From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

CD81 interacting proteins

Fertilization is a multistep and complex process culminating in the merge of gamete membranes, cytoplasmic unity and fusion of genome. CD81 is a tetraspanin protein that participates in sperm-oocyte interaction, being present at the oocyte surface. CD81 has also been implicated in other biological processes, however its specific function and molecular mechanisms of action remain to be elucidated. The interaction between CD81 and its binding partner proteins may underlie the CD81 involvement in a variety of cellular processes and modulate CD81/interactors specific functions. Interestingly, in a Yeast two Hybrid system previously performed in our lab, CD81 has emerged as a putative interactor of the Amyloid Precursor Protein (APP). In the work here described, bioinformatics analyses of CD81 interacting proteins were performed and the retrieved information used to construct a protein-protein interaction network, as well as to perform Gene Ontology enrichment analyses. CD81 expression was further evaluated in CHO, GC-1 and SH-SY5Y cell lines, and in human sperm cells. Additionally, its subcellular localization was analyzed in sperm cells and in the neuronal-like SH-SY5Y cell line. Subsequently, coimmunoprecipitation assays were performed in CHO and SH-SY5Y cells to attempt to prove the physical interaction between CD81 and APP. A functional interaction between these two proteins was accessed thought the analyses of the effects of CD81 overexpression on APP levels. A co-localization analysis of CD81 and some interactors proteins retrieved from the bioinformatics analyses, such as APP, AKT1 and cytoskeleton-related proteins, was also performed in sperm cells and in SH-SY5Y cells. The effects of CD81 in cytoskeleton remodeling was evaluated in SH-SY5Y cells through monitoring the effects of CD81 overexpression in actin and tubulin levels, and analyzing the colocalization between overexpressed CD81 and F-actin. Our results showed that CD81 is expressed in all cell lines tested, and also provided the first evidence of the presence of CD81 in human sperm cells. CD81 immunoreactivity was predominantly detected in the sperm head, including the acrosome membrane, and in the midpiece, where it co-localized with APP, as well as in the post-acrosomal region. Furthermore, CD81 co-localizes with APP in the plasma membrane and in cellular projections in SH-SY5Y cells, where CD81 overexpression has an influence on APP levels, also visible in CHO cells. The analysis of CD81 interacting proteins such as AKT1 and cytoskeletonrelated proteins showed that CD81 is involved in a variety of pathways that may underlie cytoskeleton remodeling events, related to processes such as sperm motility, cell migration and neuritogenesis. These results deepen our understanding on the functions of CD81 and some of its interactors in sperm and neuronal cells.

APP RERMS enriched matrix as an adhesion substrate for neuritic outgrowth

Specific domains can determine protein structural functional relationships. For the Alzheimer’s Amyloid Precursor Protein (APP) several domains have been described, both in its intracellular and extracellular fragments. Many functions have been attributed to APP including an important role in cell adhesion and cell to cell recognition. This places APP at key biological responses, including synaptic transmission. To fulfil these functions, extracellular domains take on added significance. The APP extracellular domain RERMS is in fact a likely candidate to be involved in the aforementioned physiological processes. A multidisciplinary approach was employed to address the role of RERMS. The peptide RERMS was crosslinked to PEG (Polyethylene glycol) and the reaction validated by FTIR (Fourier transform infrared spectrometry). FTIR proved to be the most efficient at validating this reaction because it requires only a drop of sample, and it gives information about the reactions occurred in a mixture. The data obtained consist in an infrared spectra of the sample, where peaks positions give information about the structure of the molecules, and the intensity of peaks is related to the concentration of the molecules. Subsequently substrates of PEG impregnated with RERMS were prepared and SH-SY5Y (human neuroblastoma cell line) cells were plated and differentiated on the latter. Several morphological alterations were clearly evident. The RERMS peptide provoked cells to take on a flatter appearance and the cytoskeletal architecture changed, with the appearance of stress fibres, a clear indicator of actin reorganization. Given that focal adhesions play a key role in determining cellular structure the latter were directly investigated. Focal adhesion kinase (FAK) is one of the most highly expressed proteins in the CNS (central nervous system) during development. It has been described to be crucial for radial migration of neurons. FAK can be localized in growth cones and mediated the response to attractive and repulsive cues during migration. One of the mechanisms by which FAK becomes active is by auto phosphorylation at tyrosine 397. It became clearly evident that in the presence of the RERMS peptide pFAK staining at focal adhesions intensified and more focal adhesions became apparent. Furthermore speckled structures in the nucleus, putatively corresponding to increased expression activity, also increased with RERMS. Taken together these results indicate that the RERMS domain in APP plays a critical role in determining cellular physiological responses. Here is suggested a model by which RERMS domain is recognized by integrins and mediate intracellular responses involving FAK, talin, actin filaments and vinculin. This mechanism probably is responsible for mediating cell adhesion and neurite outgrowth on neurons.

FTIR, a potential tool to dementia diagnosis trough analysis of plasma

Nowadays it is still difficult to perform an early and accurate diagnosis of dementia, therefore many research focus on the finding of new dementia biomarkers that can aid in that purpose. So scientists try to find a noninvasive, rapid, and relatively inexpensive procedures for early diagnosis purpose. Several studies demonstrated that the utilization of spectroscopic techniques, such as Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy could be an useful and accurate procedure to diagnose dementia. As several biochemical mechanisms related to neurodegeneration and dementia can lead to changes in plasma components and others peripheral body fluids, blood-based samples and spectroscopic analyses can be used as a more simple and less invasive technique. This work is intended to confirm some of the hypotheses of previous studies in which FTIR was used in the study of plasma samples of possible patient with AD and respective controls and verify the reproducibility of this spectroscopic technique in the analysis of such samples. Through the spectroscopic analysis combined with multivariate analysis it is possible to discriminate controls and demented samples and identify key spectroscopic differences between these two groups of samples which allows the identification of metabolites altered in this disease. It can be concluded that there are three spectral regions, 3500-2700 cm -1, 1800-1400 cm-1 and 1200-900 cm-1 where it can be extracted relevant spectroscopic information. In the first region, the main conclusion that is possible to take is that there is an unbalance between the content of saturated and unsaturated lipids. In the 1800-1400 cm-1 region it is possible to see the presence of protein aggregates and the change in protein conformation for highly stable parallel β-sheet. The last region showed the presence of products of lipid peroxidation related to impairment of membranes, and nucleic acids oxidative damage. FTIR technique and the information gathered in this work can be used in the construction of classification models that may be used for the diagnosis of cognitive dysfunction.

Association between common lipid metabolism genes polymorphisms and sporadic colorectal adenocarcinoma risk

Lipids can modulate the risk of developing sporadic colorectal adenocarcinoma (SCA), since alterations into lipid metabolism and transport pathways influence directly cholesterol and lipids absorption by colonic cells and indirectly reactive oxygen species (ROS) synthesis in rectum cells due to lipid accumulation. Lipid metabolism is regulated by several proteins APOA1, APOB, APOC3, APOE, CETP, NPY, PON1 and PPARG that could influence both metabolism and transport processes. Is been reported that several common single-nucleotide polymorphisms (SNPs) in these genes could influence their function and/or expression, changing lipid metabolism balance. Thus, genetic changes in those genes can influence SCA development, once the majority of them were never studied in this disease. Furthermore, there are contradictory results between some studied polymorphisms and SCA risk. Thus, the aim of this study was to explore and describe lipid metabolism-associated genes common polymorphisms (APOA1 -75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) status among SCA, and their relationship with SCA risk. Genotyping of common lipid metabolism genes polymorphisms (APOA1 75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) were done by PCR-SSP techniques, from formalin-fixed and paraffin-embedded biopsies of 100 healthy individuals and 68 SCA subjects. Mutant genotypes of APOA1 -75AA (32% vs 12%; p=0.001; OR=3.51; 95% CI 1.59-7.72); APOB 3500AA (7% vs 0%; p=0.01); APOC3 3175GG (19% vs 2%; p=0.0002; OR=11.58; 95% CI 2.52-53.22), APOC3 3206GG (19% vs 0%; p<0.0001); CETP 279AA (12% vs 1%; p=0.003; OR=13.20; 95% CI 1.61-108.17), CETP 451AA (16% vs 0%; p<0.0001); NPY 7CC (15% vs 0%; p<0.0001); PPARG 12GG (10% vs 0%; p=0.001); and heterozygote genotype PON1 192AG (56% vs 22%; p<0.0001; OR=4.49; 95% CI 2.298.80) were found associated with SCA prevalence. While, APOE E4/E4 (0% vs 8%; p=0.02) mutant haplotype seemed to have a protective effect on SCA. Moreover, it also been founded differences between APOB 3500GA, APOC3 3206TG, CETP 279AA genotypes and PPARG 12Ala allele prevalence and tissue localization (colon vs rectum). These findings suggest a positive association between most of common lipid metabolism genes polymorphisms studied and SCA prevalence. Dysregulation of APOA1, APOB, APOC3, CETP, NPY, PON1 and PPARG genes could be associated with lower cholesterol plasma levels and increase ROS among colon and rectum mucosa. Furthermore, these results also support the hypothesis that CRC is related with intestinal lipid absorption decrease and secondary bile acids production increase. Moreover, the polymorphisms studied may play an important role as biomarkers to SCA susceptibility.

Association between common lipid metabolism genes polymorphisms and sporadic colorectal adenocarcinoma risk

Lipids can modulate the risk of developing sporadic colorectal adenocarcinoma (SCA), since alterations into lipid metabolism and transport pathways influence directly cholesterol and lipids absorption by colonic cells and indirectly reactive oxygen species (ROS) synthesis in rectum cells due to lipid accumulation. Lipid metabolism is regulated by several proteins APOA1, APOB, APOC3, APOE, CETP, NPY, PON1 and PPARG that could influence both metabolism and transport processes. Is been reported that several common single-nucleotide polymorphisms (SNPs) in these genes could influence their function and/or expression, changing lipid metabolism balance. Thus, genetic changes in those genes can influence SCA development, once the majority of them were never studied in this disease. Furthermore, there are contradictory results between some studied polymorphisms and SCA risk. Thus, the aim of this study was to explore and describe lipid metabolism-associated genes common polymorphisms (APOA1 -75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) status among SCA, and their relationship with SCA risk. Genotyping of common lipid metabolism genes polymorphisms (APOA1 75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) were done by PCR-SSP techniques, from formalin-fixed and paraffin-embedded biopsies of 100 healthy individuals and 68 SCA subjects. Mutant genotypes of APOA1 -75AA (32% vs 12%; p=0.001; OR=3.51; 95% CI 1.59-7.72); APOB 3500AA (7% vs 0%; p=0.01); APOC3 3175GG (19% vs 2%; p=0.0002; OR=11.58; 95% CI 2.52-53.22), APOC3 3206GG (19% vs 0%; p<0.0001); CETP 279AA (12% vs 1%; p=0.003; OR=13.20; 95% CI 1.61-108.17), CETP 451AA (16% vs 0%; p<0.0001); NPY 7CC (15% vs 0%; p<0.0001); PPARG 12GG (10% vs 0%; p=0.001); and heterozygote genotype PON1 192AG (56% vs 22%; p<0.0001; OR=4.49; 95% CI 2.298.80) were found associated with SCA prevalence. While, APOE E4/E4 (0% vs 8%; p=0.02) mutant haplotype seemed to have a protective effect on SCA. Moreover, it also been founded differences between APOB 3500GA, APOC3 3206TG, CETP 279AA genotypes and PPARG 12Ala allele prevalence and tissue localization (colon vs rectum). These findings suggest a positive association between most of common lipid metabolism genes polymorphisms studied and SCA prevalence. Dysregulation of APOA1, APOB, APOC3, CETP, NPY, PON1 and PPARG genes could be associated with lower cholesterol plasma levels and increase ROS among colon and rectum mucosa. Furthermore, these results also support the hypothesis that CRC is related with intestinal lipid absorption decrease and secondary bile acids production increase. Moreover, the polymorphisms studied may play an important role as biomarkers to SCA susceptibility.

O Ofício das Rezadeiras: um estudo antropológico sobre as práticas terapêuticas e a comunhão de crenças em Cruzeta/RN

The present Master´s dissertation aims to study the practices of the rezadeiras, Brazilian women healers, through an anthropological perspective. Special attention will be given to the understanding of these practices as a dynamic process in relation to those women who heal in Cruzeta (Seridó, Rio Grande do Norte), where is located our ethnographic research. For this research, twenty four rezadeiras were contacted and colaborated with our work plan. Among them, two were pentecostal rezadeiras and another one was member of the Jurema cult, an afro-brazilian religious cult. Similarities among these women healers were perceived in the research process, mostly in terms of their learning process and the use of certain objects and ritual techniques. However, apparent differences among them gave us the chance for understanding and reflecting on the actual heterogeneity of this world of specialists. Furthermore, i tried to capture the relations between the rezadeiras and the therapeutic practices from health professionals or the religious practices of religious leaders (Catholic, pentecostal, etc). It is possible to ascertain about the complementarity between therapeutic practices from different cultural logics. This complementarity is also perceived through the religious interchanges and transits among different healers, including those who have different religious beliefs. In this work, rituals are also described and they are a crucial factor to the understanding of this particular religious and therapeutic practice conducted by women. Following these ideas, our basic aim is to understand how the rezadeiras make interpretations about health and illness, specially those ones which are particular associated with their practices, the so called "doenças de rezadeiras"

Toxicidade in vitro e in vivo de quantum dots de carbono recobertos com boronato

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.

Diseño, preparación y caracterización de hidrogeles de agarosa para liberación controlada de fármacos

En esta tesis doctoral se han abordado nuevas estrategias para la elaboración de sistemas farmacéuticos para la liberación de fármacos basados en hidrogeles de origen natural. La sustancia elegida para la fabricación de estos sistemas fue la agarosa para la que cada día se encuentran nuevas aplicaciones dentro de campos relacionados con la biomedicina, la biotecnología y la liberación controlada de fármacos. La agarosa presenta la gran ventaja, entre otras, de, gracias a su capacidad de gelificar en función de la temperatura, conformar diferentes tipos de materiales en piezas con notables prestaciones mecánicas que permiten su manipulación. En una primera aproximación se procedió a probar la capacidad de los sistemas de agarosa de permitir la inclusión de tres tipos de surfactantes: pluronic® F68, tween® 80 y lauril sulfato de sodio, con objetivo de facilitar la liberación de los fármacos incluidos. Los tensoactivos incluidos no afectaron las propiedades de los sistemas obtenidos. Se pudo comprobar cómo, incluso a los mayores porcentajes de surfactante, se obtienen sistemas manejables que, en el caso del fármaco modelo hidrosoluble, teofilina, respondían al comportamiento esperado, una más rápida liberación del fármaco con pequeñas variaciones en función de la naturaleza y porcentaje del surfactante añadido. Sin embargo, en el caso del fármaco modelo de baja solubilidad en agua, la tolbutamida, el comportamiento es radicalmente diferente ya que la liberación es más sostenida independientemente del tipo de surfactante. Con el fin de aclarar este inesperado comportamiento se procedió a caracterizar estos sistemas desde el punto de vista microestructural, considerando las interacciones establecidas entre las micelas cargadas de fármaco y la agarosa y las modificaciones en la porosidad de los hidrogeles liofilizados...

Efecto de las nanopartículas basadas en vitamina E sobre un modelo in vitro de cáncer escamoso de cabeza y cuello

Tesis inédita presentada en la Universidad Europea de Madrid. Facultad de Ciencias Biomédicas. Programa de Doctorado en Biomedicina y Ciencias de la Salud

Validez de la orina obtenida por micción directa con técnica de estimulación en el diagnóstico de infección urinaria en lactantes menores de 3 meses

Tesis inédita presentada en la Universidad Europea de Madrid. Facultad de Ciencias Biomédicas. Programa de Doctorado en Biomedicina y Ciencias de la Salud

Efecto del tratamiento con bacilo de calmette-guérin sobre un modelo in vitro 3D de carcinoma escamoso de cabeza y cuello

Tesis inédita presentada en la Universidad Europea de Madrid. Facultad de Ciencias Biomédicas. Programa de Doctorado en Biomedicina y Ciencias de la Salud

“¡Pero al fin y al cabo muñecas!”: Tránsitos, genitalidades y nombramientos en las trayectorias de vida de mujeres transexuales en ejercicio de prostitución en Bogotá

El presente trabajo comprende la construcción de las trayectorias de vida de cinco mujeres transexuales en ejercicio de prostitución en Bogotá a partir de la identificación de los desplazamientos en el terreno corporal, de auto-reconocimiento y de genitalidad en sus procesos de transformación y/o tránsito dentro del espacio generizado. La identificación de lo que he denominado “agentes de transformación específicos” y “condiciones de posibilidad existentes” guía el proceso de la caracterización y análisis de sus experiencias dentro de la(s) transexualidad(es). A diferencia de una línea cronológica o de avance en el tránsito, la noción de espacio generizado me permite reconocer la importancia de las diferencias, la complejidad y la variedad de velocidades y direcciones que pueden presentarse en las experiencias con el cuerpo.