Cell cycle regulation after pharmacological inhibition of the enzyme fatty acid synthase in murine melanoma line derived from, B16-F10

Orientador: Karina Gottardello ZecchinDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Ácido graxo sintase (FASN - fatty acid synthase) é a enzima metabólica responsável pela síntese endógena do ácido graxo saturado palmitato, a partir dos precursores acetil-CoA e malonil-CoA. Diversos estudos mostram que, em contraste com a maioria das células normais, FASN é altamente expressa em vários tipos de neoplasias malignas humanas, tais como as de próstata, mama e melanoma sendo que, em alguns destes tumores, a alta expressão de FASN está associada a um pior prognóstico. Anteriormente demonstramos que a inibição específica da atividade de FASN reduz significativamente a proliferação celular e promove a apoptose em linhagem celular de melanoma murino, B16-F10. O objetivo deste trabalho foi investigar de que maneira a inibição farmacológica de FASN reduz a proliferação de células B16-F10, utilizando a cerulenina, um produto natural do fungo Cephalosporium caerulens, como inibidor de FASN. O tratamento com cerulenina reduziu significativamente a proliferação das células B16-F10 de maneira dose-dependente. Ensaios de lipogênese utilizando 3H2O confirmaram a inibição da atividade de FASN pelo tratamento com cerulenina. Tal inibição resultou em significativo bloqueio da proliferação, conforme evidenciado pelo aumento do número de células nas fases G0/G1, assim como redução de células na fase S, em comparação com os controles. Paralelamente, o tratamento com cerulenina também aumentou o número de células em apoptose. Western blottings, feitos a partir de extratos de células tratadas com cerulenina, mostraram aumento significativo da proteína supressora de tumor p21WAF1/Cip1, assim como redução de cdk2, uma Ser/Thr necessária para a transição G1/S, e Skp2, uma proteína necessária para a degradação proteossômica de p27Kip1. Apesar de não ter alterado o conteúdo total de p27Kip1, a inibição de FASN aumentou a quantidade de p27Kip1 co-imunoprecipitada com cdk2. Por outro lado, o tratamento com cerulenina não alterou o conteúdo de outras proteínas envolvidas na progressão das fases G1-S do ciclo celular, tais como cdk4, cdk6, Rb total, ciclina D1 e ciclina E. Em conjunto estes resultados demonstram que a inibição de FASN primeiramente altera os níveis de proteínas envolvidas na transição de G1 para S, tais como p21WAF1/Cip1, p27Kip1 e Skp2, e posteriormente induz apoptose em células de melanoma murino B16-F10Abstract: Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid palmitate, from the precursors acetyl-CoA and malonyl-CoA. In contrast to most normal cells, the overexpression of FASN in several human malignancies, such as those of prostate, breast, ovary, melanoma, and soft tissue sarcomas has been associated with poor prognosis. We have previously shown that the specific inhibition of FASN activity significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16-F10. Here we investigated the events involved in cell cycle arrest subsequent to pharmacological FASN inhibition with cerulenin, a natural antifungal antibiotic obtained from Cephalosporium caerulens, in B16-F10 cells. Cerulenin treatment significantly reduced melanoma cells proliferation in a dose dependent manner. Lipogenesis using 3H2O confirmed inhibition of FASN activity after cerulenin treatment. Such enzymatic inhibition culminated in cell cycle arrest, evidenced by a significant increase in G0/G1 phase, as well as decline of the S phase, in comparison with untreated cells. Cerulenin treatment also induced apoptosis in B16-F10 tumor cells. Western blotting analysis of cerulenin-treated cells showed a significant accumulation of the tumor suppressor proteins p21WAF1/Cip1 and p27Kip1, together with decreased amounts of cdk2, a Ser/Thr protein kinase necessary for the G1/S transition, and Skp2, essential for the proteasomal degradation of p27Kip1. Cerulenin treatment increased the levels of p27Kip1 co-immunoprecipitated with cdk2, despite western blotting analysis showed similar content of total p27Kip1. The levels of other proteins involved in G1/S cell cycle progression, such as cdk4, cdk6, total Rb, cyclins A and E, were not affected by FASN inhibition. Collectively these findings suggest that FASN inhibition first modify the levels of proteins involved in transition G1-S, as p21WAF1/Cip1, p27Kip1 and Skp2, to finally induce apoptosis in mouse melanoma B16-F10 cellsMestradoEstomatologiaMestre em Estomatopatologi

Efecto de los ácidos eicosapentaenoico y docosahexaenoico sobre marcadores de obesidad en ratas macho con una dieta alta en grasa

Eicosapentaenoic acids (EPA) and docosahexaenoic (DHA) are omega-3 polyunsaturated fatty acids With the beneficial effects on preventing various diseases in which the inflammation plays a fundamental role. Since these omega - 3 fatty acids are ligands for PPARs, they could have an impact on metabolic activity. Therefore, they could exert a beneficial effect on individuals with obesity. The objective of this project is to evaluate the effect of EPA and DHA on obesity markers in rats with a high-fat diet. For this purpose, 25 male Wistar rats were fed a standard diet (n = 5) and a high-fat diet (n = 20) for 16 weeks. Subsequently, the group with a high-fat diet was randomly segmented into 4 subgroups to receive the following treatment for 8 weeks: HFD (vehicle); HFD + Omega-3; HFD + Fenofibrate and HFD + Pioglitazone. The biochemical profile (glucose, triglycerides and cholesterol), the zoometric parameters (weight, waist diameter, snout-anus length and index lee) and the weight of the adipose tissue (mesenteric, epididymal, retroperitoneal and subcutaneous) were measured. Furthermore, the concentration of insulin and adiponectin in the blood was determined and the HOMA –IR index was calculated. The administration of EPA and DHA promoted a decrease in the serum concentration of triglycerides and glucose. Moreover, it decreased the HOMA-IR index, the percentage of adiposity and the weight of the subcutaneous adipose tissue. Finally, when contrasting the effect of Omega-3 with the positive controls (fenofibrate and pioglitazone) on the variables, it can be observed that its administration modifies the concentration of triglycerides and glucose in a similar way, without exerting an effect on the zoometric parameters.Los ácidos eicosapentaenoico (EPA) y docosahexaenoico (DHA) son ácidos grasos poliinsaturados omega – 3 con potencial para prevenir o tratar de una serie de patologías. Especialmente aquellas donde la inflamación juega un papel preponderante en su desarrollo. Debido a que estos ácidos grasos de omega -3 son ligandos de los PPARs podrían tener impacto sobre la actividad metabólica. Por tanto, podrían ejercer un efecto benéfico sobre individuos con obesidad. El objetivo del presente proyecto, es evaluar el efecto del EPA y el DHA sobre marcadores de obesidad en ratas con dieta alta en grasa. Para tal propósito, 25 ratas Wistar macho fueron alimentadas durante 16 semanas con una dieta estándar (n=5) y una dieta alta en grasa (n=20). Posteriormente el grupo con dieta alta en grasa fue segmentado aleatoriamente en 4 subgrupos para recibir el siguiente tratamiento vía oral durante 8 semanas: HFD (vehículo); HFD + Omega3; HFD + Fenofibrato y HFD + Pioglitazona. Para evaluar el efecto de la administración de EPA y DHA, se midió el perfil bioquímico (glucosa, triglicéridos y colesterol), los parámetros zoométricos (peso, diámetro de cintura, longitud hocico – ano e índice de lee) y el peso del tejido adiposo (mesentérico, epididimal, retroperitoneal y subcutáneo). Asimismo, se determinó la concertación de insulina y adiponectina en sangre. Finalmente se calculó el índice de HOMA–IR. La administración de EPA y DHA promovió una disminución de la concentración sérica de triglicéridos y glucosa. Asimismo, disminuyó el índice HOMA-IR, el 4 porcentaje de adiposidad y el peso del tejido adiposo subcutáneo. Los cambios observados sugieren modificaciones en la actividad metabólica, no obstante, no se observaron cambios sobre el peso, diámetro de cintura e índice de lee. Finalmente, al contrastar el efecto del Omega-3 con los controles positivos (fenofibrato y pioglitazona) sobre las variables, se puede observar que su administración modifica la concentración de triglicéridos y glucosa de manera semejante, sin ejercer un efecto sobre los parámetros zoométricos

Interactional patterns in the online language classroom: a quantitative analysis across proficiency levels and lesson types

Online second language instruction has boomed in recent years, aided by technological affordances and the forced changes in instructional modality resulting from the COVID-19 pandemic. This transformation has underscored the critical role of interaction in online pedagogy. Research suggests that increasing opportunities for interaction between students and instructors is essential for fostering second language acquisition (SLA). However, little research exists quantifying the production of diverse types of interaction in online language instruction, particularly among experienced instructors. The present study utilizes an interactionist framework to perform a quantitative analysis of interaction in online Spanish language coursework, categorized according to interaction initiation type: Instructor-Prompted Participation (IPP), Unprompted Oral Participation (UOP), Unprompted Text Participation (UTP), i.e., chat usage, and interaction length (as exhibited by both the number of student turns in an interaction and the average length of those turns). Data includes 18 h of video recordings of synchronous L2 Spanish language instruction across proficiency levels and lesson types at a distance-learning university in the UK. Lesson types included grammar workshops and exam preparation. Results show that the interactional patterns in online language courses are influenced by proficiency level and lesson type. Lower proficiency students engaged in interaction routines more frequently, while the ability to engage in extended discourse was contingent upon the specific activity/lesson type. The study helps address the dearth of research on interaction and language teaching in languages other than English (LOTE)

Usefulness of circulating microRNAs miR-146a and miR-16-5p as prognostic biomarkers in community-acquired pneumonia

Introduction Patients with community-acquired pneumonia (CAP) undergo a dysregulated host response that is related to mortality. MicroRNAs (miRNAs) participate in this response, but their expression pattern and their role as biomarkers in CAP have not been fully characterized. Methods A prospective observational study was performed in a cohort of 153 consecutive patients admitted to hospital with CAP. Clinical and analytical variables were collected, and the main outcome variable was 30-day mortality. Small RNA was purified from plasma of these patients obtained on the first day of admission, and miRNA expression was analyzed by RTPCR. Univariate and multivariate analyses were carried out through the construction of a logistic regression model. The proposed model was compared with established prognostic clinical scales using ROC curve analysis. Results The mean age of the patients included was 74.7 years [SD 15.9]. Their mean PSI was 100.9 [SD 34.6] and the mean modified Charlson index was 2.9 [SD 3.0]. Both miR-146a and miR- 16-5p showed statistically significant association with 30-day mortality after admission due to CAP (1.10 vs. 0.23 and 51.74 vs. 35.23, respectively), and this association remained for miR-16-5p in the multivariate analysis adjusted for age, gender and history of bronchoaspiration (OR 0.95, p = 0.021). The area-under-the-curve (AUC) of our adjusted multivariate model (AUC = 0.954 95%CI [0.91–0.99]), was better than those of prognostic scales such as PSI (AUC = 0.799 [0.69–0.91]) and CURB-65 (AUC = 0.722 [0.58–0.86]). Conclusions High levels of miR-146a-5p and miR-16-5p upon admission due to CAP are associated with lower mortality at 30 days of follow-up. Both miRNAs could be used as biomarkers of good prognosis in subjects hospitalized with CAPThis work has been funded by the Carlos III Health Institute (ERDF, European Regional Development Fund), by the Spanish Society of Pneumology and Thoracic Surgery and by the Ministry of Science, Innovation and Universities of Spain

Articulación de las TIC en docentes en formación en educación matemática

The objective of this article is to analyze the process of articulation of information and communication technologies (ICT) carried out by teachers in training in their pedagogical practices of the degree program in Mathematics of the University of the Atlantic, As these technological tools provide us with endless advantages when it comes to addressing topics in math classes, we decided to ask the following question: How are teachers in training in Mathematics at the University of the Atlantic carrying out the process of articulating ICTs in pedagogical practices?. In our work, a descriptive-comparative design was used, with a qualitative approach, in the instruments we applied a survey directed to teachers in training and a rubric directed to their current advisory teachers, in which a triangulation was made in order to compare the results obtained with the theoretical framework previously reflected in the present investigation. During the implementation of these instruments done through the virtual platform "Google Form", the information that was collected allowed us, through a descriptive-comparative design, to clearly know how this articulation process is being carried out in different educational institutions of the Atlántico department. El objetivo de este artículo es analizar el proceso de articulación de las Tecnologías de la Información y Comunicación (TIC) que llevan a cabo los docentes en formación en sus Prácticas Pedagógicas del programa de Licenciatura en Matemáticas de la Universidad del Atlántico, dado que estas herramientas tecnológicas brindan un sinfín de ventajas al momento de abordar una temática en las clases de Matemáticas. En este trabajo se abordó un diseño Descriptivo-Comparativo, con un enfoque cualitativo, en los instrumentos se aplicó una encuesta dirigida a docentes en formación y una rúbrica dirigida a sus actuales docentes asesores de las instituciones educativas donde desempeñan la labor de docentes en formación, en la que se hizo una triangulación con el fin de comparar los resultados obtenidos con el marco teórico. Durante la implementación de estos instrumentos realizados en la plataforma virtual “Formulario de Google” se fue recolectando la información que nos permitió por medio de un diseño implementado obtener con claridad cómo se está llevando a cabo este proceso de articulación en distintas Instituciones Educativas del Departamento del Atlántico. Infortunadamente, muchos docentes en formación tienen la disposición de articular las TIC en su Práctica Pedagógica, pero por falta de recursos tecnológicos en las Instituciones Educativas no les fue posible hacer uso de estas.&nbsp

Fatores associados ao atraso no diagnóstico e tratamento do câncer bucal: revisão integrativa de literatura

Oral cancer is a relevant health problem and has a high incidence and mortality rate. Tobacco and alcohol consumption are considered the main risk factors for its occurrence. Despite advances related to detection, diagnosis, and treatment most patients are still diagnosed with the disease at an advanced stage. The aim of this paper is to establish the main factors related to the delay in the diagnosis of oral cancer. The articles were searched in the MEDLINE/PubMed, Scielo and Lilacs databases, considering the following DeCS: delayed oral cancer; delayed diagnosis of oral cancer; oral cancer young patients and hpv oral cancer patients. As a criterion of inclusion, full-text articles were delimited. Publications unrelated to the theme delimitation and aim of the research were excluded. As a result, 513 articles were found: 118 were entirely read and 96 were finally selected for the research, based on the inclusion and exclusion criteria. The data indicate that the delay in the diagnosis of oral cancer is common and occurs due to patient delay, delay by health professional and treatment, but specially by patient delay. Early diagnosis is the most effective way of reducing mortality rates and improving the prognosis and quality of life of patients with oral cancer. Strategies with the aim to reduce late diagnosis should be implemented.Introdução: O câncer bucal é apontado como um problema de saúde relevante e apresenta alta taxa de incidência e mortalidade. O consumo de tabaco e álcool são considerados os principais fatores de risco para sua ocorrência. Apesar dos avanços relacionados a detecção, diagnóstico e tratamento, grande parte dos pacientes ainda é diagnosticada com a doença em estágio avançado. Objetivo: Estabelecer os principais fatores associados ao atraso no diagnóstico do câncer bucal. Material e Métodos: Foram pesquisados artigos nas bases de dados MEDLINE/PubMed, SciELO e Lilacs, com os descritores: delayed oral cancer; delayed diagnosis oral cancer; oral cancer young patients; e HPV oral cancer patients. Como critérios de inclusão, foram delimitados artigos completos e disponíveis integramente. Publicações não relacionadas a delimitação do tema e ao objetivo do estudo foram excluídas. Resultados: Foram encontrados 513 artigos, dos quais 118 foram lidos na íntegra e 96 por fim selecionados, conforme critérios de inclusão e exclusão. Os dados indicaram que o atraso no diagnóstico do câncer bucal é frequente e relacionado ao atraso pelo paciente, atraso pelo profissional de saúde e atraso no tratamento, com destaque ao atraso pelo paciente. Conclusão: O diagnóstico precoce é o meio mais eficaz de reduzir as taxas de mortalidade e melhorar o prognóstico e qualidade de vida de pacientes com câncer bucal. Dessa forma, estratégias que objetivam a redução de diagnósticos realizados tardiamente devem ser consideradas

Adaptaciones: las obras que vuelven a nacer

Desde que existe el celuloide, han sido muchísimos los directores que han tratado de plasmar otro tipo de representación artística a través de la pantalla. Algunas adaptaciones fueron grandes sucesos en la historia del cine, otras no gozaron de una aceptación generalizada. En estos textos se comentan algunas adaptaciones cinematográficas: desde la historieta, el manga, la música, la pintura o el teatro

History Games

El presente proyecto de investigación consiste en el desarrollo del aplicativo History Games, el cual consiste en la creación del juego con temática de historia del Perú como una nueva metodología de aprendizaje. Este trabajo nos permite ver la viabilidad del proyecto a corto, mediano y largo plazo, a través de los distintos estudios ejecutados en Lima metropolitana, a niños y jóvenes de 8 a 17 años del sector socioeconómico A y B.  Por ello se pudo evidenciar que el público objetivo desea aprender un poco más de nuestro Perú a través de la experiencia de este aplicativo. Al desarrollar este proyecto se realizaron entrevistas online, encuestas y marketing digital en redes sociales. De manera que fue una investigación a fondo sobre el mercado de juegos educativos para poder llevar a cabo este proyecto, donde se ha podido ver interés por parte de las instituciones educativas que desean adoptar History Games como metodología de enseñanza. Analizamos a profundidad el proyecto ya que buscamos que sea sostenible en todos los aspectos a largo plazo y que pueda cumplir con los objetivos planteados desde el inicio. Todos los conocimientos adquiridos durante nuestra etapa universitaria fueron aplicados en cada parte del trabajo como es la parte de marketing, financiera, administrativa, entre otras. Al ser un equipo de diversas carreras, nos brindó un panorama más amplio de los puntos que debemos seguir mejorando e implementando conforme el proyecto siga creciendo. History Games es una idea que sin duda ayudará a muchas personas en su aprendizaje y en la nueva forma de ver la historia en cada una de sus clases.This research project consists of the development of the History Games application, which consists of the creation of the game with the theme of Peruvian history as a new learning methodology. This work allows us to see the viability of the project in the short, medium and long term, through the different studies carried out in metropolitan Lima, to children and young people from 8 to 17 years of age from the socioeconomic sector A and B. Therefore, it was possible to show that The target audience wants to learn a little more about our Peru through the experience of this application. When developing this project, online interviews, surveys and digital marketing were carried out on social networks. So it was an in-depth investigation of the educational games market in order to carry out this project, where interest has been seen on the part of educational institutions that want to adopt History Games as a teaching methodology. We analyze the project in depth as we seek to make it sustainable in all aspects in the long term and that it can meet the objectives set from the beginning. All the knowledge acquired during our university stage was applied in each part of the work such as the marketing, financial, and administrative part, among others. Being a diverse racing team, it gave us a broader picture of the points that we must continue to improve and implement as the project continues to grow. History Games is an idea that will undoubtedly help many people in their learning and in the new way of seeing history in each of their classes.Trabajo de investigació

SARS-CoV-2 recombinant proteins stimulate distinct cellular and humoral immune response profiles in samples from COVID-19 convalescent patients

OBJECTIVES: In this preliminary study we investigated cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in blood samples from 14 recovered coronavirus disease 2019 (COVID-19) patients and compared them to those in samples from 12 uninfected/unvaccinated volunteers. METHODS: Cellular immunity was assessed by intracellular detection of IFN-γ in CD3+ T lymphocytes after stimulation with SARS-CoV-2 spike (S1), nucleocapsid (NC), or receptor-binding domain (RBD) recombinant proteins or overlapping peptide pools covering the sequence of SARS-CoV-2 spike, membrane and nucleocapsid regions. The humoral response was examined by ELISAs and/or chemiluminescence assays for the presence of serum IgG antibodies directed to SARS-CoV-2 proteins. RESULTS: We observed differences between humoral and cellular immune profiles in response to stimulation with the same proteins. Assays of IgG antibodies directed to SARS-CoV-2 NC, RBD and S1/S2 recombinant proteins were able to differentiate convalescent from uninfected/unvaccinated groups. Cellular immune responses to SARS-CoV-2 protein stimuli did not exhibit a specific response, as T cells from both individuals with no history of contact with SARS-CoV-2 and from recovered donors were able to produce IFN-γ. CONCLUSIONS: Determination of the cellular immune response to stimulation with a pool of SARS-CoV-2 peptides but not with SARS-CoV-2 proteins is able to distinguish convalescent individuals from unexposed individuals. Regarding the humoral immune response, the screening for serum IgG antibodies directed to SARS-CoV-2 proteins has been shown to be specific for the response of recovered individuals

Diverse mutations and structural variations contribute to Notch signaling deregulation in paediatric T-cell lymphoblastic lymphoma

Background T-cell lymphoblastic lymphoma (T-LBL) is an aggressive neoplasm closely related to T-cell acute lymphoblastic leukaemia (T-ALL). Despite their similarities, and contrary to T-ALL, studies on paediatric T-LBL are scarce and, therefore, its molecular landscape has not yet been fully elucidated. Thus, the aims of this study were to characterize the genetic and molecular heterogeneity of paediatric T-LBL and to evaluate novel molecular markers differentiating this entity from T-ALL. Procedure Thirty-three paediatric T-LBL patients were analyzed using an integrated approach, including targeted next-generation sequencing, RNA-sequencing transcriptome analysis and copy-number arrays. Results Copy number and mutational analyses allowed the detection of recurrent homozygous deletions of 9p/CDKN2A (78%), trisomy 20 (19%) and gains of 17q24-q25 (16%), as well as frequent mutations of NOTCH1 (62%), followed by the BCL11B (23%), WT1 (19%) and FBXW7, PHF6 and RPL10 genes (15%, respectively). This genetic profile did not differ from that described in T-ALL in terms of mutation incidence and global genomic complexity level, but unveiled virtually exclusive 17q25 gains and trisomy 20 in T-LBL. Additionally, we identified novel gene fusions in paediatric T-LBL, including NOTCH1-IKZF2, RNGTT-SNAP91 and DDX3X-MLLT10, the last being the only one previously described in T-ALL. Moreover, clinical correlations highlighted the presence of Notch pathway alterations as a factor related to favourable outcome. Conclusions In summary, the genomic landscape of paediatric T-LBL is similar to that observed in T-ALL, and Notch signaling pathway deregulation remains the cornerstone in its pathogenesis, including not only mutations but fusion genes targeting NOTCH1.We thank the centres of the Sociedad Espanola de Hematologia y Oncologia Pediatricas that submitted cases for consultation, to Noelia Garcia, Silvia Martin and Helena Suarez for their excellent technical assistance and to Nerea Dominguez for updating clinical data. We are indebted to the IDIBAPS Genomics Core Facility and to the HCB-IDIBAPS, the HospitaI Infantil Sant Joan de Deu and the Hospital Universitari Vall d'Hebron Tumour Biobanks, all integrated in the National Network Biobanks of ISCIII for the sample and data procurement. This work was supported by Asociacion Espanola Contra el Cancer (AECC CICPFI6025SALA and 'Ayudas Clinico Formacion AECC 2020' to Jaime Verdu-Amoros), Asociacion de aitas y amas para la humanizacion, socializacion e investigacion del Cancer Infantil y la divulgacion de la donacion de medula osea-La Cuadri del Hospi, Fondo de Investigaciones Sanitarias Instituto de Salud Carlos III (Miguel Servet Program I and II CP13/00159 and MSII18/00015; Itziar Salaverria), Generalitat de Catalunya Suport Grups de Recerca (2017-SGR-1107; Itziar Salaverria), and the European Regional Development Fund 'Una manera de fer Europa'. Joan Enric Ramis-Zaldivar was supported by a fellowship AGAUR FI-DGR 2017 (2017 FI_B01004) from Generalitat de Catalunya. Noelia Garcia has been continuously supported by Accio instrumental d'incorporacio de cientifics i tecnlegs PERIS 2016 (SLT002/16/00336) and PERIS 2020 (SL017/20/000204) from Generalitat de Catalunya. Julia Salmeron-Villalobos was supported by a fellowship from La Caixa (CLLEvolution-HR17-00221). This work was developed partially at the Centro Esther Koplowitz, Barcelona, Spain