Search

A Odontologia está cada dia mais avançada no uso de biomateriais em várias áreas de atuação. A fibrina rica em plaquetas (PRF) é um exemplo desse avanço, mas apesar disso ainda existem muitas lacunas acerca de todo o potencial biológico desse material. A questão crucial para a qualidade, aplicabilidade e função do PRF está ligada à forma de obtenção desse material. Alterações no processo de centrifugação podem alterar a composição desse biomaterial e também a sua forma de ativação dos tecidos. Objetivo desse estudo foi avaliar a influência da velocidade e força centrífuga na cinética de liberação de fatores de crescimento pela PRF humana. Materiais e métodos: Para o desenvolvimento do estudo, utilizou-se amostras de sangue periférico de pacientes saudáveis e submeteu esse material a três protocolos com forças de centrifugação diferentes: 400g, 800g e 200g. Os PRFs obtidos foram cultivados em meio estéril de cultura de células e avaliados nos tempos: 15 minutos, 1 dia, 3 dias e 7 dias. Os fatores de crescimento foram detectados pela método ELISA e quantificados em todos os protocolos. Dos resultados obtidos foi visto que apenas o fator de crescimento EGF apresentou diferenças significativas (p=0,044) nas alterações da força centrifuga nos tempos avaliados. Os outros fatores apresentaram mudanças na detecção ao longo dos dias. Conclusão: Essas variações no protocolo não interferem na detecção da maioria dos fatores de crescimento, o que traz segurança ao profissional na obtenção do potencial do material, mesmo com pequenas variações em sua forma de processamento. Dentistry is increasingly advanced in the use of biomaterials in various areas of activity. Platelet-rich fibrin (PRF) is an example of this advance, but despite this, there are still many gaps regarding the full biological potential of this material. The crucial issue for the quality, applicability and function of PRF is linked to the way in which this material is obtained. Changes in the centrifugation process can change the composition of this biomaterial and also its form of tissue activation. The aim of this study was to evaluate the influence of velocity and centrifugal force on the kinetics of growth factor release by human PRF. Materials and methods: For the development of the study, peripheral blood samples from healthy patients were used and this material was subjected to three protocols with different centrifugation forces: 400g, 800g and 200g. The PRFs obtained were grown in sterile cell culture medium and evaluated at times: 15 minutes, 1 day, 3 days and 7 days. Growth factors were detected by the ELISA method and quantified in all protocols. From the results obtained, it was seen that only the growth factor EGF showed significant differences (p=0.044) in changes in centrifugal force at the times evaluated. The other factors showed changes in detection over the days. Conclusion: These variations in the protocol do not interfere in the detection of most growth factors, which gives the professional confidence in obtaining the potential of the material, even with small variations in its processing form. La odontología está cada vez más avanzada en el uso de biomateriales en diversas áreas de actividad. La fibrina rica en plaquetas (PRF) es un ejemplo de este avance, pero a pesar de ello, todavía existen muchas lagunas en cuanto al potencial biológico completo de este material. El tema crucial para la calidad, aplicabilidad y función de PRF está relacionado con la forma en que se obtiene este material. Los cambios en el proceso de centrifugación pueden modificar la composición de este biomaterial y también su forma de activación tisular. El objetivo de este estudio fue evaluar la influencia de la velocidad y la fuerza centrífuga en la cinética de liberación del factor de crecimiento por PRF humana. Materiales y métodos: Para el desarrollo del estudio se utilizaron muestras de sangre periférica de pacientes sanos y este material fue sometido a tres protocolos con diferentes fuerzas de centrifugación: 400g, 800g y 200g. Las PRF obtenidas se cultivaron en medio de cultivo celular estéril y se evaluaron en tiempos: 15 minutos, 1 día, 3 días y 7 días. Los factores de crecimiento fueron detectados por el método ELISA y cuantificados en todos los protocolos. De los resultados obtenidos se observó que solo el factor de crecimiento EGF mostró diferencias significativas (p=0.044) en los cambios de fuerza centrífuga en los tiempos evaluados. Los otros factores mostraron cambios en la detección a lo largo de los días. Conclusión: Estas variaciones en el protocolo no interfieren en la detección de la mayoría de los factores de crecimiento, lo que da confianza a los profesionales en la obtención del potencial del material, incluso con pequeñas variaciones en la forma de procesamiento.

Background: Vegetable oils have antimicrobial activity and promote cell proliferation. Sunflower oil is used as an alternative for treating skin wounds, particularly in underdeveloped or developing countries. Objective: To characterize sunflower oil and evaluate the in vitro effects on cell proliferation and antimicrobial activity. Methodology: The study was carried out using gas chromatography-mass spectrometry (GC-MS) analysis and cell proliferation and antimicrobial activity tests. Results: The chromatographic analysis identified the main components of sunflower oil, namely: unsaturated fatty acids (82.2%) with linoleic (47.8%), oleic (28.7%), and linolenic (3.9%) acids as the main lipids, followed by saturated (12.70%), palmitic (8.8%) and stearic (3.6%) acids. A difference (p < 0.001) in cell proliferation was found between treatments with sunflower oil (100 and 10 μg/ ml) and the negative controls. It failed in antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, and Klebsiella pneumoniae. Conclusion: Sunflower oil contains a high concentration of essential fatty acids and promotes cell proliferation but fails to inhibit bacterial activity., Marco contextual: Los aceites vegetales tienen acción antimicrobiana y promueven la proliferación celular. El aceite de girasol se utiliza como alternativa para tratar las heridas cutáneas, especialmente en los países subdesarrollados o en vías de desarrollo. Objetivo: Caracterizar el aceite de girasol y evaluar los efectos in vitro sobre la proliferación celular y la actividad antimicrobiana. Metodología: Análisis por cromatografía de gases acoplado a espectrometría de masas, pruebas de proliferación celular y actividad antimicrobiana. Resultados: En el análisis cromatográfico del aceite de girasol, se identificaron los compuestos mayoritarios - ácidos grasos insaturados (82,2%), los principales lípidos son el ácido linoleico (47,8%), oleico (28,7%) y linolénico (3,9%), seguidos del ácido saturado (12,70%), palmítico (8,8%) y esteárico (3,6%). Hubo una diferencia (p < 0,001) entre los tratamientos con aceite de girasol (100 y 10 μg/ml) y los controles negativos en la proliferación celular. Actividad antimicrobiana ineficaz contra las bacterias Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis e Klebsiella pneumoniae. Conclusión: La composición del aceite de girasol mostró una alta concentración de ácidos grasos esenciales, promovió la proliferación celular, pero no inhibió la actividad bacteriana., Enquadramento: Óleos vegetais apresentam ação antimicrobiana e promovem a proliferação celular. O óleo de girassol é usado como alternativa para o tratamento de feridas cutâneas, especialmente nos países subdesenvolvidos ou em desenvolvimento. Objetivo: Caracterizar o óleo de girassol e avaliar os efeitos in vitro na proliferação celular e na atividade antimicrobiana. Metodologia: Análises por cromatografia a gás acoplada à espectrometria de massas, testes de proliferação celular e atividade antimicrobiana. Resultados: Na análise cromatográfica do óleo de girassol identificaram-se os compostos maioritários - ácidos gordos insaturados (82,2%) tendo como principais lípidos os ácidos linoleico (47,8%), oleico (28,7%) e linolênico (3,9%), seguidos pelos ácidos saturados (12,70%), palmítico (8,8%) e esteárico (3,6%). Houve diferença (p < 0,001) entre os tratamentos com óleo de girassol (100 e 10 μg/ml) e controlos negativos na proliferação celular. Ineficácia na atividade antimicrobiana frente às bactérias Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis e Klebsiella pneumoniae. Conclusão: A composição do óleo de girassol mostrou elevada concentração de ácidos gordos essenciais, promoveu proliferação celular, mas não inibiu atividade bacteriana.

BMP´s are signaling proteins that belong to the Transforming Growth Factor-β (TGF-β) superfamily. These proteins promote the recruitment and differentiation of mesenchymal progenitor cells into bone forming cells, the osteoblasts and increase the rate of bone formation. The carrier systems to release rhBMP-2 to the action site are based on the use of free and soluble BMP incorporated into biopolymers such as collagen, gelatin, chitosan, hyaluronic acid and silk. The fused rhBMP-2-thioredoxin could be an interesting approach for new advances in the field of carrying systems of these growth factors. The fused protein thioredoxin can be useful as a coupling agent of BMP-2 to the carrier system, binding it to the surface of the matrix and it is one of the main aims of this work. The recombinant protein rhBMP-2 was produced by IPTG induction obtaining a soluble protein without the need for refolding process. The immobilization of rhBMP-2 at the surface of the TEMPO modified cellulose nanofibrils was indicated by FTIR spectroscopy. The cellular viability tests indicated increased proliferative behavior of both, C2C12 and stem cells from rats, when seeded in presence of rhBMP2 when compared to the free rhBMP2 substrate. The calcified extracellular matrix confirmed the increased activity of the rhBMP2-cellulose substrate, indicating the success of the proposed method. The cell proliferation assays indicated the method used to immobilize rhBMP2 onto the surface of the TEMPO modified cellulose was successful. The cells growth increased when compared to the reference sample free of rhBMP2. Las BMP son proteínas de señalización que pertenecen a la superfamilia del factor de crecimiento transformante beta (TGF-β). Estas proteínas promueven el reclutamiento y la diferenciación de células progenitoras mesenquimales en células formadoras de hueso, osteoblastos, y aumentan la tasa de formación de hueso. Los sistemas portadores para la liberación de rhBMP-2 en el sitio de acción se basan en el uso de BMP libre y soluble incorporado en biopolímeros como colágeno, gelatina, quitosano, ácido hialurónico y seda. La rhBMP-2-tioredoxina fusionada podría ser un enfoque interesante para futuros avances en el campo de los sistemas de transporte de estos factores de crecimiento. La proteína fusionada con tiorredoxina puede ser útil como agente de acoplamiento de BMP-2 al sistema transportador, uniéndola a la superficie de la matriz y es uno de los principales objetivos de este trabajo. La proteína recombinante rhBMP-2 se produjo por inducción de IPTG obteniendo una proteína soluble sin necesidad de un proceso de replegamiento. La inmovilización de rhBMP-2 en la superficie de las nanofibrillas de celulosa modificadas con TEMPO se indicó mediante espectroscopia FTIR. Las pruebas de viabilidad celular indicaron un aumento en el comportamiento proliferativo de las células madre C2C12 y de rata, cuando se sembraron en presencia de rhBMP2 en comparación con el sustrato rhBMP2 libre. La matriz extracelular calcificada confirmó el aumento de la actividad del sustrato de celulosa rhBMP2, lo que indica el éxito del método propuesto. Los ensayos de proliferación celular indicaron que el método utilizado para inmovilizar rhBMP2 en la superficie de celulosa modificada con TEMPO fue exitoso. El crecimiento celular aumentó en comparación con la muestra de referencia sin rhBMP2. As BMP's são proteínas sinalizadoras que pertencem à superfamília Fator de Crescimento Transformador Beta (TGF-β). Essas proteínas promovem o recrutamento e a diferenciação de células progenitoras mesenquimais em células formadoras de osso, os osteoblastos e aumentam a taxa de formação óssea. Os sistemas carreadores para liberação de rhBMP-2 no local de ação são baseados no uso de BMP livre e solúvel incorporada em biopolímeros como colágeno, gelatina, quitosana, ácido hialurônico e seda. A rhBMP-2-tioredoxina fundida pode ser uma abordagem interessante para novos avanços no campo de sistemas de transporte desses fatores de crescimento. A proteína fundida tiorredoxina pode ser útil como agente de acoplamento da BMP-2 ao sistema carreador, ligando-a à superfície da matriz e é um dos principais objetivos deste trabalho. A proteína recombinante rhBMP-2 foi produzida por indução de IPTG obtendo-se uma proteína solúvel sem a necessidade de processo de redobragem. A imobilização de rhBMP-2 na superfície das nanofibrilas de celulose modificadas por TEMPO foi indicada por espectroscopia FTIR. Os testes de viabilidade celular indicaram aumento do comportamento proliferativo tanto de C2C12 quanto de células-tronco de ratos, quando semeadas na presença de rhBMP2 quando comparadas ao substrato rhBMP2 livre. A matriz extracelular calcificada confirmou o aumento da atividade do substrato rhBMP2-celulose, indicando o sucesso do método proposto. Os ensaios de proliferação celular indicaram que o método usado para imobilizar rhBMP2 na superfície da celulose modificada por TEMPO foi bem-sucedido. O crescimento das células aumentou quando comparado com a amostra de referência isenta de rhBMP2.

Background: Breast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear. Methods: In this study we used antibody microarrays and phosphoproteomics to reveal a dynamic global signalling map that reveals new key regulated proteins and phosphor-sites and links between previously known and novel pathways. T47D breast cancer cells were used, and phospho-sites and pathways highlighted were validated using specific antibodies and phenotypic assays. Bioinformatic analysis revealed an enrichment in novel signalling pathways, a coordinated response between cellular compartments and protein complexes. Results: Detailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, epithelial to mesenchyme transition (EMT), cell adhesion, as well as transcription factors previously not associated with breast cancer cell proliferation. Pathway analysis confirmed the key role of the MAPK signalling cascade following progesterone and additional hormone regulated phospho-sites were identified. Full network analysis shows the activation of new signalling pathways previously not associated with progesterone signalling in T47D breast cancer cells such as ERBB and TRK. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins. Conclusions: This study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management. This research was supported by European Research Council (Project “4D Genome” 609989), the Ministerio de Economía y Competitividad (Project G62426937) and the Generalitat de Catalunya (Project AGAUR SGR 575 and AGAUR 2019PROD00115/IU68-016733), European Research Council -Proof Of Concept (Project “Impacct” 825176).

Oral squamous cell carcinoma is a common malignant lesion. ROCKs proteins are associated with pathogenesis and progression of human tumours. This study aimed to evaluate the functional role of ROCKs in the regulation of cell proliferation of oral squamous cell carcinoma in vitro. BrdU incorporation assays and KI-67 immunoexpression were performed by using SCC-4 cell line from oral squamous cell carcinoma. Control and treated cells: HA-1077 (25, 50 and 100 μmol/l), 50 μmol HA-1077 and Y-27632 30 μmol/l, Y-27632 30 μmol/l were cutured for 6 h. The number of SCC-4 cells treated with: HA-1077 (25, 50 and 100 μmol/l), HA-1077 50 μmol/l and/or Y-27632 30 μmol/l was significantly lower than control cells in BrdU assay [F (5.17) = 443.818, p

Resumen Introducción. Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación. Objetivo. Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos. Materiales y métodos. Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias. Resultados. Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados. Conclusión. Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares. Abstract Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics.

The synthetic compound fasentin has been described as a modulator of GLUT-1 and GLUT-4 transporters, thus inhibiting glucose uptake in some cancer cells. Endothelial glucose metabolism has been recently connected to angiogenesis and it is now an emerging topic in scientific research. Indeed, certain compounds with a known effect on glucose metabolism have also been shown to inhibit angiogenesis. In this work we tested the capability of fasentin to modulate angiogenesis in vitro and in vivo. We show that fasentin inhibited tube formation in endothelial cells by a mechanism that involves a negative effect on endothelial cell proliferation and invasion, without affecting other steps related to the angiogenic process. However, fasentin barely decreased glucose uptake in human dermal microvascular endothelial cells and the GLUT-1 inhibitor STF-31 failed to inhibit tube formation in these cells. Therefore, this modulatory capacity on endothelial cells function exerted by fasentin is most likely independent of a modulation of glucose metabolism. Taken together, our results show a novel biological activity of fasentin, which could be evaluated for its utility in cancer and other angiogenesis-dependent diseases.

This work aims to evaluate the effect of Low Power Laser at red wavelength of 660nm on osteogenic proliferation of human umbilical cord mesenchymal stem cells. Processing of the cord was performed, consisting of sectioning the cord into fragments; washing with the solution; separation and discarding of the stromal vessels; sectioning of the remaining jelly and placement in culture bottles with DMEM medium supplemented with 15% SFB, 20% Ham F-12 Gibco® and antibiotics and kept in a CO2 oven at 37º C and 5% CO2; The culture medium was changed in 24h and 7 days, after this period it was changed every 72h, until the spontaneous migration of cells to the surface; when 80% confluence was reached. The cells were used in the third passage, where they were trypsinized, resuspended, and the number of cells was counted for plating in 96 wells. 24 hours after plating, the differentiation inducers were inserted, and the cells belonging to the LBP groups were irradiated. The proliferation curve was performed by the MTT assay. Solution containing 0.5 mg MTT per mL of medium was added, after incubation at 37 °C for 4 h protected from light, DMSO will be added to the cells. The absorbance was read by spectrophotometer at 562 nm at 24, 48 and 72 h to establish cell proliferation curve. The proliferation of MSCs was higher in cells submitted to the regular medium and LBP showed a positive effect on the proliferation of MSCs associated with the osteogenic medium. Este trabajo tiene como objetivo evaluar el efecto del láser de baja potencia en la longitud de onda roja de 660nm en la proliferación osteogénica de las células madre mesenquimales del cordón umbilical humano. Se realizó el procesamiento del cordón, que consistió en la sección del cordón en fragmentos; el lavado con la solución; la separación y el descarte de los vasos estromales; la sección de la gelatina restante y la colocación en frascos de cultivo con medio DMEM suplementado con 15% de SFB, 20% de Ham F-12 Gibco® y antibióticos y mantenido en una estufa de CO2 a 37º C y 5% de CO2; El medio de cultivo se cambió a las 24h y a los 7 días, después de este periodo se cambió cada 72h hasta la migración espontánea de las células a la superficie; cuando se alcanzó el 80% de confluencia. Las células se utilizaron en el tercer pasaje, en el que se tripsinizaron, se resuspendieron y se contó el número de células para su colocación en 96 pocillos. 24 horas después de la colocación, se introdujeron los inductores de diferenciación y se irradiaron las células pertenecientes a los grupos LBP. La curva de proliferación se realizó mediante el ensayo MTT. Se añadió una solución que contenía 0,5 mg de MTT por mL de medio, tras incubar a 37 °C durante 4 h protegido de la luz, se añadirá DMSO a las células. La absorbancia se leyó en el espectrofotómetro a 562 nm a las 24, 48 y 72 h para establecer la curva de proliferación celular. La proliferación de las MSC fue mayor en las células sometidas al medio regular y el LBP mostró un efecto positivo en la proliferación de las MSC asociadas al medio osteogénico. Este trabalho tem como objetivo avaliar o efeito do Laser de Baixa Potência no comprimento de onda vermelho de 660nm na proliferação osteogênica das células-tronco mesenquimais do cordão umbilical humano. Foi realizado o processamento do cordão, que consiste na secção do cordão em fragmentos; lavagem com a solução; separação e descarte dos vasos do estroma; secção da geleia remanescente e colocação em garrafas de cultura com Meio DMEM suplementado com 15% de SFB, 20% de Ham F-12 Gibco® e antibióticos e mantidas em estufa de CO2 a 37º C e 5% de CO2; o meio de cultura foi trocado em 24h e 7 dias, após esse período ficou sendo trocado a cada 72h, até a migração espontânea das células para a superfície; ao atingir a confluência de 80%. As células foram utilizadas na terceira passagem, onde foram tripsinizadas, ressuspendidas e realizado a contagem do número de células para plaqueamento em 96 poços. 24 horas após o plaqueamento foram inseridos os indutores de diferenciação; e as células pertencentes aos grupos de LBP foram irradiadas. A curva de proliferação foi realizada pelo ensaio de MTT. Foi adicionado solução contendo 0,5 mg MTT por mL de meio, após incubação a 37 ° C por 4 h protegido da luz, o DMSO será adicionado às células. A absorbância foi lida através do espectrofotômetro a 562 nm com 24, 48 e 72 horas para estabelecer curva de proliferação das células. A proliferação das CTMs foi maior nas células submetidas ao meio regular e o LBP apresentou efeito positivo na proliferação das CTMs associadas ao meio osteogênico.

Objective: The role of diffusion-weighted MRI in differential diagnostics and predicting the survival of patients with spine metastases was studied. Methods: The study included data from MRI and morphological studies of 23 patients with spine metastases. Results: The values obtained for the apparent diffusion coefficient (ADC) of tumors were compared with their histological type, cell density and Ki-67 proliferation index. The effect of ADC values on overall patient survival was also assessed. A reliable inverse correlation was established between ADC values and Ki-67 proliferation index for various types of spine metastases (r=-0.753, p=0.017). The dependence of ADC values and overall survival of patients with metastases in the spine is shown. Conclusion: The technique of diffusion-weighted MRI can be used as part of a comprehensive assessment in the preoperative planning of surgical treatment, and as a prognostic factor of overall survival for this group of patients. Level of Evidence II. Prognostic retrospective study, RESUMO Objetivos: O papel da RM ponderada por difusão nos diagnósticos diferenciais e na previsão da sobrevida dos pacientes com metástases da coluna vertebral foram analisados. Métodos: O estudo incluiu dados da RM e estudos morfológicos de 23 pacientes com metástases da coluna vertebral. Resultados: Os valores obtidos para o coeficiente de difusão aparente (ADC) dos tumores foram comparados com seu tipo histológico, densidade celular e índice de proliferação Ki-67. Além disso, avaliou-se o efeito dos valores de ADC na sobrevida global dos pacientes. Foi estabelecida uma correlação inversa confiável entre os valores de ADC e o índice de proliferação Ki-67 para diversos tipos de metástases da coluna vertebral (r =-0,753, p=0,017). A dependência dos valores de ADC e a sobrevida global dos pacientes com metástases na coluna vertebral é demostrada. Conclusão: A técnica da RM ponderada por difusão pode ser utilizada como parte de uma avaliação abrangente do planejamento pré-operatório do tratamento cirúrgico e como fator prognóstico da sobrevida global para esse grupo de pacientes. Nível de Evidência II. Estudo retrospectivo prognóstico. RESUMEN Objetivos: Fueron analizados el papel de la RM ponderada por difusión en los diagnósticos diferenciales y en la previsión de la sobrevida de los pacientes con metástasis de la columna vertebral. Métodos: El estudio incluyó datos de RM y estudios morfológicos de 23 pacientes con metástasis de la columna vertebral. Resultados: Los valores obtenidos para el coeficiente de difusión aparente (ADC) de los tumores se compararon con su tipo histológico, densidad celular e índice de proliferación Ki-67. Además, se evaluó el efecto de los valores de ADC en la sobrevida global de los pacientes. Se estableció una correlación inversa confiable entre los valores de ADC y el índice de proliferación Ki-67 para diversos tipos de metástasis de la columna vertebral (r =-0,753, p=0,017). Es demostrada la dependencia de los valores de ADC y la sobrevida global de los pacientes con metástasis en la columna vertebral. Conclusión: La técnica de RM ponderada por difusión puede utilizarse como parte de una evaluación integral de la planificación preoperatoria del tratamiento quirúrgico y como un factor pronóstico de la sobrevida global para este grupo de pacientes. Nivel de Evidencia II. Estudio retrospectivo pronóstico.

O desenvolvimento de tecnologias a base de concentrado de plaquetas obtidos por meio de centrifugação e métodos de manipulação a partir de uma amostra de sangue tem a finalidade de otimizar a cicatrização tecidual e promover a regeneração de tecidos. Objetivo: foi abordar considerações a respeito dos concentrados plaquetários autólogos e sua aplicabilidade na Odontologia. Metodologia: realizou-se análise retrospectiva da literatura do período de 2001 a 2022 focando a evolução e uso dos hemoconcentrados autólogos. Foram usados como termos de busca: plasma rico em plaquetas, plasma rico em fibrina, hemoconcentrados autólogos, regeneração e cicatrização tecidual. A busca na literatura se deu por meio de pesquisa nas bases de dados PubMed, Google Scholar, SciELO e banco de dados da BVS. Revisão Bibliográfica: o plasma rico em plaquetas (PRP) foi a primeira geração de derivados de amostras de sangue humano, utilizados com este propósito. Dependendo da técnica para a preparação dos concentrados plaquetários, eles são divididos em quatro categorias, de acordo com seu conteúdo de leucócitos e fibrina. Conclusão: Os agregados plaquetários podem induzir a ativação de fibroblastos e a formação de depósitos de colágeno, além de plaquetas e fatores de crescimento usados na reparação tecidual. Assim otimizam os resultados de tratamentos na odontologia, como preenchimento cutâneo, tratamento de cicatrizes de acne, implantes dentais e cirurgias dento alveolares. The development of technologies based on platelet concentrate obtained through centrifugation and manipulation methods from a blood sample aims to optimize tissue healing and promote tissue regeneration. Objective: to address considerations about autologous platelet concentrates and their applicability in Dentistry. Methodology: a retrospective analysis of the literature from 2001 to 2022 was carried out, focusing on the evolution and use of autologous blood concentrates. The following search terms were used: platelet-rich plasma, fibrin-rich plasma, autologous hemoconcentrates, tissue regeneration and healing. The literature search was carried out through a search in PubMed, Google Scholar, SciELO and BVL databases. Bibliographic Review: Platelet-rich plasma (PRP) was the first generation of derivatives from human blood samples, used for this purpose. Depending on the technique for preparing platelet concentrates, they are divided into four categories according to their leukocyte and fibrin content. Conclusion: Platelet aggregates can induce the activation of fibroblasts and the formation of collagen deposits, in addition to platelets and growth factors used in tissue repair. Thus, they optimize the results of treatments in dentistry, such as skin filling, treatment of acne scars, dental implants and dentoalveolar surgeries. El desarrollo de tecnologías basadas en concentrado de plaquetas obtenido mediante métodos de centrifugación y manipulación a partir de una muestra de sangre tiene como objetivo optimizar la cicatrización de los tejidos y favorecer la regeneración tisular. Objetivo: abordar las consideraciones sobre los concentrados plaquetarios autólogos y su aplicabilidad en Odontología. Metodología: se realizó un análisis retrospectivo de la literatura desde 2001 hasta 2022, centrándose en la evolución y uso de concentrados de sangre autóloga. Se utilizaron los siguientes términos de búsqueda: plasma rico en plaquetas, plasma rico en fibrina, hemoconcentrados autólogos, regeneración tisular y cicatrización. La búsqueda bibliográfica se realizó a través de una búsqueda en las bases de datos PubMed, Google Scholar, SciELO y BVS. Reseña bibliográfica: El plasma rico en plaquetas (PRP) fue la primera generación de derivados de muestras de sangre humana, utilizados para este fin. Según la técnica de preparación de los concentrados de plaquetas, se dividen en cuatro categorías según su contenido en leucocitos y fibrina. Conclusión: Los agregados plaquetarios pueden inducir la activación de fibroblastos y la formación de depósitos de colágeno, además de plaquetas y factores de crecimiento utilizados en la reparación tisular. Así, optimizan los resultados de tratamientos en odontología, como rellenos cutáneos, tratamiento de cicatrices de acné, implantes dentales y cirugías dentoalveolares.

Simple Summary The treatment of oral squamous cell carcinoma (OSCC) represents a significant problem worldwide. Among cancers with the highest incidence, OSCC renders one of the worst prognoses. Therefore, novel prognostic biomarkers and therapeutic tools to tackle OSCC are urgently needed. Somatostatin-analogues (SSA) are an invaluable therapeutic option in the treatment of several cancers. We aimed to determine the expression levels of all somatostatin-receptors (SSTs) in OSCC, compared to adjacent healthy control tissues, to analyze the relationship of SSTs expression with key clinical and histopathological data, and to explore the direct in vitro effect of different SSAs on OSCC cancer cells. Our findings highlight a potential role of SST2 as a good prognostic biomarker for recurrence and metastasis in OSCC and unveil that SSA exerts antitumoral effects on OSCC cells, providing a relevant clinical conclusion, which should be soon tested for their use in humans. Abstract Oral squamous cell carcinoma (OSCC) incidence has increased by 50% over the last decade. Unfortunately, surgery and adjuvant radiotherapy and chemotherapy are still the mainstream modality of treatment, underscoring the need for alternative therapies. Somatostatin-analogues (SSA) are efficacious and safe treatments for a variety of tumors, but the presence of somatostatin-receptors (SSTs) and pharmacological effects of SSA on OSCC are poorly known. In this study, we demonstrated that SST2 and SST3 levels were significantly higher in OSCC, compared to adjacent healthy control tissues. SST2 expression was associated with less regional metastasis and a lower recurrence rate. Moreover, SST2 was elevated in OSCC and associated with histopathological good prognosis factors, such as high peritumoral inflammation, smaller depth of invasion, and expansive vs. infiltrative front of tumor invasion. Importantly, treatment with different SSA (octreotide, lanreotide, and pasireotide) significantly reduced cell-proliferation in OSCC primary cell cultures. Altogether, this study demonstrated that SST2 is overexpressed in OSCC vs. healthy tissues and could represent a novel prognostic biomarker, since its expression is associated with tumors that show better prognostic factors and less recurrent rate. Moreover, our data unveil clear antitumoral effects of SSAs on OSCC, opening new avenues to explore their potential as targeting therapy to OSCC.

Introduction: Appendix neoplasms are rare and identified in about 1% of all appendectomies, with the most common etiologies being first of epithelial origin and second, neuroendocrine. Neuroendocrine tumors are derived from enterochromaffin cells, producing the granules Chromogranin A and Synaptophysin and are the second most common histological type of appendix neoplasm, often found incidentally after appendectomy due to their indolent presentation, but are sometimes the causes of acute appendicitis. Objective: To report the experience of a patient diagnosed with neuroendocrine tumor of the appendix after acute perforated appendicitis who had previously presented abdominal vessel thrombosis. Case report: Male patient, 28 years old, with abdominal pain in mesogastrium and epigastrium for 2 months, being initially diagnosed with intra-abdominal thrombosis. Hospitalized presenting a diffuse abdominal pain, more prominent in the right iliac fossa with painful decompression, submitted to exploratory laparoscopy and evidenced grade 4 appendicitis. Histopathological analysis revealed a differentiated neuroendocrine tumor in appendage G1 with a size of 2 mm, mitotic index with less than 2 mitoses, pT1 pNx pathological staging, free margins, lymphovascular and perineural invasion. Immunohistochemistry with cell proliferation index (Ki-67) of 1% and identification of the components Chromogranin A polyclonal clone and Synaptophysin clone DARKSYNAP positive. Conclusion: We report a case of a patient with Neuroendocrine Tumor of the appendix with initial presentation as acute appendicitis and, to the pathology, limited to the appendix without lymphovascular invasion, with total appendectomy as a curative procedure. Las neoplasias del apéndice son raras y se identifican en aproximadamente el 1% de todas las apendicectomías, siendo las etiologías más comunes la primera de origen epitelial y la segunda, la neuroendocrina. Los tumores neuroendocrinos se derivan de células enterocromafines, que producen gránulos de cromogranina A y sinaptofisina y son el segundo tipo histológico más común de neoplasia de apéndice, que a menudo se encuentra de manera incidental después de la apendicectomía debido a su presentación indolente, pero a veces son la causa de apendicitis aguda. Objetivo: Informar la experiencia de un paciente diagnosticado de tumor neuroendocrino de apéndice tras apendicitis aguda perforada que previamente había presentado trombosis de vasos abdominales. Caso clínico: Paciente de sexo masculino, 28 años, de 2 meses con dolor abdominal en mesogastrio y epigastrio, siendo diagnosticado inicialmente de trombosis intraabdominal. En esta internación presentaba dolor abdominal difuso más prominente en fosa ilíaca derecha con descompresión dolorosa, sometido a videolaparoscopia exploradora y evidenciaba apendicitis grado 4. El análisis histopatológico mostró un tumor neuroendocrino en un apéndice G1 bien diferenciado con un tamaño de 2mm en la eje mayor, índice mitótico con menos de 2 mitosis, estadificación patológica pT1 pNx, márgenes libres, invasión linfovascular y perineural. Inmunohistoquímica con índice de proliferación celular (Ki-67) del 1% e identificación de los componentes Clon policlonal Cromogranina A y clon Sinaptofisina DARKSYNAP positivo. Conclusión: Presentamos el caso de un paciente con Tumor neuroendocrino de apéndice con presentación inicial como apendicitis aguda y, en patología, limitado al apéndice sin invasión linfovascular y sometido a apendicectomía total como procedimiento curativo. Introdução: As Neoplasias de apêndice são raras e identificadas em cerca de 1% de todas as apendicectomias, sendo as etiologias mais comuns em primeiro de origem epitelial e em segundo, os neuroendócrinos. Os tumores neuroendócrinos são derivados das células enterocromafins, produzindo os grânulos Cromogranina A e Sinaptofisina e são o segundo tipo histológico mais comum de neoplasia em apêndice, muitas vezes, encontrados incidentalmente após apendicectomia devido sua apresentação indolente, mas, por vezes, são os causadores da apendicite aguda. Objetivo: Relatar a experiência sobre o caso de um paciente diagnosticado com tumor neuroendócrino de apêndice após apendicite aguda perfurada que havia apresentado, anteriormente, tromboses de vasos abdominais. Relato de caso: Paciente do sexo masculino, 28 anos, há 2 meses com dor abdominal em mesogástrio e epigástrio, sendo diagnosticado inicialmente com trombose intra-abdominal. Neste internamento, apresentou dor abdominal difusa mais proeminente em fossa ilíaca direita com descompressão dolorosa, submetido à videolaparoscopia exploradora e evidenciado apendicite grau 4. À análise histopatológica evidenciado tumor neuroendócrino em apêndice bem diferenciado G1 com tamanho de 2mm em maior eixo, índice mitótico com menos de 2 mitoses, estadiamento patológico pT1 pNx, margens livres, invasão linfovascular e perineural. Imuno-histoquímico com índice de proliferação celular (Ki-67) de 1% e identificação dos componentes Cromogranina A clone policlonal e Sinaptofisina clone DARKSYNAP positivo. Conclusão: Relatamos um caso de um paciente com Tumor Neuroendócrino de apêndice com apresentação inicial como apendicite aguda e, à patologia, limitada ao apêndice sem invasão linfovascular e teve a apendicectomia total como procedimento curativo.

© The Author(s) 2021., [Background]: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, requiring novel treatments to target both cancer cells and cancer stem cells (CSCs). Altered splicing is emerging as both a novel cancer hallmark and an attractive therapeutic target. The core splicing factor SF3B1 is heavily altered in cancer and can be inhibited by Pladienolide-B, but its actionability in PDAC is unknown. We explored the presence and role of SF3B1 in PDAC and interrogated its potential as an actionable target. [Methods]: SF3B1 was analyzed in PDAC tissues, an RNA-seq dataset, and publicly available databases, examining associations with splicing alterations and key features/genes. Functional assays in PDAC cell lines and PDX-derived CSCs served to test Pladienolide-B treatment effects in vitro, and in vivo in zebrafish and mice. [Results]: SF3B1 was overexpressed in human PDAC and associated with tumor grade and lymph-node involvement. SF3B1 levels closely associated with distinct splicing event profiles and expression of key PDAC players (KRAS, TP53). In PDAC cells, Pladienolide-B increased apoptosis and decreased multiple tumor-related features, including cell proliferation, migration, and colony/sphere formation, altering AKT and JNK signaling, and favoring proapoptotic splicing variants (BCL-XS/BCL-XL, KRASa/KRAS, Δ133TP53/TP53). Importantly, Pladienolide-B similarly impaired CSCs, reducing their stemness capacity and increasing their sensitivity to chemotherapy. Pladienolide-B also reduced PDAC/CSCs xenograft tumor growth in vivo in zebrafish and in mice. [Conclusion]. SF3B1 overexpression represents a therapeutic vulnerability in PDAC, as altered splicing can be targeted with Pladienolide-B both in cancer cells and CSCs, paving the way for novel therapies for this lethal cancer., This work has been supported by Spanish Ministry of Economy [MINECO; BFU2016–80360-R (to JPC)] and Ministry of Science and Innovation [MICINN; PID2019-105201RB-I00 (to JPC), PID2019-105564RB-I00 (to RML)]. Instituto de Salud Carlos III, co-funded by European Union (ERDF/ESF, “Investing in your future”) [FIS Grants PI17/02287 and PI20/01301 (to MDG), PI18/00757 (to BS,Jr); DTS Grant DTS20/00050 (to RML)); Postdoctoral Grant Sara Borrell CD19/00255 (to AIC); Predoctoral contract FI17/00282 (to EAP)]. Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (to BS,Jr). Spanish Ministry of Universities [Predoctoral contracts FPU14/04290 (to SPA); FPU16/06190 (to VHA); FPU18/02275 (to RBE)]. Boehringer Ingelheim Fonds travel grant (EAP). Junta de Andalucía (BIO0139). GETNE2016 and GETNE2019 Research grants (to JPC); and CIBERobn. Associazione Italiana Ricerca Cancro (AIRC 5×1000 n. 12182); Fondazione Italiana Malattie Pancreas – Ministero Salute [FIMPCUP_J38D19000690001]; Fondazione Cariverona: Oncology Biobank Project “Antonio Schiavi” (prot.203885/2017).

Until recently, postoperative periods, both in medicine and dentistry, were considered factors that prevented some patients from performing the necessary procedures. With technological and scientific advancement, it was possible to develop platelet concentrates that assist in the postoperative period, since, when used, they help to accelerate the healing and tissue regeneration processes, which reduces the discomfort time of the postoperative period. The first platelet concentrate was Plasma Rich in Platelets; this, in turn, is considered a first generation platelet concentrate that, due to certain limitations, ended up making professionals stop using it. It was only in 2001 that the second generation platelet concentrate, also known as Fibrin Rich in Platelets, was introduced to the world, which had several advantages over the first generation platelet concentrates and, since then, has received several modifications, which have given rise to other varieties of it , which have been widely used until today, together with the original Fibrin Rich in Platelets. The present work aims to carry out, in a didactic way, an aggregate of information about the PRF, in order to present it to the dental community. At the end of this study, it was concluded that the PRF was a major milestone for Dentistry, generating security for the professional and for the patient. Articles obtained through the online platforms: VHL, Pubmed and Lilacs were used as bibliographic bases. With this, after the analysis and absorption of the material used as a reference, this work was written. Hasta hace poco tiempo, los postoperatorios tanto en medicina como en odontología se consideraban factores que impedían a algunos pacientes realizar los procedimientos necesarios. Con el avance tecnológico y científico, fue posible desarrollar concentrados de plaquetas que asisten en el postoperatorio, ya que, cuando se utilizan, ayudan a acelerar los procesos de cicatrización y regeneración tisular, lo que reduce el tiempo de molestias postoperatorias. El primer concentrado de plaquetas fue plasma rico en plaquetas; este a su vez es considerado un concentrado de plaquetas de primera generación que, por ciertas limitaciones, terminó haciendo que los profesionales dejen de usarlo. Recién en el año 2001 se introdujo al mundo el concentrado de plaquetas de segunda generación, también conocido como fibrina rica en plaquetas, que tenía varias ventajas sobre los concentrados de plaquetas de primera generación y, desde entonces, ha recibido modificaciones, que han dado lugar a variedades de la misma, que han sido ampliamente utilizadas hasta hoy, junto con la original Fibrina Rica en Plaquetas. El presente trabajo tiene como objetivo hacer, de manera didáctica, un agregado de información sobre el PRF, con el fin de presentarlo a la comunidad dental. Al final de este estudio, se concluyó que el PRF fue un gran hito para la Odontología, generando seguridad para el profesional y para el paciente. Se utilizaron como base bibliográfica los artículos obtenidos a través de las plataformas online: BVS, Pubmed y Lilacs. Con esto, luego del análisis y absorción del material utilizado como referencia, se redactó este trabajo. Até pouco tempo atrás, os pós operatórios tanto na Medicina quanto na odontologia, eram considerados como fatores que faziam com que alguns pacientes deixassem de realizar procedimentos necessários. Com o avanço tecnológico e científico, foi possível o desenvolvimento de concentrados plaquetários que auxiliam no pós operatório, uma vez que, quando utilizados, ajudam a acelerar os processos de cicatrização e de regeneração tecidual, o que reduz o tempo de desconforto pós operatório. O primeiro concentrado plaquetário foi o Plasma Rico em Plaquetas; este por sua vez é considerado um concentrado plaquetário de primeira geração que, por possuir certas limitações, acabou fazendo com que os profissionais deixassem de utiliza-lo. Somente em 2001 foi apresentado ao mundo o concentrado plaquetário de segunda geração, também conhecido como Fibrina Rica em Plaquetas, o qual apresentava diversas vantagens sobre os concentrados plaquetários de primeira geração e, desde então, recebeu modificações, que deram origem a variedades dele, as quais vem sendo amplamente utilizadas até os dias de hoje, juntamente com a Fibrina Rica em Plaquetas original. O presente trabalho tem como objetivo relizar, de forma didática, um agregado de informações a respeito do PRF, a fim de apresentá-las à comunidade odontológica. Ao final deste estudo, concluiu-se que a PRF foi um grande marco para a Odontologia, gerando segurança para o profissional e para o paciente. Foram utilizados como base bibliográficas, artigos obtidos pelas plataformas on-line: BVS, Pubmed e Lilacs. Com isso, feita a análise e a absorção do material utilizado como referência, foi escrito este trabalho.

Resumen Introducción. Se ha demostrado que el caseinato de sodio y sus componentes (caseínas α, β y κ) inhiben la proliferación de la línea celular hematopoyética de ratón 32D clone 3 (32Dcl3) e inducen su diferenciación hacia macrófagos. Se sabe que la caseína α induce la producción de IL-1β y que esta última citocina inhibe la proliferación celular mediante la producción del factor de necrosis tumoral alfa (TNF-α), pero se desconoce si el caseinato de sodio y las caseínas inducen la producción de TNF y si este es el responsable de la inhibición de la proliferación. Objetivo. Evaluar si el caseinato de sodio y las caseínas α, β y κ inhiben la proliferación de la línea celular 32Dcl3 mediante la producción de TNF-α. Materiales y métodos. Se usaron diferentes concentraciones de caseinato de sodio y de las caseínas α, β y κ en las células 32Dcl3. Posteriormente, se evaluaron la viabilidad celular mediante una prueba con el MTT [3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol], la inducción de apoptosis con la citometría de flujo y la síntesis del TNF-α con el ELISA. Además, se hicieron pruebas de neutralización con anti-TNF-α en células 32Dcl3 tratadas con caseinato de sodio y caseína α, y se evaluó la proliferación celular. Resultados. Se encontró que el caseinato de sodio y las caseínas α, β y κ reducían la proliferación de la línea celular 32Dcl3 sin afectar la viabilidad, y que solo el caseinato y la caseína α inducían la apoptosis y la liberación al medio de TNF-α. La proliferación de células 32Dcl3 tratadas con caseinato y caseína α se restableció al usar anticuerpos anti-TNF-α. Conclusión. El TNF-α fue el principal responsable de la inhibición de la proliferación en las células 32Dcl3 tratadas con caseinato de sodio o caseína α. Abstract Introduction: Sodium caseinate (CS) and its components (alpha-casein, beta-casein, and kappa-casein) have been shown to inhibit the proliferation of the mouse hematopoietic 32D clone 3 (32Dcl3) cell line and induce its differentiation into macrophages. It is well-known that alpha-casein induces IL-1β production and that this cytokine inhibits the proliferation via the production of tumor necrosis factor alpha (TNF-alpha), but it is not known if CS and the caseins inhibit the proliferation via TNF-alpha production. Objective: To evaluate if CS and alpha-casein, beta-casein and kappa-casein inhibit the proliferation on 32Dcl3 cell line via TNF-alpha. Materials and methods: We used different concentrations of CS, alpha-casein, beta-casein and kappa-casein in 32Dcl3 cells to evaluate cell proliferation. We assessed cell viability by MTT, induction to apoptosis by flow cytometry, and TNF-alpha synthesis by ELISA. Additionally, we performed anti-TNF-alpha neutralization assays on 32Dcl3 cells treated with CS and alpha-casein and we evaluated proliferation. Results: The results showed that CS, alpha-casein, beta-casein, and kappa-casein reduced proliferation of the 32Dcl3 cell line without affecting the viability and that only CS and alpha-casein induced apoptosis and the release of TNF-alpha. The 32Dcl3 cells treated with CS and alpha-casein reestablished their proliferation by using anti-TNF-alpha antibodies. Conclusion: TNF-alpha was the main responsible for the inhibition of proliferation in 32Dcl3 cells treated with CS or alpha-casein.

La lesión central de células gigantes (LCCG) es una lesión osteolítica benigna que en algunos casos presenta un comportamiento agresivo, con recidiva y mal pronóstico. Ki-67 es una proteína nuclear cuya función general es la regulación de la proliferación celular. Este marcador es utilizado para el reconocimiento de células en proliferación y como herramienta de pronóstico en el diagnóstico de neoplasias. El objetivo de este estudio fue cuantificar la inmunoexpresión de Ki-67 en las diferentes poblaciones celulares de las LCCG y analizar su asociación con las características clínicas, demográficas y radiográficas. Se evaluó la inmunoexpresión de Ki-67 de 17 casos de LCCG en dos poblaciones celulares: células gigantes multinucleadas (CGM) y células mesenquimatosas estromales (CME). El análisis estadístico se efectuó con el programa SAS 9.0 y SPSS versión 23.0, con un nivel alfa impuesto de P

La formación de nuevas células se genera a partir de células preexistentes a través de una serie ordenada de eventos denominada ciclo celular. El control de este es fundamental para la integridad del genoma, por lo que hay múltiples proteínas regulando este proceso. Actualmente, se conoce que el complejo Mantenimiento de Minicromosoma (MCM2-7) tiene un rol esencial en la replicación del ADN, específicamente involucrado en la proliferación, durante el ciclo celular. La identificación inmunohistoquimica de las proteínas de este complejo a nivel tisular, podría ser una herramienta muy útil, para usarlos como biomarcadores y así comprender uno de los mecanismos biológicos que se ven afectados en el cáncer. Nuestro objetivo es realizar una revisión del complejo MCM2-7, ya que estas proteínas como se ha descrito, podrían comportarse como buenos marcadores biológicos de proliferación celular, y así poder realizar un buen diagnóstico, pronóstico y futuros blancos terapéuticos de aquellas lesiones principalmente neoplásicas, principalmente las que asientan a nivel de la mucosa bucal. New cells are formed from preexisting cells through an ordered series of events called cell cycle. As the control of this cycle is fundamental for genome integrity, multiple proteins regulate this process. We currently know that the Minichromosome maintenance (MCM2-7) complex has a major role in DNA replication in the cell cycle, in particular regarding proliferation. The immunohistochemical identification of the proteins in this complex on tissues may be useful, as they could be used as biomarkers and would help us understand one of the biological mechanisms affected in cancer processes. Our aim is to collect the existing evidence regarding the members of the MCM2-7 complex, since these proteins could be effective biological cell proliferation markers, which would help practitioners make accurate diagnosis, prognosis and future therapeutic targets of lesions that are mainly neoplastic, especially in the buccal mucosa.

The study of cancer metabolism is regaining center stage and becoming a hot topic in tumor biology and clinical research, after a period where such kind of experimental approaches were somehow forgotten or disregarded in favor of powerful functional genomic and proteomic studies [...]

The seeds of Euphorbia lathyris have been used in traditional medicine to treat various medical conditions. However, neither all of their active biocompounds nor the molecular mechanisms underlying their therapeutic effects have been described. A new ethanolic extract of defatted flour from mature seeds of Euphorbia lathyris showed a high total polyphenol content and significant antioxidant activity. Chromatographic analysis showed that esculetin, euphorbetin, gaultherin, and kaempferol-3-rutinoside were the most abundant polyphenolic bioactive compounds. Antiproliferative assays showed a high and selective antitumor activity against colon cancer cell lines (T84 and HCT-15). In addition, a significant antiproliferative activity against glioblastoma multiforme cells was also demonstrated. Its mechanism of action to induce cell death was mediated by the overexpression of caspases 9, 3, and 8, and by activation of autophagy. Interestingly, a reduction in the migration capacity of colon cancer cells and a significant antiangiogenic effect on human umbilical vein endothelial cells were also demonstrated. Finally, the extract significantly reduced the subpopulations of cancer stem cells. This extract could be the basis to develop new therapeutic strategies for the treatment of colon cancer, although further experiments will be necessary to determine its in vivo effects., Granada University (project PSE/17/002), Ministerio de Ciencia e Innovación (project RTC-2017-6540-1), CTS-107 and AGR145 Groups

[Resumen] En la actualidad, el autoensamblaje de moléculas a través de interacciones covalentes se ha postulado como una herramienta de gran utilidad para la síntesis de nuevos receptores moleculares, que son de gran interés en biomedicina por su versatilidad y múltiples aplicaciones. Específicamente, los complejos host-guest resultan especialmente interesantes, ya que pueden responder a estímulos externos como cambios en el medio celular externo y ser modulados para su utilización como sistemas de liberación controlada de fármacos. Entre estos compuestos, destaca el macrociclo red box cuya capacidad aceptora puede ser modulada mediante reacciones redox y cambios de pH, con el fin de maximizar su potencial como vehículo de liberación controlada de fármacos en aplicaciones biomédicas. Como primer paso para evaluar el potencial de la red box como sistema de liberación de fármacos, en el presente estudio se evaluó su biocompatibilidad mediante la realización de ensayos biológicos basados en medidas de la viabilidad celular y citotoxicidad en una línea celular normal (HFF-1) y otra tumoral (HeLa). Además, para confirmar que el macrociclo es capaz de atravesar la membrana plasmática, se llevó a cabo un estudio de internalización celular. Los resultados mostraron que la red box afectó en mayor medida a la viabilidad y proliferación celular de la línea no tumoral, lo cual podría estar relacionado con la mayor velocidad de división de las células cancerosas y/o una mayor permeabilidad al compuesto de la línea no tumoral. Asimismo, el estudio preliminar de internalización celular en células HeLa reveló la entrada de un pequeño porcentaje de red box en el interior de las células siendo mayor a las 24 h de exposición. [Resumo] Na actualidade, a autoensamblaxe de moléculas a través de interaccións covalentes postulouse como unha ferramenta de gran utilidade para a síntese de novos receptores moleculares, que son de gran interese en biomedicina pola súa versatilidade e múltiples aplicacións. Especificamente, os complexos host-guest resultan especialmente interesantes, xa que poden responder a estímulos externos, coma cambios no medio celular externo e ser modulados para a súa utilización como sistemas de liberación controlada de fármacos. Entre estes compostos, destaca o macrociclo red box cuxa capacidade aceptora pode ser modulada mediante reaccións redox e cambios de pH, co fin de maximizar o seu potencial como vehículo de liberación controlada de fármacos en aplicacións biomédicas. Como primeiro paso para avaliar o potencial da red box como sistema de liberación de fármacos, no presente estudo avaliouse a súa biocompatibilidade mediante a realización de ensaios biolóxicos baseados en medidas da viabilidade celular e citotoxicidade nunha liña celular normal (HFF-1) e outra tumoral (HeLa). Ademais, para confirmar que o macrociclo é capaz de atravesar a membrana plasmática, levouse a cabo un estudo de internalización celular. Os resultados mostraron que a red box afectou en maior medida á viabilidade e proliferación celular da liña non tumoral, o cal podería estar relacionado coa maior velocidade de división das células cancerosas e/ou unha maior permeabilidade ao composto da liña non tumoral. Así mesmo, o estudo preliminar de internalización celular en células HeLa revelou a entrada dun pequeno porcentaxe de red box no interior das células sendo maior ás 24 h de exposición. [Abstract] Nowadays, self-assembly of molecules through covalent interactions has been postulated as a very useful tool for the synthesis of new molecular receptors, being of great interest in biomedicine due to their versatility and multiple applications. Specifically, hostguest complexes are especially interesting, since they can respond to external stimuli as those from the external cellular environment and be modulated for their use as controlled drug delivery systems. Among these compounds, the macrocycle red box, whose acceptor capacity can be modulated by redox reactions and changes in pH to maximize its potential as a vehicle for controlled drug delivery in biomedical applications, is of great interest. As a first step to evaluate its potential as a drug delivery system, in the present study the biocompatibility of red box was evaluated by conducting biological assays based on measurements of cell viability and cytotoxicity in a normal cell (HFF-1) and in a tumor cell (HeLa) lines. Furthermore, to confirm the macrocycle capability to cross the cell membrane, a cell internalization study was carried out. The results showed that the red box affected the cell viability and proliferation of the non-tumor line to a greater extent, which could be related to the higher division rate of cancer cells and/or the major permeability to the compound of the non-tumor line. Likewise, the preliminary study of cellular internalization in HeLa cells revealed the entry of a small percentage of red box inside the cells, being higher after 24 h of exposure. Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2020/2021

[spa] En Ia levadura de gemación el tamaño celular en START o tamaño crítico está determinado por Ia velocidad de crecimiento en G1. Durante esta fase, Saccharomyces cerevisiae presenta una disminución de Ia relación area superficie/volumen con el tamaño. En cambio, en el memento de Ia gemación se observa Ia existencia de un pico máximo en Ia velocidad de síntesis de Ia superficie celular, necesitando un gran aumento en Ia síntesis de materiales de Ia membrana plasmática y Ia pared celular, y un óptimo transporte de esos componentes hacia Ia yema para el correcto crecimiento de ésta. Por todo ello, postulamos Ia hipótesis de que Ia determinación del tamaño celular en START tendría importantes implicaciones en el óptimo crecimiento y desarrollo de Ia yema en las fases S-G2 del ciclo celular de levaduras. Para comprobar esta idea, realizamos un cuidadoso estudio delcrecimiento en S-G2 y observamos que Ia velocidad de crecimiento de Ia yema no aumenta de manera proporcional al tamaño de Ia célula madre,especialmente a volúmenes muy grandes. Ella no sucedía durante G1, donde observamos que Ia velocidad de crecimiento es proporcional al tamaño celular, según las !eyes del crecimiento exponencial. De modo muy notable, dicho efecto tampoco fue observado en células diploides y tetraploides, donde el tamaño es progresivamente mayor, pero proporcional al DNA genómico. Estos datos sugerían que Ia baja eficiencia de crecimiento de las células madre haploides de gran tamaño podría ser causada por algún tipo de insuficiencia génica. Por ejemplo, si Ia expresión de algunos genes importantes en crecimiento no escalase con el tamaño, se producirían cantidades limitantes de las proteínas codificadas y, por tanto, Ia eficiencia de los procesos correspondientes se vería seriamente comprometida. El crecimiento de Ia yema depende del transporte de vesículas desde Ia célula madre,y observamos que dicho transporte no aumentaba proporcionalmente al volumen de Ia célula madre.Sin embargo, el transportador de vesículas Myo2 no mostró una expresión disminuida y tampoco observamos una deficiencia en el número de cables de actina en células grandes, indicando que el sistema de transporte en sí no era limitante. Por dichas razones concluimos que el problema podría residir en el contenido de las vesículas, el cargo.Por tanto,estudiamos proteínas representativas del crecimiento en G1/S,y descubrimos que su nivel disminuye con el tamaño. Como pieza central de este trabajo, comparamos los transcriptomas de células pequeñas y grandes en G1/S, y encontramos muchos genes de categorías funcionales específicas que se regulan al alza o a Ia baja en células grandes en comparación con células pequeñas.Entre ellos encontramos genes relacionados con el crecimiento apical, el transporte de vesículas,el citoesqueleto,y Ia biogénesis ribosómica, que representa un coste energético extraordinario y debe estar muy bien regulada para un crecimiento óptimo. Finalmente, para genes paradigmáticos en estas categorías,realizamos un análisis cuidadoso de Ia insuficiencia génica y confirmamos sus efectos limitantes sobre el crecimiento de Ia yema. En resumen, nuestros resultados demuestran que S. cerevisiae ha establecido un tamaño crítico para lograr el crecimiento óptimo de Ia yema. A tamaños inusualmente mayores, Ia expresión de genes relacionados con proteínas ribosómicas, así como componentes esenciales del crecimiento apical, se reduce claramente produciendo una seria limitación del crecimiento celular., [eng] In budding yeast,cell size at START or the critical size is determined by the growth rate in G.l During this phase, Saccharomyces cerevisiae shows a decrease in the surface area/volume ratio as it increases in size.When the cell shifts from isotropic growth during Gl to polarized growth from the mother cell to the bud,surface synthesis rate peaks right after budding,where a great increase in plasma membrane and cell wall synthesis,and an optimal transport of these components to the bud,is necessary to grow as efficiently as possible. Therefore, we hypothesized that cell size set at START would have important implications for the optimal growth and development of the bud in the S-G2 phases of the yeast cell cycle. To test this notion,we carefully analyzed growth kinetics in S-G2 phases and observed that the growth rate of the bud does not increase proportionally to the mother cell size, particularly at very large cell volumes.This effect did not take place during Gl,where we found that growth rate is proportional to cell size, exquisitely following the rules of exponential growth. Notably, the abovementioned effect was not observed either in diploid or tetraploid cells where mother cell size is progressively larger,but proportional to genomic DNA. These data suggested that low growing efficiencies of large haploid mother cells could be caused by some sort of gene insufficiency.For instance,if expression of some genes playing prominent roles in bud growth would not scale with size,rate-limiting amounts of the encoded proteins would be produced and,hence,the efficiency of the corresponding processes would be seriously compromised. One of the most important mechanisms for bud growth is the transport of vesicles from the mother cell. Indeed,we observed problems in transport,because it did not occur at a rate proportional to mother cell volume.However, it was not a matter of the transport machinery itself, since the Myo2 vesicle transporter did not show a decreased expression. Moreover,we did not observe either a deficiency in the number of actin cables at large cell volumes, indicating that the actin-based transport system was not limiting. For all these reasons, we concluded that the problem could reside in the vesicle contents, the cargo. Therefore, we studied representative proteins needed for active growth in the Gl/S transition and found that their cell concentration was reduced at large volumes. As a central set of experiments of this work, we compared the transcriptomes of small and large cells while performing the Gl/S transition, and found many genes belonging to specific functional categories that were upregulated or downregulated in large cells compared to small cells.Among those we found genes related to apical growth, vesicle transport, cytoskeleton, but especially to ribosomal biogenesis, which represents an extraordinary energetic cost, and must be extremely well regulated for optimal growth. Finally, for paradigmatic genes in these categories, we performed a careful analysis of gene insufficiency and confirmed their limiting effects on bud growth. In all, our results demonstrate that S.cerevisiae has established a critical size to achieve an optimal bud growth. At unusually larger sizes,the expression of ribosomal protein-related genes, as well as essential components for apical growth, is clearly reduced and, as a consequence,results in serious cell growth limitation.

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells., Graphical abstract Graphical Abstract. Sorafenib reduced ROS/RNS cytoplasmic concentrations which were associated with decreased Nrf2 nuclear translocation, Trx1 expression/activity, and SNO–NOS3 expression and NO intracellular generation in HepG2. The reduction of SNO–NOS3 expression and NO generation might also be influenced by the induction of transient GSNOR expression/activity and reduced NOS3 expression by Sorafenib. The diminution of SNO-CD95 expression by Sorafenib involved reduction of caspase-8 activity and cell proliferation, and increased downstream caspase-3 activity in liver cancer cells.Image 1, Highlights • Sorafenib induces mitochondrial ROS generation, but also acts as nucleophilic scavenger. • Sorafenib reduces Nrf2-depenent Trx1 expression, and SNO–NOS3 and SNO-CD95 ratios. • Sorafenib-related antitumoral in vivo activity involves diminution of Trx1 and SNO-CD95.

Resumen Objetivo: Reportar un caso sobre el uso de la terapia de presión negativa como tratamiento alternativo en el manejo de una úlcera vascular arterial de miembros inferiores. Métodos: Por medio de la historia clínica, se revisó los registros previos de una paciente en un centro de atención médica de tercer nivel en Popayán, Colombia. Posteriormente, se realizó una revisión sistemática bibliográfica a través de las bases de datos de Pubmed, ScienceDirect y Scielo. Resultados: La terapia de presión negativa favoreció el proceso de cicatrización, permitiendo el cierre de la herida. Conclusiones: Actualmente, este sistema se considera un apoyo fundamental en el tratamiento de las heridas complejas, las cuales suponen un reto terapéutico importante. Abstract Objective: To report a case about the use of the Negative-pressure wound therapy as an alternative treatment for arterial vascular ulcers presented in the lower limbs. Methods: The patient’s records were reviewed based on her medical history in a tertiary referral care center in Popayan, Colombia. Subsequently, a systematic literature review was conducted through the Pubmed database, ScienceDirect, and Scielo. Results: The use of Negative-pressure wound therapy favored the lesion healing process. Conclusions: Nowadays, this system is considered as an essential treatment for complex wounds, which represent a major therapeutic challenge.

La endometriosis (EDT) es una de las enfermedades ginecológicas benignas más comunes en mujeres en edad reproductiva y se define por la presencia de tejido endometrial fuera de la cavidad uterina. Debido a la cuestionada eficiencia de la terapéutica actual y basándonos en trabajos previos de nuestro grupo, orientamos la búsqueda de terapias alternativas hacia compuestos naturales que puedan administrarse por períodos prolongados sin causar efectos adversos. El objetivo general de esta tesis consistió en evaluar el efecto del ácido elágico y dos de sus metabolitos, urolitina A y B; sobre el crecimiento de lesiones endometriósicas en un modelo murino de EDT. Asimismo, se evaluaron sus efectos sobre el crecimiento y comportamiento de células epiteliales y estromales endometriales in vitro. Los resultados obtenidos resultan prometedores ya que todos los tratamientos, especialmente con los metabolitos, provocaron efectos inhibitorios sobre el comportamiento celular y el desarrollo de la EDT, in vitro; así como las urolitinas demostraron tener efectos antiproliferativos y proapoptóticos in vivo. Estos resultados avalan continuar investigando los mecanismos de acción de estos compuestos, ya que podrían representar una nueva estrategia terapéutica potencial para el tratamiento de la EDT. Endometriosis (EDT) is one of the most common benign gynecological diseases in women of reproductive age and is defined by the presence of endometrial tissue outside the uterine cavity. Due to the questioned efficiency of the current therapeutics, and based on previous work made by our research group, we focused our searching of alternative therapies towards natural compounds that can be administered for prolonged periods without causing adverse effects. The general objective of this thesis was to evaluate the effect of ellagic acid and two of its metabolites, urolithin A and B, on the growth of endometriotic lesions in a murine EDT model. Its effects on the growth and behavior of endometrial stromal and epithelial cells were also evaluated in vitro. The results obtained are promising because all treatments, especially metabolites, caused inhibitory effects on cell behavior and the development of EDT in vitro; Urolithins also showed antiproliferative and proapoptotic effects in vivo. These results support further research into the mechanisms of action of these compounds, as they could represent a potential new therapeutic strategy for the treatment of EDT. Fil: Mc Cormack, Bárbara Andrea. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Los tumores de células de Leydig (TCL) son tumores endócrinos que se originan principalmente en el intersticio testicular, y en menor proporción en ovario, glándula adrenal y cordón espermático. Según reportes recientes asociados a una mejora en las técnicas de diagnóstico, la incidencia de los TCL testiculares se halla en aumento. Estos tumores se asocian a un 48% de infertilidad masculina, y si bien suelen ser benignos, cuando malignizan no responden a las terapias disponibles actualmente, dando como resultado una sobrevida promedio de solamente dos años. Más aun, múltiples trabajos reportan la reaparición de TCL hasta diez años después de su remoción, sugiriendo así la presencia de células remanentes en otros órganos al momento del tratamiento. Los TCL se caracterizan por una sobreproducción de esteroides, cuyo resultado es la pseudo-pubertad precoz en niños y la disfunción eréctil o ginecomastia, en adultos. En conjunto, lo expuesto evidencia la necesidad de establecer nuevos enfoques terapéuticos. La histamina (HA), amina biogénica sintetizada únicamente por la enzima histidina decarboxilasa (HDC), ha sido descripta en múltiples patologías como factor de crecimiento autócrino, además de su conocido rol como neurotransmisor y modulador de la inflamación. A su vez, ha sido relacionada con el síndrome carcinoide, en donde en forma secundaria a la patología de origen, como ser los tumores secretores de HA, el paciente experimenta diarrea, hipotensión, taquicardia, vasoconstricción, disnea, fibrosis retroperitoneal e incluso falla cardíaca. En nuestro laboratorio hemos descripto la sobreexpresión de HDC en células de Leydig (CL) tumorales MA-10, las cuales también presentan un aumento significativo en la actividad de la enzima en comparación con las CL normales. A su ez, observamos que en dichas células la HA es capaz de estimular la proliferación celular, y que ejerce un efecto modulador sobre la esteroidogénesis. En base a las consideraciones descriptas, el objetivo de este trabajo fue investigar el papel de la HA en la progresión de los TCL, su efecto sobre la neoangiogénesis tumoral, y la inhibición de HDC como potencial estrategia terapéutica, tanto para disminuir el tamaño tumoral como para evitar las complicaciones asociadas al tumor y a los tratamientos actuales. En este sentido, la orquiectomía radical y/o la quimioterapia utilizadas de rutina en la clínica conllevan efectos secundarios físicos y psicológicos, que afectan la calidad de vida de los pacientes. En primer término, realizamos ensayos in vitro en dos líneas celulares de TCL con diferente fenotipo (las CL de ratón MA-10, que secretan como esteroide mayoritario progesterona ante la estimulación con LH, y las CL de rata R2C, que secretan estradiol de forma constitutiva), y observamos que la HA incrementó la proliferación celular, la neoangiogénesis y la síntesis de esteroides. Además, las CL R2C presentaron elevada expresión de HDC, al igual que habíamos reportado previamente para las CL MA-10. En concordancia con estos resultados, observamos intensa marcación positiva para HDC en cortes de testículo de pacientes portadores de TCL, no así en controles normales de la misma edad. A su vez, la inhibición de HDC utilizando dos inhibidores, α-MHD y EGCG (sintético y natural, respectivamente) resultó en una disminución de la proliferación celular y la síntesis de esteroides, así como también disminuyó la producción de factores pro-angiogénicos por parte de las CL tumorales. Para los estudios in vivo se establecieron dos modelos de TCL murinos a partir de las líneas de CL tumorales citadas. En ambos modelos se evaluó el efecto de α-MHD y EGCG, solos o en combinación con Carboplatino (CP). En el caso de los TCL generados a partir de las CL MA-10, α-MHD fue capaz de disminuir el tamaño tumoral, solo y en combinación con CP, a diferencia de EGCG que no mostró efecto sobre el crecimiento del tumor en los tiempos y esquema de dosis estudiados. Por su parte, el crecimiento de los TCL generados a partir de las CL R2C sólo disminuyó post tratamiento con α-MHD o EGCG en combinación con CP; los inhibidores administrados solos no tuvieron efecto. Puntualmente la combinación de EGCG con CP fue la más eficaz, dado que el crecimiento de los tumores se detuvo a tiempos tempranos. A pesar de no observarse efecto de ninguno de los inhibidores solos sobre el crecimiento tumoral en los tiempos evaluados, se registró una disminución en los niveles de esteroides plasmáticos, demostrando así su capacidad de contrarrestar el desbalance hormonal asociado la presencia de un tumor endócrino. En conjunto, los resultados de estos ensayos dan cuenta de la necesidad de establecer a futuro a qué fenotipo pertenecen los pacientes con TCL, a fin de definir el inhibidor de HDC que será más efectivo. Con base en los hallazgos que aquí se describen, el presente trabajo demuestra por primera vez el rol de la HA en el crecimiento de los TCL, y sienta las bases para el desarrollo de una estrategia terapéutica novedosa, menos agresiva que las utilizadas actualmente, que apunte a la inhibición de HDC. Las publicaciones relacionadas con el efecto protector del EGCG sobre las gametas masculinas en pacientes sometidos a tratamientos oncológicos subrayan la importancia del presente trabajo, y validan el uso de EGCG en combinación con las terapias de rutina. Leydig cell tumors (LCT) are endocrine tumors that originate primarily in the testicular interstitium, and to a lesser extent in the ovary, adrenal gland and spermatic cord. According to recent reports associated with an improvement in diagnostic techniques, the incidence of testicular LCT is increasing. These tumors are associated with 48% male infertility, and although they tend to be benign, when they become malignant they do not respond to currently available therapies, resulting in an average survival of only two years. Moreover, multiple studies have reported the reappearance of LCT up to ten years after its removal, suggesting the presence of remaining cells in other organs at the time of treatment. LCTs are characterized by an overproduction of steroids, the result of which is early pseudo-puberty in children and erectile dysfunction or gynecomastia in adults. All in all, the evidence above demonstrates the need to establish new therapeutic approaches. Histamine (HA), a biogenic amine synthesized solely by histidine decarboxylase (HDC) enzyme, has been described in multiple pathologies as an autocrine growth factor, in addition to its well-known role as a neurotransmitter and modulator of inflammation. Also, HA has been related to the carcinoid syndrome, a paraneoplastic syndrome which causes the patient to experience diarrhea, hypotension, tachycardia, vasoconstriction, dyspnea, retroperitoneal fibrosis and even heart failure. In our laboratory we have described the overexpression of HDC in MA-10 tumor Leydig (LC) cells, which also have significantly increased enzyme activity compared to normal LC. Also, we observed that in these cells HA is capable of stimulating cell proliferation, and that it can modulate steroidogenesis. Based on the above, the objective of this thesis was to investigate the role of HA in the progression of LCT, its effect on tumor neo-angiogenesis, and the inhibition of HDC as a potential therapeutic strategy not only to reduce tumor growth but also to minimize complications related to the tumor itself and to current treatments. In this sense, radical orchiectomy and / or chemotherapy routinely used in the clinic have physical and psychological side effects that affect the life quality of the patients. Firstly, we performed in vitro assays in two LCT cell lines with different phenotype (MA-10 mouse LCs, which secrete progesterone upon stimulation with LH as a major steroid, and R2C rat LCs, which secrete estradiol in a constitutive manner), and we observed that HA increased cell proliferation, neo-angiogenesis and steroid synthesis. In addition, R2C LCs showed high HDC expression, as we had previously reported for MA-10 LCs. In accordance with these results, we observed intense positive HDC staining in testis sections of patients with LCT, but not in normal controls of the same age. Additionally, inhibition of HDC using two inhibitors, α-MHD and EGCG (synthetic and natural, respectively) resulted in a decrease in cell proliferation and steroid synthesis, as well as in a decreased production of pro-angiogenic factors by tumor LC. For in vivo studies, two murine LCT models were established based on the aforementioned tumor LC lines. In both models we evaluated the effect of α-MHD and EGCG, as single agents or in combination with Carboplatin (CP). In the case of the LCT generated from MA-10 LCs, α-MHD was able to decrease the tumor size, alone and in combination with CP, unlike EGCG which showed no effect on tumor growth over the time and dosing scheme studied. On the other hand, the growth of the LCT generated from R2C Lcs only decreased after treatment with α-MHD or EGCG in combination with CP; the inhibitors administered alone had no effect. Particularly, the combination of EGCG with CP was the most effective, since the growth of the tumors stopped at early stages. In spite of not observing an effect of any of the inhibitors alone on tumor growth in the evaluated times, we did see a decrease in plasma steroid levels, thus demonstrating their ability to counteract the hormonal imbalance associated with the presence of an endocrine tumor. Together, the results of these work evidence the need to establish the phenotype to which patients baring LCT belong, in order to define which HDC inhibitor would be most effective. Based on the findings described herein, this work demonstrates for the first time the role of HA in the growth of TCL, and sets the basis for the development of a novel therapeutic strategy, less aggressive than the ones currently used, directed to HDC inhibition. Recent publications related to the protective effect of EGCG on male gametes in patients undergoing cancer treatments underline the importance of this work, and validate the use of EGCG in combination with current therapies. Fil: Abiuso, Adriana María Belén. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Las células madre amnióticas epiteliales (hAECs) son aisladas del amnios de la placenta humana a término. Son pluripotentes, no son tumorigénicas y poseen propiedades inmunomoduladoras. Estas características las vuelven candidatas ideales para la medicina regenerativa. La falla hepática es una de las mayores causas de morbilidad y mortalidad en el mundo. Si bien el trasplante representa el mejor tratamiento, existen varios obstáculos al respecto. Recientemente, las hAECs han sido propuestas como fuente alternativa de hepatocitos debido a su potencial de diferenciación hepatogénica. Por otro lado, el amnios presenta potencial terapéutico para tratar diferentes enfermedades, incluido el cáncer. Varios estudios indican que la membrana amniótica posee propiedades antiproliferativas, antiangiogénicas y proapoptóticas, concertando de esta manera una actividad antitumoral. El carcinoma hepatocelular (HCC) es el tercer tipo de cáncer que más muertes causa a nivel mundial. Actualmente, se buscan tratamientos alternativos a la radio o quimioterapia, con mayor efectividad. El presente trabajo de Tesis ha sido desarrollado con el objetivo de estudiar y caracterizar a las hAECs durante su diferenciación hepática, evaluando los mecanismos celulares relativos a la proliferación y a los caminos de transducción de señales activados, así como su potencial regenerativo. Por otro lado, se analizaron las propiedades antiproliferativas del medio condicionado de la membrana amniótica (MC-MA) en modelos de hepatocarcinoma, evaluando su potencial antitumoral. En primer lugar, demostramos utilizando un protocolo con factores de diferenciación (DH), que las hAECs se diferencian eficientemente a células símil-hepáticas, incrementándose la expresión de marcadores hepáticos y la síntesis de glucógeno, así como la proliferación y la progresión del ciclo celular; y activándose las vías de MAPK y de PI3K. El tratamiento de las hAECs con medio condicionado de hepatocitos en cultivo (MC) no resultó eficiente para inducir la diferenciación hepática y la proliferación de las hAECs. En este contexto, nos planteamos evaluar la capacidad regenerativa de las hAECs diferenciadas con el medio DH, en un modelo murino de daño hepático inducido con CCl4. Observamos que el trasplante de hAECs diferenciadas disminuye parámetros característicos de la fibrosis hepática como la necrosis, los depósitos de colágeno y los niveles de enzimas hepáticas. En conjunto, nuestros resultados evidencian la potencial aplicación de las hAECs diferenciadas en el tratamiento de enfermedades hepáticas en el contexto de la medicina regenerativa. Por otro lado, demostramos que el MC-MA disminuye significativamente la proliferación y la viabilidad de células HepG2 y HuH-7 y regula la expresión de microARNs involucrados en el desarrollo del HCC, contribuyendo a su posible efectiva acción antitumoral. Nuestros estudios amplían el conocimiento acerca de las propiedades antitumorales de la membrana amniótica, representando un avance para los futuros tratamientos alternativos contra el HCC. Amniotic epithelial stem cells (hAECs) can be isolated from the amnion of the human placenta at term. They are pluripotent, they are not tumorigenic and they have immunomodulatory properties. These characteristics would make it ideal candidates for regenerative medicine. Hepatic failure is one of the major causes of morbidity and mortality in the world. Although liver transplantation is the best treatment, there are several obstacles. Recently, hAECs have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. On the other hand, the amnion has therapeutic potential to treat different diseases, including cancer. Several studies indicate that the amniotic membrane possesses antiproliferative, antiangiogenic and proapoptotic properties, thus leading to an antitumoral activity. Hepatocellular carcinoma (HCC) is the third type of cancer that causes more deaths worldwide. Currently, alternative treatments to radio- or chemotherapy have been searched. This work has been developed in order to study and to characterize the hAECs during their hepatic differentiation. In particular, we evaluated the cellular mechanisms regarding proliferation and the signaling pathways involved, as well as their regenerative potential. Moreover, we analyzed the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma models, as part of its antitumoral potential. First, we demonstrated by applying a protocol with differentiation factors (HD), that hAECs efficiently differentiate to hepatic like cells, increasing hepatic markers expression and glycogen synthesis, as well as proliferation and cell cycle progression. The MAPK and PI3K pathways are activated during this differentiation process. The treatment of hAECs with conditioned medium of cultured hepatocytes (CM) was not efficient to induce neither hepatic differentiation nor proliferation of hAECs. In order to validate the regenerative capacity of the differentiated hAECs, we evaluated these hepatic like cells in a CCl4-induced mouse liver injury model. We observed that the differentiated hAECs transplantation decreases liver fibrosis characteristic parameters such as necrosis, collagen deposition and liver enzyme levels. Taken together, our results show the potential application of hepatocyte differentiated hAECs in the treatment of liver diseases. On the other hand, we demonstrated that AM-CM significantly decreases the proliferation and viability of HepG2 and HuH-7 cells and regulates the microRNAs expression involved in the HCC physiology, all together contributing to a possible effective anticancer action. Our studies increase the knowledge about the amniotic membrane antitumoral properties, representing an innovation for future alternative treatments against HCC. Fil: Riedel, Rodrigo Nicolás. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Podoplanin (Pdpn), a brain-tumor-related glycoprotein identified in humans and animals, is endogenously expressed in several organs critical for life support such as kidney, lung, heart and brain. In the brain, Pdpn has been identified in proliferative nestin-positive adult neural progenitor cells and in neurons of the neurogenic hippocampal dentate gyrus (DG), a structure associated to anxiety, critical for learning and memory functions and severely damaged in people with Alzheimer’s Disease (AD). The in vivo role of Pdpn in adult neurogenesis and anxiety-like behavior remained however unexplored. Using mice with disrupted Pdpn gene as a model organism and applying combined behavioral, molecular biological and electrophysiological assays, we here show that the absence of Pdpn selectively impairs long-term synaptic depression in the neurogenic DG without affecting the CA3-Schaffer’s collateral-CA1 synapses. Pdpn deletion also enhanced the proliferative capacity of DG neural progenitor cells and diminished survival of differentiated neuronal cells in vitro. In addition, mice with podoplanin gene disruption showed increased anxiety-like behaviors in experimentally validated behavioral tests as compared to wild type littermate controls. Together, these findings broaden our knowledge on the molecular mechanisms influencing hippocampal synaptic plasticity and neurogenesis in vivo and reveal Pdpn as a novel molecular target for future studies addressing general anxiety disorder and synaptic depression-related memory dysfunctions., C, PU and FM were supported by the Austrian Science Fund [FWF: Project Number P_27551].

Resumen El interés y la necesidad de estudiar las células madre en terapias regenerativas han aumentado en los últimos años debido a su capacidad de proliferación y diferenciación hacia múltiples linajes. Desafortunadamente, el número de células demandado para un trasplante satisfactorio es mayor al que se logra extraer directamente del paciente, por lo que se deben realizar cultivos in vitro de células madre. Sin embargo, con el tiempo las células de estos cultivos se vuelven senescentes, disminuyendo así su número de divisiones celulares y su capacidad proliferativa. Para solucionar esta problemática se han propuesto factores de crecimiento como agentes potenciadores de la proliferación celular. Entre estos se encuentra el factor de crecimiento fibroblástico 2, que al agregarse al medio podría promover la proliferación y aumentar el tiempo de vida de las células en cultivo. En la siguiente revisión se recopila información sobre la biología de este factor de crecimiento y el tipo de señalización que utiliza, al igual que sus aplicaciones en terapia regenerativa y su efecto en la proliferación celular y senescencia. Abstract The interest and need to study stem cells in regenerative therapies has increased in recent years due to their ability to proliferate and differentiate into multiple lineages. Unfortunately, the number of cells needed for a successful transplant is greater than that obtained directly from the patient, so in vitro cultures of stem cells must be done. However, over time the cells in these cultures become senescent, decreasing their number of cellular divisions and their proliferative capacity. To solve this problem, different growth factors have been proposed as promoting agents for cell proliferation. Among these is the fibroblast growth factor 2 which, when added to the medium, could promote proliferation and increase the life time of the cells in the culture. The following review compiles information about the biology of this growth factor and the type of signaling it uses, as well as its applications in regenerative therapy and its effect on cell proliferation and senescence.

A high adherence to a Mediterranean diet has been related to numerous beneficial effects in human health, including a lower incidence and mortality of prostate cancer (PCa). Olive oil is an important source of phenolic bioactive compounds, mainly hydroxytyrosol (HT), of this diet. Be-cause of the growing interest of this compound and its derivatives as a cancer chemopreventive agent, we aimed to compare the in vitro effect of HT isolated from olive mill wastewaters and five semisynthetic alkyl ether, ester, and nitro-derivatives against prostate cancer (PCa) cell lines. The effect in cell proliferation was determined in RWPE-1, LNCaP, 22Rv1, and PC-3 cells by resazurin assay, the effect in cell migration by wound healing assay, and tumorsphere and colony formation were evaluated. The changes in key signaling pathways involved in carcinogenesis were assessed by using a phosphorylation pathway profiling array and by Western blotting. Antiproliferative effects of HT and two lipophilic derivatives [hydroxytyrosyl acetate (HT-Ac)/ethyl hydroxytyrosyl ether (HT-Et)] were significantly higher in cancerous PC-3 and 22Rv1 cells than in non-malignant RWPE-1 cells. HT/HT-Ac/HT-Et significantly reduced migration capacity in RWPE-1 and PC-3 and prostatosphere size and colony formation in 22Rv1, whereas only HT-Ac and HT-Et reduced these functional parameters in PC-3. The cytotoxic effect in 22Rv1 cells was correlated with modifications in the phosphorylation pattern of key proteins, including ERK1/2 and AKT. Consistently, HT-Ac and HT-Et decreased p-AKT levels in PC-3. In sum, our results suggest that HT and its lipophilic derivatives could be considered as potential therapeutic tools in PCa. Junta de Andalucía PI-0152-2019, BIO-0139, RH-0084-2020 Ministerio de Economía y Competitividad PID2019-105564RB-I00, PID2019-105201RB-I00, FPU17/00263, FPU16/06190, FPU18/02485

The scaffolds and their interaction with mesenchymal stem cells are objects of study in bioengineering and tissue repair. Mechanisms such as surface adhesion, proliferation, viability, and cytotoxicity are essential for the development of therapies. The present study analyzed the influence of platelet-rich fibrin (PRF) in viability, cytotoxicity, and proliferation of stem cells from human exfoliated deciduous teeth (SHED) exposed to bovine biomaterial surfaces. The studied groups were divided and analyzed as follows: (S) only SHED as control Group; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. Analyses of cells seeded in 24-well plates were performed after 24, 48 and 72 hours. Individual groups were subjected to viability, cytotoxicity and cell proliferation tests using neutral red, MTT and crystal violet, respectively; and in the 72-hour group, scanning electron microscopy (SEM) was performed to record cell ultra-morphology. Data were submitted to statistical analysis by two-factor ANOVA with a significance level of 5%. The results demonstrated a better performance in the viability/cytotoxicity and proliferation of stem cells for the group (SBP) in comparison to the group (SB) and the group (S). The applied statistical tests showed that the biomaterial factor, time, and interaction between them gave rise to results with statistical significance. SHED submitted to bovine biomaterial were more viable, proliferative and with lower toxicity when associated with PRF. PRF seemed to activate the metabolism of stem cells in culture, indicating that such an association can bring an effective benefit in clinical outcome. El marco y su interacción con las células madres mesenquimales son objetos de estudio en bioingeniería y reparación de tejidos. Mecanismos como la adhesión superficial, la proliferación, la viabilidad y la citotoxicidad son fundamentales para el desarrollo de terapias. El presente estudio analizó la influencia de la fibrina rica en plaquetas (PRF) sobre la viabilidad, citotoxicidad y proliferación de células madre mesenquimales de dientes primarios humanos exfoliados (SHED) en contacto con biomateriales bovinos. Los grupos estudiados se dividieron y analizaron de la siguiente manera: (S) solo SHED como grupo de control; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. Se realizaron análisis de células sembradas en placas de 24 pocillos después de 24, 48 y 72 horas. Los grupos individuales se sometieron a pruebas de viabilidad, citotoxicidad y proliferación celular utilizando rojo neutro, MTT y violeta cristal, respectivamente, y en el grupo de 72 horas, se realizó microscopía electrónica de barrido (SEM) para registrar la morfología celular. Los datos se sometieron a análisis estadístico mediante ANOVA de dos factores con un nivel de significancia del 5%. Los resultados demostraron un mejor desempeño en la viabilidad / citotoxicidad y proliferación de células madre para el grupo (SBP) en comparación con el grupo (SB) y el grupo (S). Las pruebas estadísticas aplicadas mostraron que el factor biomaterial, el tiempo y la interacción entre ellos dieron lugar a resultados con significación estadística, las SHED sometidas a biomaterial bovino fueron más viables, proliferativos y con menor toxicidad cuando se asociaron con PRF. PRF parecio activar el metabolismo de las células madres en cultivo, lo que indica que tal asociación puede aportar un beneficio efectivo en los resultados clínicos. O arcabouço e a sua interação com as células-tronco mesenquimais são objetos de estudo em bioengenharia e reparo de tecidos. Mecanismos como adesão superficial, proliferação, viabilidade e citotoxicidade são fundamentais para o desenvolvimento de terapias. O presente estudo analisou a influência da fibrina rica em plaquetas (PRF) na viabilidade, citotoxicidade e proliferação de células-tronco mesenquimais de dentes decíduos humanos esfoliados (SHED) em contato com biomaterial de origem bovina. Os grupos estudados foram divididos e analisados ​​da seguinte forma: (S) apenas SHED como Grupo controle; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. As análises das células semeadas em placas de 24 poços foram realizadas após 24, 48 e 72 horas. Grupos individuais foram submetidos a testes de viabilidade, citotoxicidade e proliferação celular usando vermelho neutro, MTT e cristal violeta, respectivamente, e no grupo de 72 horas foi realizada a microscopia eletrônica de varredura (MEV) para registrar a ultra morfologia celular. Os dados foram submetidos à análise estatística por ANOVA de dois fatores com nível de significância de 5%. Os resultados demonstraram um melhor desempenho na viabilidade / citotoxicidade e proliferação de células-tronco para o grupo (SBP) em relação ao grupo (SB) e ao grupo (S). Os testes estatísticos aplicados mostraram que o fator biomaterial, o tempo e a interação entre eles deram origem a resultados com significância estatística. As SHED submetidas ao biomaterial bovino se apresentaram mais viáveis, proliferativas e com menor toxicidade quando associadas à PRF. A PRF pareceu ativar o metabolismo das células-tronco em cultivo, indicando que tal associação pode trazer um benefício efetivo nos desfechos clínicos.