fibroblastos da polpa humana. Os grupos foram: pasta Guedes- Pinto, pasta Óxido de Zinco e Eugenol (OZE), Vitapex®, Calen® e Calen PMCC®. Os extratos brutos dos fármacos foram testados na concentração 0,2g/ml de meio DMEM/F12 (ASTM, 1992), nas diluições 10, 100 e 1000x. A citotoxicidade foi analisada pela viabilidade (24hs) e sobrevivência celular (24, 48 e 72hs) que se baseou na atividade mitocondrial de fibroblastos da polpa humana (FP5) pelo método de redução do MTT. O grupo controle foi utilizado como 100% de células viáveis. Os resultados foram submetidos à análise de variância, e teste de Tukey como contraste. O efeito dos fármacos na distribuição in vitro de proteínas da matriz extracelular (fibronectina, tenascina, colágeno I) e de citoesqueleto (vimentina) nas FP5 também foi analisado por imunofluorescência. Os resultados demonstraram que na viabilidade celular, as pastas Guedes-Pinto, pasta OZE, e Calen® foram mais citotóxicas que o grupo controle somente na maior concentração (p<0,05), sendo que esse efeito perdurou para a pasta OZE nas menores concentrações em relação ao grupo controle (p<0,05). As diferenças de citotoxicidade entre os fármacos só foram observadas na maior concentração, onde a pasta Guedes-Pinto teve maior efeito tóxico que a pasta Calen®, Calen PMCC® e Vitapex® (p<0,05), mas similar à pasta OZE. Na análise da sobrevivência celular em 72hs, todos os grupos apresentaram mesma capacidade proliferativa que o grupo controle em 24 e 48hs (p>0,05). As diferenças entre os fármacos foram observadas ao final do tempo experimental quando a Pasta Guedes-Pinto não manteve a mesma capacidade de proliferação celular que o grupo controle na maior e menor concentração (p<0,05). Na imunofluorescência, não houve diferença entre os grupos para a distribuição de proteínas da matriz extracelular e citoesqueleto nas FP5. A vimentina, proteína do citoesqueleto de células mesenquimais, encontrou-se distribuída na forma de filamentos ao longo do citoplasma celular. A fibronectina obteve marcação positiva, formando uma rede reticular no citoplasma. A tenascina e colágeno I apareceram como pequenos pontos (vesículas) distribuídos homogeneamente no citoplasma e na região perinuclear. Concluiu-se que, todos os fármacos estudados foram biocompatíveis em relação à citotoxicidade e à distribuição in vitro de proteínas da matriz extracelular e citoesqueleto de fibroblastos da polpa humana. / When electing a material to be used in deciduous pulp therapy, it is essential to acquire knowledge regarding the materials potential toxicity, histological reactions and clinical properties. This study aims at analyzing the effect of different drugs used in pulp therapy in relation to their cytotoxicity and in vitro protein distribution of extracellular matrix and cytoskeleton from human dental pulp fibroblasts. The groups were: Guedes-Pinto Paste, Zinc Oxide and Eugenol paste (ZOE), Vitapex®, Calen® e Calen PMCC®. The materials were tested using the following concentration: 0.2g/mL of culture medium (DMEM/F12) (ASTM, 1992), diluted in 10x, 100x and 1000x. The cytotoxicity was evaluated by cellular viability (24hs) and survival (24, 48 and 72hs), which was based on mitochondrial activity (MTT reduction test) of human dental pulp fibroblasts (FP5). The control group was considered to have 100% of viable cells. Data were submitted to variance analysis, using Tukey test as contrast. The drugs effect on in vitro expression of extracellular matrix proteins (fibronectin, tenascin, type I collagen) and cytoskeleton (vimentin) from FP5 were also evaluated using immnunofluorescence. The results demonstrated that, concerning cellular viability, Guedes-Pinto paste, ZOE paste, and Calen® were more cytotoxic than the control group only in their highest concentration (p<0.05), an effect observed for ZOE paste in all dilutions (p<0.05). Differences regarding cytotoxicity between groups were only observed in the highest concentration where Guedes-Pinto Paste was more toxic than Calen®, Calen PMCC® e Vitapex® (p<0.05), but was similar to ZOE paste (p>0.05). Cellular survival analysis after 72 h showed that all groups presented a similar proliferative capacity compared to the control group at 24 and 48h (p>0.05). Differences were only observed in the end of experimental period (72hs), when Guedes-Pinto paste did not maintain the same proliferative capacity than the control group in its lowest and highest concentrations (p<0.05). In immnunofluorescence tests, there was no difference between all groups for extracellular matrix and cytoskeleton proteins distribution from FP5. Vimentin, a protein from the cytoskeleton of mesenchymal cells, was distributed as filaments throughout the cytoplasm. Fibronectin was positively marked, forming a reticular net in the cytoplasm. Tenascin and Collagen I appeared punctually (as vesicles) and were homogeneously distributed in the cytoplasm and peri-nuclear region. It was concluded that all studied drugs investigated were biocompatible regarding cytotoxicity and in vitro distribution of extracellular matrix proteins and cytoskeleton proteins from human dental pulp fibroblasts.

A homeostase da mama normal depende das interações entre células epiteliais e o estroma a elas associado. Estudos anteriores mostraram que no carcinoma mamário o estroma é constituído por células com diferentes funções. Estes elementos do estroma incluem fibroblastos, os quais modulam o comportamento tumoral, fornecendo fatores de crescimento e componentes de matriz extracelular. Nosso objetivo foi investigar a expressão gênica diferencial entre fibroblastos derivados de tecido mamário neoplásico ou não neoplásico e analisar a influência de células epiteliais normais (MCF10A) no perfil de expressão gênica de fibroblastos obtidos de tecido mamário neoplásico. Culturas primárias de fibroblastos foram estabelecidas e a expressão de vimentina e actina de músculo liso foi positiva. Foi realizada a co-cultura destas células com separação por insertos, o que permite a passagem de fatores solúveis, e o RNA foi extraído. Após a amplificação do RNAm foram sintetizadas sondas de cDNA, as quais foram marcadas com fluorocromos conjugados a deoxinucleotídeo, hibridizadas competitivamente sobre lâminas de vidro contendo 4.608 ORESTES criadas no Instituto Ludwig de Pesquisa sobre o Câncer/FAPESP e os sinais fluorescente gerados foram quantificados. Após a normalização destes dados, os genes diferencialmente expressos, com False Discovery Ratio (FDR) menor que 0,05, foram selecionados para análises posteriores. Encontramos 283 genes diferencialmente expressos em fibroblastos derivados de tecido mamário neoplásico quando comparados àqueles derivados de tecido mamário não-neoplásico. Dentre estes genes, 187 foram quantitativamente regulados negativamente (com variação de expressão de 1,05 a 4,14) contra 96 regulados positivamente (variação de expressão de 1,17 a 7,73). A maioria destas alterações foram relacionadas ao transporte entre membranas, transdução de sinal e biosíntese. Estes resultados podem sugerir uma redução na expressão gênica durante o processo de transformação. Após a co-cultura com células MCF10A, encontramos 566 genes diferencialmente expressos nos fibroblastos derivados de tecido mamário neoplásico, 323 foram regulados negativamente (expressão variando de 1,09 a 10,62) e 243 regulados positivamente (variação de expressão de 1,03 a 16,62). A influência das células MCF10A na expressão gênica destes fibroblastos pôde ser vista através da desregulação da expressão de genes relacionados com a proliferação celular, adesão, apoptose e sobrevivência. / The homeostasis of normal breast depends on interactions between epithelial cells and their associated stroma. Previous studies indicated that in breast cancer carcinoma, tumor associated stromal cells with different functions appear to be emerged. These stromal elements include fibroblasts which modulate tumor behavior providing various growth factors and extracellular matrix components. Our aim was to evaluate the differential gene expression between fibroblasts derived from mammary tissue neoplasic or not and to analyze the influence of normal epithelial cells (MCF10A) on gene expression profile of fibroblasts obtained from neoplasic mammary tissue. Fibroblast primary cultures were established and expression of vimentin and smooth cell actin was positive. Co-culture of these cell types separated by inserts, which allow the passage of soluble factors, was done and total RNA was extracted. After mRNA amplification using a template-switching prime, cDNA probes were synthesized, labeled with fluorochrome conjugated deoxynucleotide, a competitive hybridization was undertaken onto cDNA microarray glass slides in which 4,608 ORESTES (open reading frame expressed sequence tags) from Instituto Ludwig de Pesquisa sobre o Câncer/FAPESP bank were spotted and fluorescent signals were quantified. After normalization, the differentially expressed genes, at a False Discovery Ratio (FDR) less then 0.05, were selected for further analysis. We found 283 differentially expressed genes in fibroblasts obtained from neoplasic mammary tissue when compared with non neoplasic derived fibroblasts. Among these genes, 187 were quantitatively down regulated (fold ranging from 1.05 to 4.14) against 96 up regulated (fold ranging from 1.17 to 7.73). The majority of alterations were related to membrane transport, signaling transduction and biosynthesis. Overall these results could suggest a reduced gene expression along transformation process After coculture with MCF10A cells, we found 566 differentially expressed genes in neoplasic mammary tissue derived fibroblasts, 323 were down regulated (fold ranging from 1.09 to 10.62) and 243 up regulated (fold ranging from 1.03 to 16.62). MCF10A influence in mammary tissue neoplasic derived fibroblasts gene expression could be seen trough the deregulation of expression of some genes possibly related cell proliferation, adhesion, apoptosis and survival.

O quelóide é um tumor fibroso benigno que ocorre durante a cicatrização da pele em indivíduos geneticamente predispostos. A cicatrização é um processo biológico complexo e depende da interação de diferentes estruturas teciduais e de um grande número de tipos celulares residentes e infiltrativos, que produzem citocinas. A interleucina 8 (IL-8), citocina pró-inflamatória, é super-expressa pelos fibroblastos durante o desenvolvimento do tecido de granulação, acelerando o processo de cicatrização. Como o quelóide resulta de uma reparação tecidual anormal após lesão da pele, o presente estudo teve por objetivo determinar a expressão dos receptores da IL-8, CXCR1 e CXCR2, e a capacidade proliferativa, pelo ciclo celular, dos fibroblastos queloideanos cultivados e extraídos ex vivo, por citometria de fluxo. Fibroblastos de cicatriz queloideana e de pele normal foram obtidos de 21 pacientes da raça negra, com idade variando entre 10 e 40 anos, de lesões com até 2 anos de evolução. Em nosso estudo constatamos expressão reduzida dos receptores para a IL-8, CXCR1(35,7%±11,2) e CXCR2 (27,8%±11,3), em fibroblastos de cicatriz queloideana cultivados, comparando com a pele normal (44,1±16,2 e 46,3±27,1 respectivamente). Entretanto, essa diferença só foi significante para o receptor CXCR2. A baixa expressão desses receptores poderia ser decorrente da atividade de metaloproteinases, que regulam a expressão de proteínas da superfície celular, através de clivagem enzimática, ou a capacidade reduzida de internalização e a reciclagem de receptores, mantida por filamentos de actina do citoesqueleto, que nos fibroblastos do quelóide estão diminuídos. Em relação ao ciclo celular de fibroblastos cultivados do quelóide e da pele normal, verificamos diferenças não significantes da capacidade de replicação (fase S do ciclo celular) e de apoptose. No quelóide observamos significante aumento de células na fase G2/M, indicando aumento da velocidade de divisão celular. Para confirmar esses achados estudamos o ciclo celular de fibroblastos extraídos ex vivo, da porção periférica e central do quelóide e da pele normal. Os fibroblastos da porção periférica apresentaram porcentagem significantemente maior de células com capacidade replicativa, fase S do ciclo (22,9% ± 11,6), em relação à porção central (4,7% ± 2,9) e à pele normal (6,8% ± 4,9), e maior velocidade de divisão celular, fase G2/M (18,6 ± 12,0), em relação à porção central (35,6 ± 7,0) e pele normal (32,3 ± 6,9). Verificamos que a porção central apresentou maior porcentagem de células em apoptose (7,0% ± 2,1), comparado à porção periférica (4,9% ± 1,9) e pele normal (2,0% ± 0,86). Esses dados indicam que as células da porção periférica do quelóide parecem ser responsáveis pela elevada taxa de proliferação, justificando o crescimento expansivo a partir das margens da cicatriz queloideana, com desenvolvimento de lesão semelhante a tumor, bem como a porção central ser responsável pela fibrose, contendo células quiescentes e apoptóticas. Esses resultados sugerem modulação diferencial das reações celulares através das vias de sinalização para proliferação ou morte celular programada. Neste sentido, a baixa expressão dos receptores da IL-8, CXCR1 e principalmente de CXCR2, nos fibroblastos do quelóide sugere capacidade reduzida da IL-8 em promover cicatrização acelerada. A baixa atividade da IL-8 sobre os fibroblastos queloideanos estaria promovendo desregulação da resposta inflamatória e com isso atraindo novas células inflamatórias para o local e produzindo sinais alterados, como grande produção da citocina TGF&#946;. Essa desregulação do processo de cicatrização, com alteração de citocinas e da matriz extracelular, poderia ser responsável pelas duas populações de fibroblastos, uma proliferativa na periferia e outra quiescente e apoptótica na porção central. Finalizando, podemos concluir que nossos resultados correspondem às alterações histológicas e clínicas do quelóide que se expande nos limites da lesão. / A keloid is a benign fibrous tumor that occurs during wound healing in genetically predisposed individuals. Healing is a complex biological process and depends on the interaction of different tissue structures and a great number of resident and infiltrative cell types. The interleukin-8 (IL-8), a proinflammatory chemokine, showed higher expression in fibroblasts during the development of the granulation tissue, promoting more rapid tissue maturation. Since keloids result from abnormal wound healing, the objective of the present study was to determine the expression of CXCR1 and CXCR2, IL-8 receptors, and the proliferation capacity, throughout the cell cycle, of the keloid fibroblasts extracted ex vivo and those submitted to in vitro cultivation. Normal skin and keloid scar fibroblasts were obtained from 21 African-Brazilian patients, aged from 10 to 40 years, whose lesions had evolved for no longer than 2 years. Expression of receptors and the cell cycle was assessed by flow cytometry. We showed lower expression of the CXCR1 (35,7% ± 11,2) and CXCR2 (27,8%±11,3) in keloid fibroblasts, when compared with normal skin (44,1 ± 16,2 e 46,3 ± 27,1 respectively), but the difference was not significant for the CXCR1 receptor. This lower expression of IL-8 receptors in keloid fibroblasts could be due to the action of metalloproteinases, which regulate the surface protein enzymatically, or fibroblastic cytoskeleton conditions, which influence receptor internalization and recycling. The distribution assessment of cell cycle phases of fibroblasts cultivated from keloid scars and normal skin did not show significant difference in replication capacity and apoptosis. The keloid fibroblasts presented a significantly higher proportion of cells in the G2/M phase, suggesting higher rate of cell division. To confirm these results we studied the cell cycle of fibroblasts extracted ex vivo, now separated by central and peripheral portions of keloid and normal skin. The peripheral fibroblasts showed significant high cell proportions in phase S (22,9% ± 11,6), compared with the central portion (4,7% ± 2,9) and normal skin (6,8% ± 4,9), and higher cells in division phase G2/M (18,6% ± 12,0), compared with the central portion (35,6% ± 7,0) and normal skin (32,3% ± 6,9). The central portion showed higher proportion of apoptosis (7,0% ± 2,1), compared with the peripheral portion (4,9% ± 1,9) and normal skin (2,0% ± 0,86). These results suggest that the keloid peripheral cells could be responsible for the proliferation rate, justifying the expansive keloid growth at the borders of the keloid scar, in a similar fashion to tumor development and the central portion being responsible for fibrosis, with quiescent and apoptotic cells. These results suggest a differentiated modulation of cell reactions by signal pathways for programmed cellular proliferation or death. In this sense, the low expression of the IL-8 receptors CXCR1 and CXCR2 in keloid fibroblasts suggests a diminished capacity of IL-8 to promote accelerated healing. This low expression of IL-8 receptors in keloid fibroblasts could promote the dysregulation of the inflammatory response and thus attract more inflammatory cells to the site, producing different signals, such as a high production of the TGF&#946; cytokine. This dysregulation of the healing process, with changed cytokine and extracellular matrix expression, could be responsible for two different cell populations of fibroblasts, one proliferation at the periphery and the other fibrotic at the center of the lesion, with apoptotic and quiescent cells. Finally, we conclude that our results correspond to the histological and clinical changes of keloids that grow beyond the wound boundaries.

Orientadores: Mary Ann Foglio, Ana Lúcia Tasca Gois Ruiz, João Ernesto de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:38:49Z (GMT). No. of bitstreams: 1 Zago_PatriciaMariaWiziack1982-_D.pdf: 5120519 bytes, checksum: a581f776ecdecf5822427a828d170cc1 (MD5) Previous issue date: 2014 / Resumo: A Osteonecrose Maxilar Induzida por Bisfosfonatos (OMIB) está associada à terapia com bisfosfonatos como o ácido zoledrônico (ZA), que são medicamentos importantes para o manejo de patologias ósseas. Estudos in vitro demostraram que os bisfosfonatos podem alterar a viabilidade e proliferação de células epiteliais, o que justificaria situações de deficiente cicatrização em feridas de OMIB. Arrabidaea chica (H&B.) Verlot é uma Bignoniaceae com propriedades cicatrizantes, e que apresenta as antocianidinas como principais compostos identificados. Este estudo avaliou a atividade antimicrobiana, citotóxica e citoprotetora, in vitro, do extrato hidroalcoólico de A. chica (AC) na presença de ZA. Foi realizado estudo de estabilidade nas condições de 40 °C e 75% umidade, do extrato microencapsulado em mistura de goma arábica com maltodextrina (1:1) e do extrato livre correspondente. Queratinócitos (HaCaT), fibroblastos (PT64-04HF) e osteoblastos (MC3T3-E1) foram submetidos a 2 protocolos de tratamento: a exposição a ZA 10 µM por 24h e posteriormente AC por 48h e vice-versa; ou o co-tratamento de ambos. Os parâmetros estudados foram viabilidade, ocorrência de apoptose e ativação de caspases 3/7. A viabilidade de células de câncer de próstata (C4-2B) foi avaliada após tratamento com AC. Streptococcus mutans foram submetidos ao co-tratamento ZA e AC. Microcápsulas contendo AC foram produzidas por processamento em spray dryer, aliquotadas e armazenadas em câmara climática para o estudo de estabilidade acelerada, segundo protocolo da ANVISA (RE 01/2005). Os resultados foram submetidos à análise estatística (ANOVA e Teste T de Student, a=0,05). Após 48 h, ZA reduziu a viabilidade de queratinócitos, fibroblastos e osteoblastos em, respectivamente, 34%, 47% e 51%. O co-tratamento ZA e AC 5 µg mL-1 aumentou a viabilidade celular (82%, 83% e 55%), o mesmo ocorrendo para ZA e AC 10 µg mL-1 (69%, 70% e 64%). Após 48 h de tratamento com ZA, não houve apoptose celular ou ativação de caspases em fibroblastos, mas verificou-se a ativação de caspase 3/7 em osteoblastos. As concentrações de 5 e 10 µg mL-1 de AC não alteraram a viabilidade de células C4-2B, porém observou-se uma redução de mais de 40% em concentrações maiores que 10 µg mL-1. O crescimento de S. mutans foi aparentemente aumentado após o tratamento com ZA em concentrações maiores que 50 µM, sem que AC ocasionasse qualquer efeito. Finalmente, o material formador de parede (mistura de goma arábica com maltodextrina 1:1) empregado para microencapsular AC, foi incapaz de inibir a degradação das antocinidinas, cujas concentrações relativas foram reduzidas após 15 dias de incubação. Assim, baixas concentrações de AC (5 e 10 µg mL-1) demostraram promissora atividade citoprotetora contra os efeitos prejudiciais de ZA, em células epiteliais e osteoblastos, sem estimularem o crescimento das células cancerígenas (C4-2B) e de S. mutans. Outros materiais formadores de parede serão avaliados, buscando-se aumentar a estabilidade química de AC. Mais estudos são necessários para a comprovação dos efeitos benéficos de AC como um auxiliar no tratamento de OMIB / Abstract: The bisphosphonate related osteonecrosis of the jaw (BRONJ) is a condition associated to bisphosphonates, medicines used to treat bone disorders, as zoledronic acid. In vitro studies showed epithelial cells viability and growth can be altered by bisphosphonates, and may cause a deficient wound healing in BRONJ. Arrabidaea chica (H&B.) Verlot is a Bignoniaceae with wound healing properties, however, anthocyanidins, the main compounds of the plant extract, can be easily degraded under environmental conditions. This in vitro study aimed to evaluate the antimicrobial, citotoxic and citoprotective actions of A. chica hydroalcoholic extract (AC) in the presence of ZA therapy. Stability studies at 40 °C and 75% humidity conditions, from microencapsulated into arabic gum plus maltodextrin (1:1) and free crude extract samples were conducted. Keratinocytes (HaCaT), fibroblasts (PT64-04HF) and osteoblasts (MC3T3-E1) were submitted to 2 treatment protocols: zoledronic acid 10 µM (ZA) therapy during 24 h and then AC during 48 h and vice-versa; or co-treatment with both. Parameters as viability, apoptosis and caspase 3/7 activation were evaluated. Cancer cells (C4-2B) viability was determined after AC treatment. Streptococcus mutans were submitted to co-treatment with ZA and AC. Microcapsules containing AC were obtained after spray dryer processing, aliquoted and stored into climatic chamber for stability study according to ANVISA protocol (RE 01/2005). ANOVA and T Student test, ?a=0,05, were applied for statistical analysis. After 48 h, ZA treatment decreased viability of keratinocytes, fibroblasts and osteoblasts repectively by 34%, 47% and 51%. Co-treatments ZA and AC 5 µg mL-1 (82%, 83% and 55%), and ZA and AC 10 µg mL-1 (69%, 70% and 64%) increased cell viability. No apoptosis or caspase activation were detected on fibroblasts, however, caspase 3/7 was active on osteoblasts after ZA 48 h treatment. Concentrations higher than 10 µg mL-1 of AC were toxic to cancer cells and showed a decrease by 40% in cell viability, while no effect was seen with AC 5 or 10 µg mL-1. Higher ZA concentrations (50 µM) seemed to induce S. mutans growth, while AC showed no effect on bacteria. Finally, the studied core matherial (arabic gum plus matodextrin 1:1) was ineffective on protecting AC extract against degradation, since anthocyanidins concentration was reduced after 15 days incubation. Therefore, lower AC concentrations (5 and 10 µg mL-1) were promising on treating BRONJ wounds, as they were effective in protect epithelial cells against harmfull effects of ZA, without affecting cancer cells or S. mutans growth. Different types of core matherials must be evaluated for improving AC chemical stability. More studies are required to prove AC beneficial effects on treating BRONJ / Doutorado / Farmacologia, Anestesiologia e Terapeutica / Doutora em Odontologia

Les fibroblastes activés qui composent les stromas tumoraux sont des acteurs majeurs des interactions tumeur-stroma impliquées dans la croissance et la dissémination des cellules tumorales. Ce processus d'activation des fibroblastes est caractérisé par l'expression de marqueurs protéiques spécifiques parmi lesquels figure l'alphaSMA et FAPalpha;. Le TGFbeta;, cytokine secrétée massivement par les cellules tumorales, est un des éléments impliqués dans l'activation des fibroblastes et la formation du stroma tumoral qui en résulte. L'EMMPRIN, glycoprotéine transmembranaire surexprimée dans les cellules tumorales est également un médiateur des interactions tumeur-stroma puisqu'il a la capacité d'induire la synthèse des MMPs par les fibroblastes péri-tumoraux accroissant ainsi la propagation des cellules tumorales à travers l'organisme. Nos travaux identifièrent que le TGFbeta secrété par les cellules tumorales induisait la synthèse du marqueur FAPalpha par les fibroblastes. L'EMMPRIN stromal apparaît comme récepteur de ces signaux tumoraux et est nécessaire à la synthèse du marqueur FAPalpha; par les fibroblastes. L'EMMPRIN participe donc à l'activation TGFbeta; dépendante des fibroblastes. Son inhibition dans ces cellules conduit à un dysfonctionnement de la signalisation médiée par les protéines Smad2/Smad3 aboutissant à une diminution de la synthèse du marqueur alphaSMA ainsi que de certaines protéines matricielles induites par le TGFbeta. L'étude du mécanisme d'action de l'EMMPRIN dans ce processus a permis d'identifier l'EMMPRIN comme nouvelle protéine chaperonne du récepteur de type I au TGFbeta;. / Tumor stroma activated fibroblasts are major actors of tumor stroma interactions taking to tumor growth and spreading. Activated fibroblasts are characterized by the expression of specific markers including alphaSMA and FAPalpha;. The TGFbeta;, a cytokine highly secreted by tumor cells, is one of the key factors involved in fibroblast activation and tumor stroma formation. EMMPRIN, a transmembrane glycoprotein overexpressed in tumor cells, is also a mediator of tumor-stroma interactions by its ability to induce the synthesis of MMPs by peri-tumor fibroblasts enhancing then tumor cells dissemination across the organism.Here, we demonstrate that TGFbeta; secreted by tumor cells is the tumor factor involved in the synthesis of FAPalpha; by fibroblasts. Stromal EMMPRIN appeared to be the receptor of these tumor-stroma interactions and is required for the synthesis of FAPalpha; by fibroblasts. EMMPRIN was also evidenced to take part in TGFbeta;-dependent fibroblast activation. Its inhibition in these cells correlate to a dysfunction in Smad2/Smad3 signaling leading to a decrease in the expression of alphaSMA and matrix proteins induced by TGFbeta;. The study of the mechanism used by EMMPRIN in this process evidenced this protein as a new chaperone for the type I TGFbeta; receptor.

The discovery of biomarkers for Parkinson's disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.

Elastin-like peptides (ELPs) are stimulus-responsive protein-based polymers which are attractive material for biomedical research due to their biocompatibility and unique properties. The physical properties of ELPs are dependent on the chain length and the chosen amino acid at the guest residue position. This imparts unlimited flexibility in designing ELP based biomaterials with the desired physical properties. We have shown that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. ELPs can be genetically fused to biologically active peptides or proteins. These fusions can be expressed and readily purified since they maintain the phase transitioning property of the fused ELP domain. Moreover, depending on the ELP sequence chosen the chimeric fusion sequences can self-assemble into unique structures such as nanoparticles. These structures can then be applied to the injury site where they not only provide unique topographical cues or structural support but also act as delivery vehicles for the fused bioactive protein. We developed a multifunctional nanoparticle that is comprised of PMP-D2-ELP fusion protein and different functional peptide ELP fusion proteins to preserve the bioactivity of the functional group with the existence of elastase. These heterogeneous particles will be beneficial for the delivery of combination therapies to solve multiple problems that often existed in chronic wound healing or other tissue regeneration process. In summary, this study adds to our understanding of the biological activity of ELP and the interaction mechanism that allow the regulation of cellular behavior. Furthermore this work also investigated the potential therapeutic application of ELP as a delivery platform for chronic wound healing.

Abstract Squamous cell carcinoma (SCC) of the mobile tongue is the most common type of cancer of the oral cavity, accounting for 30-40% of oral cancers. It behaves aggressively and almost half of the affected patients still die of the disease despite great advances in its medical and surgical care. Ameloblastomas are the most common clinically significant type of odontogenic tumors, constituting approximately 1% of all cysts and tumors of the jaw. They are benign but locally invasive tumors with a strong tendency to recur after surgery. Ameloblastic carcinoma combines the histological features of ameloblastoma with cytologic atypia irrespective of the presence or absence of metastasis. The effectiveness of tight junction proteins (claudins 1, 4, 5, 7 and occludin) and cancer-associated fibroblasts (CAFs) as prognostic markers in OTSCC and as markers of malignancy in ameloblastomas was studied. Abundance of CAFs and Claudin 7 derangement was found to be associated with poor disease-specific survival in oral (mobile) tongue cancer. Appearance of CAFs within the epithelial islands of ameloblastoma was found to be a marker of malignancy in the tumor. The prognostic predictability of CAF density, Ki-67 (cell proliferation marker), maspin (tumor suppressor marker) and tumor DNA content (tumor ploidy using image cytometry) in tongue cancers was also tested. CAF density was the only marker strongly predictive of prognosis. In ameloblastomas, α-SMA (for CAFs), Ki-67, epithelial membrane antigen (EMA) and DNA content (using image and flow cytometry) were assessed as markers of ameloblastic carcinoma. Only α-SMA was able to predict ameloblastic carcinoma when found in the epithelial islands. In conclusion, staining for α-SMA and claudin 7 seems to be beneficial for prognostication in tongue cancer, while α-SMA staining may be beneficial in differentiating ameloblastoma from ameloblastic carcinoma.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cigarette smoking is one of the foremost causes of chronic obstructive pulmonary diseases such as emphysema and chronic bronchitis, and although it is the most preventable causes of death, it accounts for approximately 6 million deaths worldwide each year. Cigarette smoking during pregnancy and lactation remains one of the primary modifiable risk factors for undesirable fetal, obstetrical, and developmental outcomes. Consequently, the offspring of the smoking mother is exposed to nicotine via the blood and the milk of the mother. As a result, nicotine interacts with the developing offspring and therefore interferes with normal fetal lung development. Maternal smoking during gestation and lactation has been associated with both short and long term health risks ranging from intrauterine growth restriction to physiological abnormalities. Maternal smoking has also been strongly linked to an increased risk for pulmonary diseases and respiratory morbidity in the offspring of the smoking mother. The main objectives of this study were to determine the effects of maternal nicotine exposure during gestation and lactation on the alveolar wall composition during lung development in the offspring; if maternal nicotine exposure during gestation and lactation induces premature cellular senescence in the lungs of the offspring; to clarify the role of pulmonary fibroblasts in premature senescence; and to establish whether tomato juice supplementation will prevent premature aging in the lungs of rats that were exposed to nicotine via the placenta and mother’s milk. From the data generated in this study it was evident that maternal nicotine exposure during gestation and lactation compromises the gas exchange function of the lungs of the F1 offspring. This was prevented by supplementing the mother’s diet with tomato juice which is then received by the offspring via the placenta and mother’s milk. This is conceivably achieved by maintaining the oxidant-anti-oxidant ratio of the mother and of the developing fetus and neonate, thereby averting premature senescence caused by nicotine exposure. Moreover, the present study also demonstrates that a decrease in fibroblast density is associated with emphysematous-like lesions in the lungs of the nicotine exposed F1 progeny. Since pulmonary fibroblasts are chief contributors to the extracellular matrix of the lungs, involved in alveolar multiplication and regeneration; premature aging or cessation of the metabolically active fibroblasts largely contributes to diminished lung structure and function.