Estudio molecular, celular y anatomopatológico de los tumores de la cavidad oral

Oral cavity squamous cell carcinoma (OSCC) continues to be an aggressive disease and a global challenge with a 5-year survival rate of 60%. Depending on the risk factors, surgery and adjuvant radiotherapy +/- chemotherapy continue to be the main treatment modality for local or advanced disease. Therefore, new diagnostic, prognostic biomarkers and therapeutic targets for oral cavity cancer are urgently needed. It is important to point out that it has been confirmed that there is a neuroendocrine differentiation component in some tumors that are not classically considered to be of neuroendocrine origin, including squamous cell carcinoma of the lung and esophagus, and more recently in the head and neck region. In this context, somatostatin (SST) is a well-known inhibitory neuropeptide that is produced at different locations throughout the body, both central and systemic. The inhibitory actions of SST are mediated through the so-called SST receptors (SSTs), which are widely distributed in normal and tumor tissues, and regulate, among other activities, cell alteration, differentiation, and angiogenesis in many types of tumors. This property allows the SST regulatory system to be very useful clinically as it is widely used in tumor imaging [SST scintigraphy or octreotide scintigraphy (a synthetic analog of SST that preferentially binds to SST2, the receptor most widely distributed across of the organism)]. Tumor cells usually express more than one SST, with SST2 being the most frequently expressed subtype and, therefore, the most important target of treatment. Consequently, synthetic analogs of SST (SSA) represent an attractive therapeutic target to treat tumor pathologies positive for SSTs, since it has been shown that they are capable of controlling hormonal hypersecretion and tumor growth in some types of tumor pathologies. Our current understanding of the presence of SST in squamous cell carcinoma of the oral cavity is quite scarce, and in some cases controversial. Specifically, previous studies have shown that the relative immunohistochemical expression of some SST subtypes is altered in malignant lesions in the larynx compared to healthy tissue, as well as in tumor samples from the head and neck area compared to normal samples from the larynx. oropharyngeal mucosa (obtained during uvulopalatopharyngoplasty) from other patients. However, to date, no molecular analysis has been performed to quantitatively analyze, in parallel, the expression levels (copy numbers) of all subtypes of SSTs in oral carcinoma samples compared to healthy tissue (control; within same patient) by quantitative PCR. To date, the direct effects of different SSAs on cultures of primary human oral cavity carcinoma cells have also not been studied. Therefore, based on the information mentioned above, we believe that it would be necessary to interrogate the expression patterns of SST receptors and their association with clinical, pathological and survival data of oral cavity cancer. Likewise, the molecular machinery of splicing has been shown to play a fundamental role in controlling cellular functions, and in regulating gene expression levels and tissue specificity. Furthermore, the production of splicing machinery has recently emerged as an important feature of cancer, with great potential to serve as a diagnostic, prognostic, or therapeutic tool. Thus, it has been proven that a slight alteration in some of the components of the machinery (known as a spliceosome) can significantly affect the expression pattern of many key genes and the appearance of oncogenic splicing variants. Specifically, deregulation of splicing factors that make up the spliceosome leads to dysregulation of the splicing process and aberrant appearance of variants that can promote cancer initiation and affect cancer cell phenotype, including wasting, apoptosis, invasion and metastasis of many types of cancer. In recent years, several studies have focused on the analysis and impact of some splicing factors in head and neck cancer. Their results are highly variable, even contradictory in terms of the expression pattern of some splicing factors in tumor tissues compared to healthy tissue. Some of these splicing factors have an impact on antineoplastic treatments by modulating apoptosis and allowing cancer cells to be sensitized to therapeutic treatments. These factors can act as survival factors that decrease druginduced apoptosis or, on the contrary, enhance the pro-apoptotic effects of chemotherapy drugs. For all these reasons, it is necessary to carry out a study that focuses on studying the alteration of the splicing machinery in oral cavity cancer, since this could help identify new biomarkers and molecular targets that help to better understand the behavior of these tumors and to development of new therapeutic strategies. Taken all this information together, the INITIAL HYPOTHESIS of this PhD Thesis was that the dysregulation of the SST and splicing machinery systems could directly influence OSCC, and consequently, that their levels of expression in tumor tissues could provide useful information to improve diagnosis and/or prognosis of these tumors, and to identify novel therapeutic sources to treat patients with these devastating pathologies. Based on this hypothesis, the GENERAL AIM of this Doctoral Thesis was to explore the presence, potential dysregulation, and/or functional role of components of two key cellular systems involved in critical regulatory processes [i.e., somatostatin receptors (SSTs), and by the splicing machinery (spliceosome components and splicing factors)] that could be associated with the development, progression, and aggressiveness of OSCC, with the ultimate goal of discovering novel biomarkers and therapeutic tools to improve the diagnosis, prognosis, treatment and, therefore, the management of the patients with these tumors. To achieve this main aim, we have carried out different specific objectives such as: 1) to quantitatively analyze the expression profile of SSTs in a representative battery of clinically wellcharacterized OSCC tissues in comparison with adjacent healthy tissues obtained within the same patient; 2) to assess the putative in vivo association between the expression of all SSTs in the tumor of patients with OSCC and relevant clinical and histopathological data parameters; 3) to explore and compare, side-by-side, the direct antitumor effects of different SSAs (octreotide, lanreotide, and pasireotide) in primary OSCC human cell cultures; 4) to characterize the expression pattern of key splicing-related elements (spliceosome components and splicing factors) in a representative battery of clinically well-characterized OSCC tissues in comparison with adjacent healthy tissues obtained within the same patient; 5) to assess the putative in vivo association between the expression pattern of key splicing-related elements (spliceosome components and splicing factors) in the tumor of patients with OSCC and relevant clinical and histopathological data parameters; and, 6) to assess the therapeutic potential of a splicing machinery inhibitor (pladienolide B) in primary OSCC human cell cultures.El carcinoma de células escamosas de cavidad oral continúa siendo una enfermedad agresiva y un desafío mundial con una tasa de supervivencia a los 5 años del 60%. Dependiendo de los factores de riesgo, la cirugía y la radioterapia adyuvante +/- quimioterapia siguen siendo la principal modalidad de tratamiento para la enfermedad local o avanzada. Por lo tanto, se necesitan con urgencia nuevos biomarcadores de diagnóstico, pronostico y dianas terapéuticas para el cáncer de cavidad oral. Es importante señalar que se ha comprobado que existe un componente de diferenciación neuroendocrina en algunos tumores que no se consideran clásicamente de origen neuroendocrino, incluido el carcinoma de células escamosas de pulmón y esófago, y más recientemente en la región de cabeza y cuello. En este contexto, la somatostatina (SST) es un neuropéptido inhibidor bien conocido que se produce en diferentes localizaciones a través del organismo, tanto centrales como sistémicas. Las acciones inhibitorias de SST están mediadas a través de los llamados receptores de SST (SSTs), que están ampliamente distribuidos en tejidos normales y tumorales, y regulan, entre otras actividades, la proliferación celular, la diferenciación y la angiogénesis en muchos tipos de tumores. Esta propiedad permite al sistema regulador de la SST ser muy útil clínicamente ya que es ampliamente utilizado en la obtención de imágenes tumorales [gammagrafía SST o gammagrafía con octreótido (un análogo sintético de SST que se une preferentemente al SST2, receptor más ampliamente distribuido a través del organismo)]. Las células tumorales suelen expresar típicamente más de un SSTs, siendo el SST2 el subtipo expresado con mayor frecuencia y, por tanto, el objetivo más importante del tratamiento. En consecuencia, los análogos sintéticos de SST (SSA), representan un objetivo terapéutico atractivo para tratar patologías tumorales positivas para SSTs ya que se ha demostrado que son capaces de controlar la hipersecreción hormonal y el crecimiento tumoral en algunos tipos de patologías tumorales. Nuestra comprensión actual de la presencia de SST en carcinoma de células escamosas de cavidad oral es bastante escasa, y en algunos casos controvertida. Concretamente, estudios previos han demostrado que la expresión inmunohistoquímica relativa de algunos subtipos de SSTs esta alterada en lesiones malignas en la laringe en comparación con tejido sano, así como en muestras tumorales del área de la cabeza y el cuello en comparación con muestras normales de mucosa orofaríngea (obtenidas durante la uvulopalatofaringoplastia) de otros pacientes. Sin embargo, hasta la fecha, no se han realizado análisis moleculares para analizar cuantitativamente, en paralelo, los niveles de expresión (número de copias) de todos los subtipos de SSTs en muestras de carcinoma oral en comparación con tejido sano (control; dentro de mismo paciente) mediante PCR cuantitativa. Hasta la fecha, tampoco se han estudiado los efectos directos de diferentes SSA en cultivos de células humanas primarias de carcinoma de cavidad oral. Por lo tanto, en base a la información mencionada anteriormente, consideramos que sería necesario interrogar los patrones de expresión de los receptores de la SST y su asociación con los datos clínicos, anatomopatológicos y de supervivencia del cáncer de cavidad oral. Asimismo, la maquinaria molecular de splicing se ha comprobado que juega un papel fundamental para controlar las funciones celulares, y para regular los niveles de expresión génica y la especificidad tisular. Es más, la alteración de la maquinaria de splicing ha surgido recientemente como una característica importante del cáncer, con un gran potencial para servir como herramienta diagnostica, pronostica o terapéutica. Así, se ha comprobado que una ligera alteración en algunos de los componentes de la maquinaria (conocida como spliceosoma) puede afectar significativamente el patrón de expresión de multitud de genes claves y a la aparición de variantes de splicing oncogénicas. Concretamente, la desregulación de los factores de splicing que componen el spliceosoma lleva a una mala regulación del proceso de splicing y a la aparición aberrante de variantes que puede promover la iniciación del cáncer y afectar el fenotipo de las células cancerosas, incluida la proliferación, la apoptosis, la invasión y la metástasis de muchos tipos de cáncer. En los últimos anos, varios estudios se han centrado en el análisis y el impacto de algunos factores de splicing en el cáncer de cabeza y cuello. Sus resultados son muy variables, incluso contradictorios en cuanto al patrón de expresión de algunos factores de splicing en tejidos tumorales en comparación con tejido sano. Algunos de estos factores de splicing tienen un impacto en los tratamientos antineoplásicos al modular la apoptosis y permitir la sensibilización de las células cancerosas a los tratamientos terapéuticos. Estos factores pueden actuar como factores de supervivencia que disminuyen la apoptosis inducida por fármacos o, por el contrario, potencian los efectos pro-apoptoticos de los medicamentos quimioterapéuticos. Por todo ello, es necesario realizar un estudio que se centre en estudiar la alteración de la maquinaria del splicing en cáncer de cavidad oral ya que esto podría ayudar a identificar nuevos biomarcadores y dianas moleculares que ayuden a comprender mejor el comportamiento de estos tumores y al desarrollo de nuevas estrategias terapéuticas. Teniendo en cuenta toda esta información, la HIPÓTESIS INICIAL de esta Tesis Doctoral es que la desregulación de los sistemas moleculares de la SST y de la maquinaria del splicing podría influir directamente la patofisiología de los tumores de cavidad oral y, en consecuencia, que sus niveles de expresión en los tejidos tumorales podrían proporcionar información útil para mejorar el diagnóstico y/o pronóstico de estos tumores, e identificar nuevas dianas terapéuticas para tratar a los pacientes con estas devastadoras patologías tumorales. En base a esta hipótesis, el OBJETIVO GENERAL de esta Tesis Doctoral ha sido explorar la presencia, la posible desregulación y/o el papel funcional de algunos de los componentes de estos dos sistemas celulares clave involucrados en procesos críticos de la regulación celular [los receptores de somatostatina (SST), y por la maquinaria de splicing (componentes de spliceosoma y factores de splicing)] y que podrían estar asociados con el desarrollo, progresión y agresividad de los tumores de cavidad oral, con el objetivo final de descubrir nuevos biomarcadores y herramientas terapéuticas para mejorar el diagnostico, pronostico, tratamiento y, por tanto, el manejo de los pacientes con estos tumores. Para lograr este objetivo principal, hemos llevado a cabo diferentes objetivos específicos, tales como: 1) analizar cuantitativamente el perfil de expresión de los SSTs en una batería representativa de pacientes con carcinoma escamoso de cavidad oral clínicamente bien caracterizados en comparación con tejidos sanos adyacentes obtenidos dentro del mismo paciente; 2) evaluar la asociación in vivo entre la expresión de todos los SSTs en carcinoma de cavidad oral con parámetros de datos clínicos e histopatológicos relevantes; 3) explorar y comparar los efectos antitumorales directos de diferentes SSA (octreótido, lanreótido y pasireótido) en cultivos celulares primarios de carcinoma escamoso de cavidad oral; 4) caracterizar el patrón de expresión de elementos clave relacionados con el splicing (componentes del splicing y factores de splicing) en una batería representativa de pacientes con carcinoma escamoso de cavidad oral clínicamente bien caracterizados en comparación con tejidos sanos adyacentes obtenidos dentro del mismo paciente; 5) evaluar la asociación in vivo entre el patrón de expresión de elementos clave relacionados con el splicing (componentes del splicing y factores de splicing) en carcinoma escamoso de cavidad oral con parámetros de datos clínicos e histopatológicos relevantes; y 6) evaluar el potencial terapéutico de un inhibidor de la maquinaria de splicing (pladienolida B) en cultivos celulares primarios de carcinoma de cavidad oral

Molecular and Clinical Implications of Somatostatin Receptor Profile and Somatostatin Analogues Treatment in Oral Cavity Squamous Cell Carcinoma

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

Early Cretaceous charophytes from South Dobrogea (Romania). Biostratigraphy and palaeobiogeography.

Eleven boreholes and one outcrop of the Lower Cretaceous in South Dobrogea (south-eastern Romania) were sampled for charophytes. Twenty species are described and illustrated in two non-marine rock units, the Zăvoaia Member and the Gherghina Formation. The Zăvoaia Member contains a charophyte assemblage dominated by Feistiella bijuescensis, aff. Mesochara harrisii, Nodosoclavator bradleyi, Clavator bilateralis, and Clavator grovesii var. grovesii, indicating a Berriasian age. Other less abundant species include Feistiella sp., Latochara sp., Mesochara dobrogeica sp. nov., Globator maillardii var. nurrensis, Atopochara trivolvis var. micrandra, Clavator grovesii var. discordis, Hemiclavator adnatus, and Hemiclavator neimongolensis var. posticecaptus. The occurrence of G. maillardii var. nurrensis in this assemblage suggests late Berriasian age. The Gherghina Formation is dominated by the species Atopochara trivolvis var. triquetra and Clavator grovesii var. jiuquanensis. Other less abundant species include Sphaerochara andersonii, aff. Mesochara harrisii, Globator maillardii var. trochiliscoides, Globator maillardii var. biutricularis, Clavator harrisii var. reyi, and Clavator ampullaceus var. latibracteatus var. nov. This assemblage indicates a late Barremian-early Aptian age. This study sheds new light on the palaeobiogeographical distribution of Lower Cretaceous charophytes in the Tethyan realm. Well-known western European charophyte species such as F. bijuescensis, S. andersonii, G. maillardii var. trochiliscoides, H. adnatus, and H. neimongolensis var. posticecaptus are herein described for the first time in eastern Europe. Very significantly, this is the first report in Europe of the hitherto North American taxon C. bilateralis, while the species C. ampullaceus has previously been reported only from the Middle East and eastern Africa

Condylar intramedullary intraosseous lipoma : contribution of a new case and review of the literature

Lipoma is the most common benign tumour of the human body, being intraosseous involvement very rare. Just 1 to 4% of all cases of lipoma are located in the oral cavity, only 0.1% being intraosseous. The jaw is its most uncommon bone location. Etiology of intraosseous lipoma (IOL) is unknown, although several theories have been proposed. Usually asymptomatic, the symptoms, when present, will depend on its location and size. Its origin may be intraosseous or juxtacortical. A biopsy is essential for diagnosis, and definitive treatment involves resection or curettage of the lesion. The aim of this paper is to present a new case of intramedullary intraosseous lipoma of the mandible with involvement of the left mandibular ramus and condylar neck. A case of intramedullary intraosseous lipoma (IOL) on the left mandibular ramus and condyle is presented. No history of trauma in temporomandibular joint existed. The radiology showed a radiolucent multi-lobulated lesion with values of attenuation in the range of fat. Curettage is performed and the histopathology showed a conglomerate of adipocytes without trabeculae, calcifications or atypia. According to the bibliography 24 cases of mandibular IOL have been described. This is the second reported case of condylar involvement and the first with cortical expansion. Lipoma intraosseous is a very rare benign bone neoplasm. Histology is required for the differential diagnosis from other radiolucent lesions. The IOL treatment is the curettage with a good prognosis, although malignant transformation to liposarcoma has been reported in other locations. It is a disease with a difficult differential diagnosis, therefore the publication of new cases is important

Resistive-Based Micro-Kelvin Temperature Resolution for Ultra-Stable Space Experiments

High precision temperature measurements are a transversal need in a wide area of physical experiments. Space-borne gravitational wave detectors are a particularly challenging case, requiring both high precision and high stability in temperature measurement. In this contribution, we present a design able to reach 1 μK/(squareroot of)Hz in most of the measuring band down to 1 mHz, and reaching 20 μK/(squarerooot of) Hz at 0.1 mHz. The scheme is based on resistive sensors in a Wheatstone bridge configuration which is AC modulated to minimize the 1/f noise. As a part of our study, we include the design of a test bench able to guarantee the high stability environment required for measurements. We show experimental results characterising both the test bench and the read-out, and discuss potential noise sources that may limit our measurement

Actitudes hacia la asignatura de Estadística en estudiantes de la Facultad de Ciencias Médicas “General Calixto García”

Introducción: La formación estadística del profesional de Medicina en nuestro país, ha estado encaminada a desarrollar una capacidad transformadora capaz de dar respuestas a las necesidades o demandas de la población en general y el sector de la salud en particular, siendo una de sus expresiones, el desarrollo de un pensamiento científico. El propósito de este trabajo es proporcionar información sobre las actitudes hacia la Estadística de estudiantes de la Facultad de Ciencias Médicas "Calixto García". Objetivo: Determinar la fiabilidad de un instrumento que explora las actitudes hacia la asignatura de Estadística en estudiantes de Medicina e identificar los ítems que mayor contribución tienen al desempeño de dicho instrumento. Material y Métodos: Se realizó una investigación de carácter descriptivo, se midieron las actitudes hacia la Estadística a partir de las respuestas a la escala de LIKER, del instrumento SATS (Survey of Attitudes Toward Statistics). Resultados: Se valora la actitud global de los estudiantes, que resultó positiva en su mayoría, por lo que se propone considerar este resultado como punto de partida para un aprendizaje significativo de la Estadística orientado a las  competencias profesionales en los diferentes perfiles de la carrera de Medicina. Conclusiones: La mayor puntuación correspondió a la componente del Conocimiento acerca de relacionar la estadística con situaciones de la realidad; muy próximas a este componente se encuentra la Dificultad, la Afectiva y la Valoración.Palabras clave: Competencias, comportamiento, actitudes, componentes principales, aprendizaje significativo, SATS. ABSTRACTIntroduction: The statistical formation of the professional of Medicine in our country has been conducting to develop a transforming capacity able to give answers at the needs or demands of the population in general and to the sector of the health in particular, being one of its expressions, the development of a scientific thought. The intention of this work is to provide information about the attitudes towards the students' Statistics of the faculty of Medical sciences "Calixto García". Objectives: To determine the reliability of an instrument that explores the attitudes towards the Statistics subject in Medicine students and to identify the items that have major contribution to the performance of the above mentioned instrument. Material and Methods: An investigation of descriptive character was realized, the attitudes measured themselves towards the Statistics from the answers to the scale of LIKER, of the instrument SATS (Survey of Attitudes Toward Statistics). Results: There is valued the global attitude of the students, which turned out to be positive mostly, for what he proposes to consider the above mentioned result to be a starting point for significant learning of the Statistics faced to the professional competitions in the different profiles of the career of Medicine. Conclusions: The highest scores corresponded to the component of the Knowledge about relating the statistics to situations of the reality, very next to this component there is the Difficulty, the Affective one and the Assessment.Key words: Competitions, behavior, attitudes, main components, significant learning, SATS.</p

Repercusión del cambio del programa de Informática Médica en la Facultad de Ciencias Médicas "General Calixto García"

Introducción: la asignatura Informática Médica proporciona herramientas al futuro profesional de la salud, que le permiten aplicar las tecnologías de la información y comunicación y brinda métodos científicos para desarrollar sus labores docentes, asistenciales e investigativas. Objetivo: analizar el programa vigente de dicha asignatura en cuanto a contenido y describir el nivel de desempeño de los educandos por la formación recibida a través de los resultados de evaluación, obtenidos en los dos cursos académicos. Material y Métodos: se realizó un estudio cualitativo de tipo exploratorio_descriptivo, sobre la población de los estudiantes de 1er año matriculados en la carrera de Medicina de los cursos 2010-2011 y 2011-2012. Se utilizó el análisis documental y la técnica del Positivo/Negativo/Interesante para la obtención de los datos, aplicando el método fenomenológico y se categorizaron mediante el análisis de contenido. Resultados: se plantea que el programa actual se adapta a las condiciones económicas del país. La aplicación del P.N.I. mostró que el proceso de enseñanza fue bueno, ya que los aspectos negativos no fueron imputables a este proceso. Los resultados de las evaluaciones de los dos cursos analizados, fueron satisfactorios. Conclusiones: valorando a través del test P.N.I. aplicado, pudimos constatar que los educandos nuestros han aceptado el reto que supone la migración al software Libre a pesar de los aspectos negativos detallados, ya que estos no dependen de nuestra Institución. Los resultados de las evaluaciones realizadas así lo confirman.  Palabras Clave: Software Libre, Informática Médica, migración. </p

Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca2+ elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca2+/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.This work was financed by grants from the Ministerio de Educación y Ciencia (BFU2006-13802), and the Consejería de Innovación, Ciencia y Empresa (P06-CTS-1936), Junta de Andalucía, Spain, awarded to F S