Aplicació de tècniques òmiques per a la millora del diagnòstic en la síndrome de càncer de mama i ovari hereditari.

La síndrome de càncer de mama i ovari hereditari és una de les síndromes de càncer hereditari més freqüents. Al voltant del 5-10% dels casos s’associen a variants patogèniques en els gens BRCA1 i BRCA2. La identificació d’aquestes alteracions és important per a realitzar un assessorament genètic que ajudi a prendre decisions mèdiques basades en l’avaluació del risc individual. En l’estudi genètic d’aquesta síndrome, també s’identifiquen variants de significat incert, que dificulten el procés d’assessorament genètic. Per a poder classificar aquestes variants s’utilitzen principalment anàlisis multifactorials que inclouen l’estudi de la cosegregació de la variant, la freqüència poblacional i l’anàlisi del procés d’empalmament. Tot i això, continuen quedant moltes variants sense categoritzar i per això calen altres eines per a identificar i classificar aquestes variants així com estudiar altres gens que ens puguin explicar la causa hereditària de la malaltia. Per tot això, mitjançant els diferents estudis d’aquesta tesi doctoral basats en diferents tècniques òmiques, s’ha pogut classificar algunes variants mitjançant la combinació de freqüència poblacional, anàlisi in silico i estudi de l’ARN. A més a més, s’ha descartat la hipermetilació del promotor dels gens BRCA1 i BRCA2 com a mecanisme hereditari de silenciament gènic en aquesta síndrome, i s’han identificat altres gens, principalment el gen PALB2, com a gens a incloure en l’estudi d’aquesta síndrome. Finalment s’ha fet un estudi basat en metabolòmica que ha permès identificar una empremta característica amb capacitat predictora del fenotip BRCA-like de càncer de mama.El síndrome de cáncer de mama y ovario hereditario es uno de los síndromes de cáncer hereditario más frecuentes. Alrededor del 5-10% de los casos son portadores de variantes patogénicas en los genes BRCA1 y BRCA2. La identificación de éstas es importante para realizar un asesoramiento genético que ayude en la toma de decisiones médicas basadas en la evaluación del riesgo individual. En su estudio genético, también se identifican variantes de significado incierto, que dificultan el proceso de asesoramiento genético. Para clasificarlas se usan principalmente análisis multifactoriales que incluyen el estudio de la cosegregación de la variante, frecuencia poblacional y análisis del proceso de splicing. No obstante, siguen quedando muchas variantes sin categorizar, por lo que se necesitan otras herramientas para identificar y clasificarlas y para estudiar otros genes que puedan explicar la causa hereditaria de la enfermedad. Mediante los diferentes estudios que conforman esta tesis doctoral, basados en diferentes técnicas ómicas, se han podido clasificar algunas variantes mediante la combinación de frecuencias poblacionales, análisis in silico y estudio del ARN. Además, se ha descartado la hipermetilación del promotor de los BRCA1 y BRCA2 como un mecanismo hereditario de silenciamiento génico en este síndrome, y se han identificado otros genes a incluir en el análisis de el síndrome. Finalmente, se ha llevado a cabo un estudio basado en metabolómica que ha permitido identificar una huella con capacidad predictiva del fenotipo BRCA-like de cáncer de mama.Hereditary breast and ovarian cancer syndrome is one of the most common hereditary cancer syndromes. At least 5-10% of cases are associated with pathogenic variants in the BRCA1 and BRCA2 genes. The identification of these alterations is important to carry out genetic counselling that helps to make medical decisions based on the assessment of the individual risk. In the mutational study, variants of uncertain significance are also identified, which make the process of genetic counselling difficult. To classify them, strategies based on multifactorial analysis are used, which include as cosegregation analysis of the variant, population frequency, as well as the mRNA splicing. However, there are still many variants that cannot be categorized. That’s the reason why other methods are needed to identify and classify them, as well as to study other genes that can explain the hereditary cause of cancer. Therefore, the studies included in this doctoral thesis, based on different omics techniques, allow us to classify some unclassified variants by means of a combination of population frequency, in silico analysis and mRNA study. Moreover, the hypermethylation of the promoter of the BRCA1 and BRCA2 genes has been ruled out as a hereditary mechanism of genetic silencing in this syndrome, and other genes have been identified as genes to be included in the study of this syndrome. Finally, a metabolomics-based study has been carried out to identify a characteristic fingerprint with the ability to predict the BRCA-like phenotype of breast cancer

Analysis of the mitochondrial DNA of Schizophrenia patients by next generation sequencing

Curs 2012-2013Mitochondrial DNA (mtDNA), a maternally inherited 16.6-Kb molecule crucial for energy production, is implicated in numerous human traits and disorders. It has been hypothesized that the presence of mutations in the mtDNA may contribute to the complex genetic basis of schizophreniadisease, due to the evidence of maternal inheritance and the presence of schizophrenia symptoms in patients affected of a mitochondrial disorder related to a mtDNA mutation. The present project aims to study the association of variants of mitochondrial DNA (mtDNA), and an increased risk of schizophrenia in a cohort of patients and controls from the same population. The entire mtDNA of 55 schizophrenia patients with an apparent maternal transmission of the disease and 38 controls was sequenced by Next Generation Sequencing (Ion Torrent PGM, Life Technologies) and compared to the reference sequence. The current method for establishing mtDNA haplotypes is Sanger sequencing, which is laborious, timeconsuming, and expensive. With the emergence of Next Generation Sequencing technologies, this sequencing process can be much more quickly and cost-efficiently. We have identified 14 variants that have not been previously reported. Two of them were missense variants: MTATP6 p.V113M and MTND5 p.F334L ,and also three variants encoding rRNA and one variant encoding tRNA. Not significant differences have been found in the number of variants between the two groups. We found that the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of the bioinformatics analysis and annotation step would be desirable to facilitate the application of NGS in mtDNA analysis.Director/a: Lourdes Martorell Bonet i Tutor/a: M. Luz Call

Neuroprotection with Hypothermia and Allopurinol in an animal model of hypoxic-ischemic injury: Is it a gender question?

Abstract Background Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of neonatal brain injury. Therapeutic hypothermia (TH) is the standard treatment for term newborns after perinatal hypoxic ischemic injury (HI). Despite this, TH does not provide complete neuroprotection. Allopurinol seems to be a good neuroprotector in several animal studies, but it has never been tested in combination with hypothermia. Clinical findings show that male infants with (HI) fare more poorly than matched females in cognitive outcomes. However, there are few studies about neuroprotection taking gender into account in the results. The aim of the present study was to evaluate the potential additive neuroprotective effect of allopurinol when administrated in association with TH in a rodent model of moderate HI. Gender differences in neuroprotection were also evaluated Methods P10 male and female rat pups were subjected to HI (Vannucci model) and randomized into five groups: sham intervention (Control), no treatment (HI), hypothermia (HIH), allopurinol (HIA), and dual therapy (hypothermia and allopurinol) (HIHA). To evaluate a treatment's neuroprotective efficiency, 24 hours after the HI event caspase3 activation was measured. Damaged area and hippocampal volume were also measured 72 hours after the HI event. Negative geotaxis test was performed to evaluate early neurobehavioral reflexes. Learning and spatial memory were assessed via Morris Water Maze (MWM) test at 25 days of life. Results Damaged area and hippocampal volume were different among treatment groups (p = 0.001). The largest tissue lesion was observed in the HI group, followed by HIA. There were no differences between control, HIH, and HIHA. When learning process was analyzed, no differences were found. Females from the HIA group had similar results to the HIH and HIHA groups. Cleaved caspase 3 expression was increased in both HI and HIA. Despite this, in females cleaved caspase-3 was only differently increased in the HI group. All treated animals present an improvement in short-term (Negative geotaxis) and long-term (WMT) functional tests. Despite this, treated females present better long-term outcome. In short-term outcome no sex differences were observed. Conclusions Our results suggest that dual therapy confers great neuroprotection after an HI event. There were functional, histological, and molecular improvements in all treated groups. These differences were more important in females than in males. No statistically significant differences were found between HIHA and HIH; both of them present a great improvement. Our results support the idea of different regulation mechanisms and pathways of cell death, depending on gender

Opportunistic genetic screening increases the diagnostic yield and is medically valuable for care of patients and their relatives with hereditary cancer

Background: Multigene panel testing by next-generation sequencing (MGP-NGS) enables the detection of germline pathogenic or likely pathogenic variants (PVs/LPVs) in genes beyond those associated with a certain cancer phenotype. Opportunistic genetic screening based on MGP-NGS in patients with suspicion of hereditary cancer reveals these incidental findings (IFs). Methods: MGP-NGS was performed in patients who fulfilled the clinical criteria to undergo genetic testing according to the Catalan Health Service guidelines. Variants were classified following the American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines and the Cancer Variant Interpretation Group UK guidelines. Results: IFs were identified in 10 (1.22%) of the 817 patients who underwent MGP-NGS. The mean age at cancer diagnosis was 49.4±9.5 years. Three IFs (30.0%) were detected in PMS2, two (20.0%) in ATM and TP53 and one (10.0%) in MSH6, NTHL1 and VHL. Seven (70.0%) IFs were single-nucleotide substitutions, two (20.0%) were deletions and one (10.0%) was a duplication. Three (30.0) IFs were located in intronic regions, three (30.3%) were nonsense, two (20.0%) were frameshift and two (20.0%) were missense variations. Six (60.0%) IFs were classified as PVs and four (40.0%) as LPVs. Conclusions: Opportunistic genetic screening increased the diagnostic yield by 1.22% in our cohort. Most of the identified IFs were present in clinically actionable genes (n=7; 70.0%), providing these families with an opportunity to join cancer early detection programmes, as well as secondary cancer prevention. IFs might facilitate the diagnosis of asymptomatic individuals and the early management of cancer once it develops

Aspectos didácticos de Dibujo. 4

Ponencias presentadas en el 'IX Encuentro sobre Aspectos Didácticos en la Enseñanza Secundaria', celebrado en Zaragoza del 13 al 15 de septiembre de 1993Recoge cinco ponencias de Dibujo,presentadas a un Encuentro celebrado en Zaragoza, que tratan de cinco aspectos diferentes de la enseñanza del mismo: 'Desarrollo profesional y autonomía del profesor de educación artística en un contexto de reforma educativa' revisa el concepto de profesionalidad docente y analiza los cambios de conceptualización de dicha profesión y la articulación de este cambio dentro del contexto de la reforma; 'Los contenidos en el área de educación visual y plástica en la educación secundaria obligatoria' expone las dimensiones que determinan el perfil de la disciplina y la importancia de su estructuración e interrelación, poniendo especial énfasis en el estudio de la diversidad de los contenidos propios de la educación artística; 'La introducción al diseño en la enseñanza secundaria' reflexiona acerca de los contenidos esenciales de su concepto actual; 'Los nuevos planteamientos en la enseñanza de los sistemas de representación, con aplicación a la enseñanza secundaria' aclara qué se quiere decir al hablar de sistemas de representación y hace ciertas precisiones acerca de conceptos, términos y teoría, profundizando en el tema y, por último, 'La evaluación en la formación artística'' estudia sus características y funciones, analiza el aprendizaje de los procedimientos y fija unos criterios.AragónBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín 5 -3 Planta; 28014 Madrid; Tel. +34917748000; [email protected]

Neuroprotection with Hypothermia and Allopurinol in an animal model of hypoxic-ischemic injury: Is it a gender question?

Abstract Background Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of neonatal brain injury. Therapeutic hypothermia (TH) is the standard treatment for term newborns after perinatal hypoxic ischemic injury (HI). Despite this, TH does not provide complete neuroprotection. Allopurinol seems to be a good neuroprotector in several animal studies, but it has never been tested in combination with hypothermia. Clinical findings show that male infants with (HI) fare more poorly than matched females in cognitive outcomes. However, there are few studies about neuroprotection taking gender into account in the results. The aim of the present study was to evaluate the potential additive neuroprotective effect of allopurinol when administrated in association with TH in a rodent model of moderate HI. Gender differences in neuroprotection were also evaluated Methods P10 male and female rat pups were subjected to HI (Vannucci model) and randomized into five groups: sham intervention (Control), no treatment (HI), hypothermia (HIH), allopurinol (HIA), and dual therapy (hypothermia and allopurinol) (HIHA). To evaluate a treatment's neuroprotective efficiency, 24 hours after the HI event caspase3 activation was measured. Damaged area and hippocampal volume were also measured 72 hours after the HI event. Negative geotaxis test was performed to evaluate early neurobehavioral reflexes. Learning and spatial memory were assessed via Morris Water Maze (MWM) test at 25 days of life. Results Damaged area and hippocampal volume were different among treatment groups (p = 0.001). The largest tissue lesion was observed in the HI group, followed by HIA. There were no differences between control, HIH, and HIHA. When learning process was analyzed, no differences were found. Females from the HIA group had similar results to the HIH and HIHA groups. Cleaved caspase 3 expression was increased in both HI and HIA. Despite this, in females cleaved caspase-3 was only differently increased in the HI group. All treated animals present an improvement in short-term (Negative geotaxis) and long-term (WMT) functional tests. Despite this, treated females present better long-term outcome. In short-term outcome no sex differences were observed. Conclusions Our results suggest that dual therapy confers great neuroprotection after an HI event. There were functional, histological, and molecular improvements in all treated groups. These differences were more important in females than in males. No statistically significant differences were found between HIHA and HIH; both of them present a great improvement. Our results support the idea of different regulation mechanisms and pathways of cell death, depending on gender

MCF-7 Drug Resistant Cell Lines Switch Their Lipid Metabolism to Triple Negative Breast Cancer Signature

Obesity and adipose tissue have been closely related to a poor cancer prognosis, especially in prostate and breast cancer patients. The ability of transferring lipids from the adipose tissue to the tumor cells is actively linked to tumor progression. However, different types of breast tumor seem to use these lipids in different ways and metabolize them in different pathways. In this study we have tracked by mass spectrometry how palmitic acid from the adipocytes is released to media being later incorporated in different breast cancer cell lines (MDA-MB-231, SKBR3, BT474, MCF-7 and its resistant MCF-7 EPIR and MCF-7 TAXR). We have observed that different lines metabolize the palmitic acid in a different way and use their carbons in the synthesis of different new lipid families. Furthermore, we have observed that the lipid synthesis pattern varied according to the cell line. Surprisingly, the metabolic pattern of the resistant cells was more related to the TNBC cell line compared to their sensitive cell line MCF-7. These results allow us to determine a specific lipid pattern in different cell lines that later might be used in breast cancer diagnosis and to find a better treatment according to the cancer molecular type

Neuroprotection with hypothermia and allopurinol in an animal model of hypoxic-ischemic injury: Is it a gender question?

<div><p>Background</p><p>Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of neonatal brain injury. Therapeutic hypothermia (TH) is the standard treatment for term newborns after perinatal hypoxic ischemic injury (HI). Despite this, TH does not provide complete neuroprotection. Allopurinol seems to be a good neuroprotector in several animal studies, but it has never been tested in combination with hypothermia.</p><p>Clinical findings show that male infants with (HI) fare more poorly than matched females in cognitive outcomes. However, there are few studies about neuroprotection taking gender into account in the results.</p><p>The aim of the present study was to evaluate the potential additive neuroprotective effect of allopurinol when administrated in association with TH in a rodent model of moderate HI. Gender differences in neuroprotection were also evaluated.</p><p>Methods</p><p>P10 male and female rat pups were subjected to HI (Vannucci model) and randomized into five groups: sham intervention (Control), no treatment (HI), hypothermia (HIH), allopurinol (HIA), and dual therapy (hypothermia and allopurinol) (HIHA). To evaluate a treatment’s neuroprotective efficiency, 24 hours after the HI event caspase3 activation was measured. Damaged area and hippocampal volume were also measured 72 hours after the HI event. Negative geotaxis test was performed to evaluate early neurobehavioral reflexes. Learning and spatial memory were assessed via Morris Water Maze (MWM) test at 25 days of life.</p><p>Results</p><p>Damaged area and hippocampal volume were different among treatment groups (p = 0.001). The largest tissue lesion was observed in the HI group, followed by HIA. There were no differences between control, HIH, and HIHA. When learning process was analyzed, no differences were found. Females from the HIA group had similar results to the HIH and HIHA groups.</p><p>Cleaved caspase 3 expression was increased in both HI and HIA. Despite this, in females cleaved caspase-3 was only differently increased in the HI group.</p><p>All treated animals present an improvement in short-term (Negative geotaxis) and long-term (WMT) functional tests. Despite this, treated females present better long-term outcome. In short-term outcome no sex differences were observed.</p><p>Conclusions</p><p>Our results suggest that dual therapy confers great neuroprotection after an HI event. There were functional, histological, and molecular improvements in all treated groups. These differences were more important in females than in males. No statistically significant differences were found between HIHA and HIH; both of them present a great improvement. Our results support the idea of different regulation mechanisms and pathways of cell death, depending on gender.</p></div

Water maze test.

<p>Plot representing the average escape latency in four trials performed each day. Results were expressed as mean of escape latency. Abbreviatons: ST: Sham-treated; HI: Hypoxic-ischemic, HIA: Hypoxic-ischemic allopurinol, HIH: Hypoxic-ischemic hypothermia, HIHA: Hypoxic-ischemic hypothermia allopurinol.</p