Submitted by Fernanda Rodrigues de Lima (fernanda.rlima@ufpe.br) on 2018-07-17T21:03:07Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Marcelle Aquino Rabelo.pdf: 1811490 bytes, checksum: a8883899ff40c5c405ea02f911de3b40 (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-07-19T22:19:11Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Marcelle Aquino Rabelo.pdf: 1811490 bytes, checksum: a8883899ff40c5c405ea02f911de3b40 (MD5) / Made available in DSpace on 2018-07-19T22:19:11Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Marcelle Aquino Rabelo.pdf: 1811490 bytes, checksum: a8883899ff40c5c405ea02f911de3b40 (MD5) Previous issue date: 2017-02-17 / CAPES / Staphylococcus spp. é considerado um dos agentes de maior impacto para as infecções associadas aos cuidados em saúde e também uma das principais causas de complicações infecciosas em pacientes com câncer. A gravidade das infecções estafilocócicas está relacionada à presença de fatores de resistência e de virulência, os quais favorecem a evasão bacteriana do sistema de defesa do hospedeiro. Com objetivo de comparar isolados de Staphylococcus spp. de pacientes oncológicos e não oncológicos de dois hospitais da cidade do Recife, durante o ano de 2013, foram realizados testes de susceptibilidade antimicrobiana e reações em cadeia da polimerase (PCR) para identificar a ocorrência de genes de resistência (mecA, blaZ, ermA e ermC) e virulência (icaAD e hlg). Também, identificou-se a resistência à vancomicina em Staphylococcus spp., através de métodos fenotípicos e os fatores de risco mais prevalentes para infecção em pacientes oncológicos. Observou-se um maior percentual de isolados sensíveis aos antimicrobianos testados. Em relação ao fármaco vancomicina, foram identificados 27,3% de isolados resistentes pela técnica do screening. No hospital oncológico foram positivos, 50% e 77,7% respectivamente para o gene blaZ e mecA. Observou-se percentuais de isolados positivos de 25%, 42,8%, 32,1% e 70%, respectivamente para icaAD e hlg no hospital universitário e hospital oncológico. No hospital oncológico, observou-se um isolado de fenótipo constitutivo positivo para o gene ermC. Após a realização dos testes de associação estatísticos, não foi observada diferença estatisticamente significante na presença dos genes de resistência quando comparou-se os dois hospitais. No grupo de pacientes oncológicos, observou-se diferença significativa quando comparados os indivíduos infectados com Staphylococcus spp. que albergavam o gene mecA e os indivíduos infectados com Staphylococcus spp. sem o gene mecA em relação a contagem de neutrófilos no período da infecção. Em relação aos genes de virulência, foi possível observar diferença estatisticamente significante entre os dois hospitais. Assim, não existe diferença significativa na presença de genes de resistência quando se compara grupos de pacientes, entretanto observa-se diferenças significativas em relação à presença dos genes relacionados à virulência entre diferentes grupos, o que pode estar associado às características dos pacientes e suas internações. Desta forma, concluiu-se que o monitoramento antimicrobiano é essencial para o tratamento das infecções em cada hospital e a condição do paciente parece estar ligada ao potencial patogênico do microrganismo. / Staphylococcus spp. is one of the major infection-associated agent within health care and one of principal cause of complication in cancer patients. The severity of staphylococcal infections is related to the presence of resistance and virulence factors, which favor bacterial evasion of the host defense system. In order to compare isolates of Staphylococcus spp. of oncological and non-oncological patients from two hospitals in the city of Recife, during the period of 2013, antimicrobial susceptibility and polymerase chain reaction (PCR) tests were performed to identify the occurrence of resistance genes (mecA, blaZ, ermA and ermC) and virulence genes (icaAD and hlg). Resistance to vancomycin was identified by phenotypic methods in Staphylococcus spp. and the most prevalent risk factors for infection in cancer patients. A higher percentage of antimicrobial susceptible isolates was observed. Regarding the vancomycin drug, 27.3% of resistant isolates were identified by the screening technique. At the oncology hospital they were positive, 50% and 77.7% respectively for the blaZ and mecA gene. Percentage of positive isolates of 25%, 42.8%, 32.1% and 70%, respectively, found for icaAD and hlg in the university hospital and oncology hospital, respectively. A positive constitutive phenotype isolate for the ermC gene was observed at the oncology hospital. After performing the statistical association tests, no statistically significant difference was observed in the presence of resistance genes when comparing the two hospitals. In the group of cancer patients, a significant difference was observed when compared to individuals infected with Staphylococcus spp. that harbored the mecA gene and individuals infected with Staphylococcus spp. without the mecA gene in relation to the neutrophil count at the time of infection. Regarding the virulence genes, it was possible to observe a statistically significant difference between the two hospitals. Thus, there is no significant difference in the presence of resistance genes when comparing groups of patients, however, there are significant differences regarding the presence of virulence-related genes between different groups, which may be associated to the characteristics of the patients and their hospitalizations. In conclusion, antimicrobial monitoring is essential for the treatment of infections in each hospital and the patient's condition appears to be linked to the pathogenic potential of the microorganism.

Orientadores: Sophie Françoise Mauricette Derchain, Neury Jose Boteja / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T03:38:00Z (GMT). No. of bitstreams: 1 Rezende_VeraLucia_D.pdf: 530664 bytes, checksum: fba187bc0197794eb14788bbc9347f98 (MD5) Previous issue date: 2005 / Resumo: Introdução: Em estudos que abordam as conseqüências de cuidar do paciente com câncer na fase terminal da doença, a experiência é descrita como muito pesada e negativa. Pouco se conhece sobre o estado psicológico do cuidador principal no período que antecede a morte do paciente, embora existam muitos instrumentos padronizados para avaliar o bem-estar deste cuidador. O objetivo do presente estudo foi, na primeira fase, identificar um instrumento de avaliação global do cuidador principal de pacientes com câncer na fase terminal da doença. Na segunda fase foi avaliar a ansiedade e a depressão destes cuidadores. A seguir, avaliar o bem-estar global do cuidador e verificar sua associação com os fatores sociodemográficos, os encargos, a ansiedade e a depressão. Método: Inicialmente foi realizado um estudo de revisão de literatura para selecionar o instrumento padronizado e este foi traduzido de acordo com padrões internacionais. Em seguida, foi realizado um estudo de corte transversal, com 133 cuidadores principais indicados pelas pacientes com câncer de mama ou ginecológico, sem possibilidades curativas e acamadas por mais de 50% das horas de vigília, ou incapacitadas, internadas na enfermaria de oncologia, entre agosto de 2002 a maio de 2004. Setenta e uma mulheres apresentavam câncer de mama e 62 câncer genital. Inicialmente foi aplicada a Escala de Ansiedade e Depressão (HAD) e a seguir o General Comfort Questionnaire (GCQ). Todos os instrumentos foram lidos pelo entrevistador para cada cuidador, a fim de contornar problemas de analfabetismo e baixa escolaridade. Resultados: Os dados obtidos com este estudo foram apresentados na forma de três publicações, cujos principais resultados estão descritos a seguir. No primeiro estudo foram identificados 67 resumos sobre instrumentos padronizados e, após leitura, foram selecionados 34 artigos e escolhido o General Comfort Questionnaire (GCQ). No segundo estudo constatou-se que 99 cuidadores principais (74,4%) apresentaram ansiedade e 71 (53,4%) apresentaram depressão, com grande chance de serem concomitantes (OR 5,6; IC 95% 2,2 a 15,9). Apenas o fato de o cuidador ser homem e ser marido esteve associado com a menor ansiedade. No terceiro estudo, a aplicação do GCQ identificou que as variáveis que mais se relacionaram com o bem-estar global do cuidador foi ser homem, não ter depressão e nem ansiedade. Conclusão: O instrumento selecionado para avaliar os cuidadores de pacientes com câncer na fase terminal da doença foi o GCQ. Verificou-se um alto índice e a concomitância de ansiedade e depressão entre os cuidadores. Apenas o fato de o cuidador ser homem e marido da paciente diminuiu a ansiedade. A utilização do GCQ identificou vários fatores positivos e negativos relacionados ao cuidar. As variáveis que mais se relacionaram com o bem-estar global foram o cuidador ser homem, não ter ansiedade e nem depressão / Abstract: Introduction: Studies addressing the consequences of caring for terminally-ill cancer patients generally describe that the situations experienced are usually sensed as being negative and stressful. Little is known about the psychological status of the caregiver during the period preceding the patient¿s death, but several instruments exist to assess the well-being of the caregiver. In its first phase, the prime objective of this study was to identify an evaluation instrument capable of performing a comprehensive assessment of the informal caregiver, during the terminal phase of the disease. In the second phase, the objective was to assess the prevalence of anxiety and depression in caregivers. And finally to evaluate the association between caregiver¿s global comfort being and socio-demographic factors, burdens related to caring, anxiety and depression. Method: A revision of the literature was carried out to select a standardized instrument that has been translated into Portuguese following international translating rules. Then, a cross-sectional study with 133 principal caregivers, which had been identified by the terminally-ill patients with breast or gynecologic cancer, was undertaken between August 2002 and May 2004. Seventy-one women had breast cancer and 62 gynecologic malignancies. Initially, the Hospital Anxiety and Depression (HAD) scale has been applied to the caregivers, followed by an interview using the General Comfort Questionnaire (GCQ). All instruments were read by the investigators, in order to overcome caregivers¿ illiteracy and low educational levels. Results: Sixty ¿seven abstracts were encountered and 34 selected. The General Comfort Questionnaire (GCQ) was considered the best instrument. In the second study, 99 (74.4%) caregivers presented with anxiety and 71 (53.4%) presented with depression, and concomitance of both symptons was statistically significant (OR 5.6; 95% CI 2.2 a 15.9). Male caregivers and the patient¿s spouses were less likely to present with anxiety or depression. In the third study, analysis with the GCQ disclosed that being male and not having anxiety or depression were the caregivers¿ features related to higher levels of general comfort. Conclusion: The selected instrument to evaluate comprehensively the caregivers of patients in the terminal phase of cancer was the GCQ. Anxiety and depression were frequent in the studied population and were detected concomitantly in the majority of patients. Being male or the patients¿ spouse were characteristics associated with less anxiety and depression. GCQ identified other positive and negative factors associated with caring. Male caregivers and those not diagnosed with anxiety or depression had better general comfort / Doutorado / Ciencias Biomedicas / Doutor em Tocoginecologia

Orientador: Laura Sterian Ward / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-27T20:03:24Z (GMT). No. of bitstreams: 1 Costa_SolangeCampelo_M.pdf: 11916000 bytes, checksum: 395197d06c771d1626afeb749a3d8eeb (MD5) Previous issue date: 2001 / Resumo: Mutações no gene da Neoplasia Endócrina Múltipla tipo 1 (MEN1) têm sido descritas não só em portadores da Síndrome da MEN1, mas também em tumores esporádicos como gastrinomas, insulinomas, e carcinóides brônquicos. Pesquisamos o exon 2 do gene MEN1, tendo em vista a alta prevalência de mutações descritas nesta região do gene. Foram analisados 148 tumores esporádicos, endócrinos e nãoendócrinos. Setenta e oito tumores eram de tiróide: 28 adenomas foliculares (AF), 35 carcinomas papilíferos (CP), 14 carcinomas foliculares (CF), e um carcinoma anaplásico (Ana); havia 46 lesões de adrenal: 3 hiperplasias, 3 adenomas, e 35 carcinomas de córtex adrenal, 2 feocromocitomas, 2 ganglioneuroblastomas e um linfoma; e 24 tumores de mama: 6 carcinomas não invasivos, 16 do tipo infiltrante ductal, 2 do tipo invasivo lobular. A extração de DNA de 108 tumores embebidos em parafina e de 40 tecidos frescos foi realizada utilizando a técnica de fenol-clorofórmio e precipitação com álcool. Utilizamos 5 pares de primers para amplificar todo o exon 2 do gene MEN 1 pela técnica da Reação da Polimerase em Cadeia seguida por Single Strand Conformation Polymorphism Analysis (pCRSSCP). Os produtos de PCR de cinco tumores apresentaram banda suspeita de alteração ao SSCP. Esses produtos foram seqüenciados sense e anti-sense, mas não mostraram mutações. Nossos resultados sugerem que mutações no exon 2 do gene MEN 1 não ocorrem nos tumores esporádicos de tiróide, adrenal e mama / Abstract: Besides the mutations that underlie most cases of familial or sporadic Multiple Endocrine Neoplasia syndrome, somatic mutations of the MEN1 gene has also been described in sporadic tumors like gastrinomas, insulinomas and bronchial carcinoid, where MEN 1 is the known gene most frequently mutated. In order to determine whether the gene could also be involved in other types of endocrine and non-endocrinetumors, we examined this gene for exon 2 mutations in thyroid, adrenal and breast tumors. We performed polymerase chain reaction -single strand conformation polymorphism (PCR-SSCP) technique to screen exon 2 of the MEN1 gene of 78 sporadic thyroid tumors: 28 follicular adenomas (FA), 35 papillary carcinomas (PC), 14 follicular carcinomas (FC) and 1 anaplastic thyroid carcinoma (Ana). We also examined 46 adrenal lesions (3 hyperplasias, 3 adenomas and 35 adrenocortical carcinomas, 2 pheochromocytomas, 2 ganglioneuroblastomas, 1 lymphoma) and 24 breast cancers (6 noninvasive, 16 infiltrating ductal, 2 invasive lobular). DNA was obtained using phenol/chIoroform extraction and ethanol precipitation from 108 formalin-fixed, paraffin embedded samples and other 40 fresh tumor tissues. A non-radioactive PCR-SSCP was performed using 5 pairs ofprimers to encompass the complete coding sequence of exon 2 of the MEN 1 gene where most mutations have been described. The product of PCR of 5 tumors suspected to present bands shift at SSCP was submit to direct sequencing, sense and antisense, but did not identify mutations. These results suggest that exon 2 of MEN 1 gene is not important in breast, thyroid or adrenal sporadic tumorigenesis / Mestrado / Clinica Medica / Mestre em Clinica Medica

Programa de Doctorat: Biomedicina / Este proyecto de tesis doctoral se ha centrado en tres objetivos principales: el primero es el estudio de la función de la vía de señalización de RANK-RANKL-OPG en el desarrollo y progresión del cáncer de mama en humanos. El segundo es el análisis de la expresión de RANK en muestras de tumores humanos y análisis modificaciones en el gen que afecten en la incidencia de cáncer de mama en humanos. El tercero es la generación de modelos ortotópicos de ratón de cáncer de mama humano que sirvan como herramienta para la caracterización de nuevas dianas terapéuticas así como para testar la eficacia de nuevas drogas dirigidas a pacientes de cáncer de mama resistente a los tratamientos ya existentes. Respecto al primero objetivo, los resultados obtenidos indican que la sobre-expresión de RANK en células epiteliales de mama humanas no transformadas MCF10A induce la adquisición de un fenotipo asociado al proceso de transición epitelio-mesenquina (EMT) así como la adquisición de un fenotipo característico de células madre. El mecanismo por el cual la sobre-expresión de RANK induce EMT está relacionado con el aumento de la producción del factor de crecimiento transformate beta (TGF-beta) mediante la activación de las vías de señalización del factor nuclear potenciador de las cadenas ligeras kappa de las células B activadas (NF-κB) y la proteína quinasa activada por mitógenos p38. La inducción del fenotipo de célula madre está asociado a la activación de vía de señalización de las quinasas reguladas por señales extracelulares 1 y 2 (ERK 1/2). Los resultados referentes al segundo objetivo indican que en muestras de pacientes humanos, los niveles de expresión de RANK son más elevados en tumores con un peor pronóstico clínico (fenotipo triple negativo, alto índice de proliferación y algo grado histológico) y mediante la expresión de los niveles de mARN de RANK y RANKL se puede diferenciar entre tumores metastáticos y no metastáticos a nódulo linfático. Además, se ha descrito la existencia de un polimorfismo de nucleótido simple (SNP) presente en la región 5´del gen de RANK que está asociado con una menor probabilidad de padecer cáncer de mama en pacientes que poseen mutaciones en el gen BRCA2 (“breast cancer 2”). Finalmente, en este trabajo se describe la generación de cinco modelos ortotópicos de cáncer de mama a partir de muestras clínicas de pacientes. Estos modelos mantienen las características histológicas de los tumores humanos de origen así como los patrones de metástasis de los pacientes de los que proceden. De los cinco modelos generados, dos tienen unas características histológicas relativas a tumores triples negativos (TNBC), dos a tumores luminales (positivos para la expresión del receptor de estrógenos y progesterona) y uno a tumor Her2+. Los tumores Luminales y Her2+ manifiestan diferentes comportamientos de crecimiento en respuesta a hormonal, siendo un reflejo de los distintos tipos de comportamientos que existen en clínica. Por último, se ha testado la sensibilidad de estos modelos a taxanos (Docetaxel), una de las drogas quimioterapéuticas mas empleadas en la actualidad. Los dos modelos TNBC muestran más sensibilidad a la droga mientras que los modelos hormono-dependientes son resistentes. A partir de un modelo sensible TNBC se ha desarrollado un segundo modelo de ratones resistente a Docetaxel mediante la administración continuada del fármaco. Lo que se pretende en futuros proyectos es emplear estos modelos generados en el estudio de nuevas dianas terapéuticas en cáncer de mama así como testar nuevos fármacos en su fase pre-clínica de desarrollo. / This study has been focused in three main goals: study of RANK and RANKL pathway in the development and progression of human breast cancer; analysis of RANK expression in clinical human samples and analysis in RANK gene modifications implicated in human breast cancer; generation of orthoxenopatients of human breast cancer. The results of first objective showed RANK over-expression in human mammary cells MCF10A induces epithelial-mesenchymal transition (EMT) and Stemness phenotype. The molecular mechanism though RANK induces these effects implicates the increment of transforming growth factor beta (TGF-beta) ligands expression thorugh activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen activated kinase protein p38. On the other hand, the induction of Stemnes phenotype implicates the activation of Extracellular signal-regulated kinases 1/2 (ERK 1/2). The analysis of RANK expression in clinical human samples showed that tumor with a poor prognosis (such as triple negative tumors, tumors with high proliferative index and high histological grade) has higher levels of RANK expression. Moreover, through analysis of RANK and RANKL expression is able discriminate metastatic and non metastatic tumors. In addition, a lower probability to undergo breast cancer in patients with mutations in BRCA2 gene has been correlated with a SNP in the 5´region of RANK gene. Finally, five models of ortothopic mouse models of human breast cancer have been generated. The models obtained were two triple negative, two luminal and one Her2+. These models resemble de histological characteristics of original tumors and the metastasis patterns of the patients. Also the growth rate, latency and dependence to hormones were evaluated in these models. The sensitivity of these tumors was tested to Docetaxel. The triple negative tumors were sensitive whereas Luminal and Her2+ tumors were resistant to Docetaxel. Moreover, resistant models to Docetaxel were generated from sensitive models by continued Docetaxel administration. Samples were collected during the process in order to investigate the mechanisms implicated in resistance acquisition to Decetaxel in the future.

Infections by methicillin-resistant Staphylococcus aureus (MRSA) are still an important cause of morbidity and mortality worldwide. Health-care associated infections, especially bacteremia, often represent a clinical challenge because of the severity of the infection, the associated high mortality (close to 30% in some series) and the difficulty to initiate a correct empirical (o guided) antibiotic therapy. MRSA has spread throughout Hospital Universitari de Bellvitge (HUB) for more than 20 years and, in spite of preventive measures, the percentage of methicillin resistance among S. aureus is currently close to 24%. This figure is similar to that found in many other hospitals around Spain. The epidemiology of MRSA infection has been changing over the years with the evolution of endemic clones and the emergence of new genetic lineages of MRSA with distinct virulence attributes and with the ability to spread within the community. The identification of new reservoirs and the knowledge of the MRSA genetic content will help on one hand, to better understand the pathogenesis of the disease and to facilitate the adequate therapy; and, on the other hand, to limit the spread of epidemic lineages. The aim of the work presented in this Thesis was to provide an integrated analysis of the genetic background of successive MRSA populations causing infection in the HUB since the beginning of the endemia (1990-2014), linked to the phenotypic expression of antibiotic resistance. In addition, we were the reference Microbiology Laboratory for a multicentre study that allowed us to have access to MRSA bacteremic isolates from 23 different hospitals in Spain obtained in 2008-2009. The results of the studies addressed in this Thesis showed that, on average, 20% of all S. aureus isolated in Hospital Universitari de Bellvitge (HUB) from 1990 to 2014, were methicillin resistant. Over the last 10 years, methicillin resistance percentage was around 24%. This figure is similar to other hospitals in Spain (25%). The dominant lineage in HUB for the first decade (the multiresistant Iberian clone, ST247-SCCmecI-agrI) was considered extinct. Currently, Clonal Complex 5, related to the Pediatric clone (ST5-SCCmecIV-agrII), was the dominant lineage in the HUB, as it was in other Spanish hospitals. In the HUB, clone ST8-SCCmecIV-agrI has become the second most frequent lineage in 2014. Antibiotic resistance determinants as well as genetic determinants of virulence properties were specific of particular clones. The dominant lineage in HUB for the first decade (the multiresistant Iberian clone, ST247-SCCmecI-agrI) was considered extinct. Currently, Clonal Complex 5, related to the Pediatric clone (ST5-SCCmecIV-agrII), was the dominant lineage in the HUB, as it was in other Spanish hospitals. In the HUB, clone ST8-SCCmecIV-agrI has become the second most frequent lineage in 2014. Antibiotic resistance determinants as well as genetic determinants of virulence properties were specific of particular clones. The presence of community-acquired MRSA lineages was detected in both MRSA collections (MRSA isolated from HUB and MRSA from different Spanish hospitals), being the most important clone USA300 (with positive and negative ACME) and the livestock related clone MRSA-ST398. However, the number of isolates belonging to these clones was scarce and in most cases corresponded to hospital-onset infections, caused by bacteria acquired in the community. No significant nosocomial transmission of community-acquired clones was detected. Vancomycin tolerance was only detected among isolates belonging to clone ST247-SCCmecI-agrI (Iberian clone). In addition, we could not detect any influence on vancomycin susceptibility expression of agr polymorphism or rpoB mutations. / SARM se ha diseminado en el Hospital Universitari de Bellvitge (HUB) durante más de 20 años y a pesar de la aplicación de medidas preventivas, actualmente el porcentaje de resistencia a meticilina entre los aislamientos de SARM está cerca al 24%. Esta situación es similar a muchos otros hospitales españoles. El objetivo del trabajo presentado en esta tesis fue realizar un análisis integrado del perfil genético de las sucesivas poblaciones de SARM que han causado infección en el HUB desde el principio de la endemia (1990-2014), y un estudio de la expresión fenotípica de la resistencia antibiótica. En, este trabajo, se incluye también los resultados de un estudio multicéntrico en el que el Servicio de Microbiología del HUB actuó como centro de referencia, para el estudio de aislamientos procedentes de pacientes con bacteriemia por SARM ingresados en 23 hospitales españoles aislados durante el período 2008-2009. Los resultados de los estudios presentados en esta tesis muestran que la resistencia media a meticilina en S. aureus alcanzó un valor del 20% en el HUB en el período 1990-2014. Durante los últimos 10 años, el porcentaje de resistencia a meticilina fue próximo al 24%. Esta situación fue similar en los demás hospitales (25%). El linaje dominante en el HUB durante la primera década del estudio (el clon multiresistente — clon Ibérico, ST247-SCCmecl-agr1), desapareció posteriormente. Actualmente el complejo clonal 5, relacionado con el clon Pediátrico (ST5-SCCmecIV-agrIII) es el linaje dominante en el HUB, así como en los demás hospitales en España. En el HUB, el clon ST8-SCCmecIV-agrl se ha convertido en el segundo clon más frecuente en 2014. Los determinantes de resistencia antibiótica así como los determinantes genéticos de virulencia fueron específicos para cada clon. La presencia de linajes de origen comunitaria se detectó en ambas colecciones de SARM (SARM aislado en el HUB y SARM aislado de diferentes hospitales españoles), siendo los más importantes los clones USA300 (ACME positivo y negativo) y el clon SARM asociado al ganado - SARM-ST398. Sin embargo, el número de aislados que pertenecieron a estos clones fue bajo y en la mayoría de los casos se asociaron a infecciones que debutaron en el hospital, causadas por bacterias adquiridas en la comunidad. No se detectó transmisión nosocomial de los clones asociados a la comunidad.

Se trata de un estudio observacional, retrospectivo, de tipo analítico de todas las biopsias realizadas en la Unidad de Patología Mamaria del Servicio de Radiología Diagnóstica y Terapéutica del Hospital Universitario General de Castellón entre Enero de 2011 y Diciembre de 2014. Durante estos 4 años se llevaron a cabo 997 biopsias mamarias para diagnosticar 804 lesiones de 761 pacientes. Mediante este estudio se pretende evaluar la utilidad de la biopsia por aspiración al vacío (BAV) en el diagnóstico de las lesiones mamarias sospechosas de malignidad y compararla con la BAG y el arpón que son las otras dos técnicas de biopsia mamaria que se utilizan en nuestro Servicio. Realizaremos también un estudio económico de estos tres tipos de biopsia mamaria para averiguar cual es la opción más costo efectiva. Las tres técnicas de biopsia mamaria evaluadas se realizan guiadas por ecografía o por estereotáxia vertical. Tras realizar el estudio se ve que no hay diferencias estadísticamente significativas en cuanto al porcentaje de diagnósticos correctos conseguidos con la BAG (91.75 %), la BAV (94.03 %) y la biopsia por arpón (100%) con una p = 0.3485. La BAG es la opción dominante en el estudio de coste efectividad comparado con la BAV y la biopsia por arpón para el diagnóstico de las lesiones mamarias sospechosas de malignidad en su conjunto. No obstante, el bajo porcentaje de diagnósticos correctos de la BAG (50 %) en la biopsia de microcalcificaciones desaconsejan su uso para biopsiarlas y colocan a la BAV como técnica indicada para la biopsia de microcalcificaciones tanto por su elevado porcentaje de diagnósticos correctos (96.77 %) como por ser la opción más costo efectiva frente a la biopsia con arpón, que sería la otras técnica indicada para biopsia de microcalcificaciones.

Tesi vinculada a l'Institut de Recerca Biomèdica de Barcelona (IRBB) / The human Tousled-like kinases 1 and 2 (TLKs) are predicted serine/threonine kinases that show maximal activity in S phase and are transiently inhibited by the DNA damage response (DDR). Both TLKs interact with and phosphorylate each other and the histone chaperone Asf1. Asf1 plays a critical role in regulating histone pools during several cellular processes, suggesting that the primary function of TLKs could be in the regulation of chromatin assembly during transcription, replication and repair processes. Thus, we hypothesize that reduction of TLK activity will impact on the function of Asf1, and perhaps other chromatin modulators, affecting key cellular processes that maintain genome integrity and proliferative capacity. In order to examine the in vivo consequences of TLK1 or TLK2 loss of function, we generated mice harboring genetraps that inhibit the expression of either gene. Surprisingly, mice lacking TLK1 were born normally, showed no overt pathology and aged normally over 18 months. Examination of developmental processes, such as lymphocyte maturation, revealed no abnormalities, and the DNA replication and cell cycle progression were normal, even following DNA damage. To determine if TLK2 provided redundant functions, we performed transient siRNA depletion of TLK2 in wild type and TLK1 null cell cultures. While this led to no defects in survival in WT cells, TLK1 mutants were profoundly sensitized to DNA damaging agents. We next generated mice harboring a genetrap allele to block the expression of TLK2. In contrast to TLK1, no liveborn homozygous mutants have been observed and embryos isolated for MEFs are severely runted, displaying heterogeneous defects including failure to close the neural tube and placental impairment. These data indicated that TLK1 and TLK2 do not play equivalent roles during development. Given that we see an acute response to DNA damage under conditions where total TLK activity is reduced and it is unlikely that cells lacking all TLK activity can support proliferation, we believe that the modulation of TLK activity represents a potentially valuable therapeutic approach. Collectively, the work I have presented represents a significant advance in our understanding of TLK function in cells and in mammalian development and supports the possibility that TLKs represent a potentially valuable target for the treatment of human disease.

Antecedentes: La alimentación del recién nacido (RN) sano, prematuro y enfermo con leche materna (LM), mejora su cociente de desarrollo, trasmite factores inmunoprotectores y previene complicaciones como enterocolitis necrotizante y sepsis. Los RN que no disponen leche de su propia madre, la leche materna donada (LMD) es la mejor opción. Por esta razón, la apertura de Bancos de Leche Humana (BLH) continúa en expansión. Hasta el momento en las guías de actuación de los diferentes BLH no existen protocolos que incluyan el cribado de drogas en LMD. La mayoría de las sustancias que inhala o ingiere la madre pasan parcialmente a la leche. Es importante que la LMD sea segura y se encuentre en las mejores condiciones para ser proporcionada a los RN. Los objetivos del presente trabajo son: (1) evaluar la seguridad toxicológica de la LMD; (2) analizar la concentración de tóxicos encontrados en la LMD y su posible repercusión en el neonato; y (3) justificar la realización de un cribado de drogas en la selección de donantes y durante el procesamiento de la leche. Metodología: En una primera fase (febrero a julio de 2009), se incluyeron 63 mujeres donantes del BLH del Hospital 12 de Octubre en Madrid. Se analizaron 400 muestras de leche pasteurizada además de 34 muestras antes y después del proceso de pasteurización. En un segundo período (agosto de 2010 a febrero de 2012), participaron 36 mujeres para el cribado de sustancias de abuso en LM y pelo. En el mismo periodo se analizaron 54 muestras de orina recogida durante la selección de donantes. Se compararon los resultados obtenidos a partir de 3 matrices biológicas con los datos recogidos en el cuestionario de hábitos de vida. Para el estudio bioquímico de leche y orina se utilizó la cromatografía líquida acoplada a espectrometría de masas en tándem (LC-MS/MS) y cromatografía de gases para cabello. Resultados: El 45,3% de las muestras contenían cafeína (CAF) (496 ng/ml). En el análisis de la leche y el pelo de las 36 mujeres se encontró: CAF (272,76 ng/ml) en el 50% de las muestras de leche. En pelo CAF (1.769 ng/mg) en 77,7% de las muestras; nicotina (NIC) (1.805 ng/mg) y su metabolito cotinina (COT) (0,148 ng/mg) en el 33,3%. En el análisis de orina se determinó: CAF (343,70 ng/ml) en el 57,4% de las muestras; COT (0,52 ng/ml) y 3-OH-COT (2,01 ng/ml) en el 35,12%. No se identificaron sustancias de abuso ilegales. El proceso de pasteurización no modificó la concentración de CAF. Discusión: Hasta el momento no existen guías en BLH donde se incluya algún método objetivo para el cribado de sustancias de abuso. Se toma como válida la información declarada mediante un cuestionario de hábitos de vida. Las donantes del BLH están sensibilizadas para evitar el consumo de sustancias de abuso, por lo que los resultados no pueden extrapolarse a la población general. Conclusiones: Los cuestionarios empleados durante la selección de donantes y el proceso de pasteurización no constituyen una medida de seguridad toxicológica absoluta. No obstante, los niveles de CAF encontrados en la LMD no son lo suficientemente altos para ocasionar sintomatología en el RN. Actualmente se cuenta con la tecnología necesaria para el cribado de tóxicos en BLH. Incluir el estudio de orina y pelo durante la selección de donantes y un cribado en la leche acumulada de cada madre previo al proceso de pasteurización y alicuotado puede garantizar que la LM dispensada sea de calidad y libre de tóxicos. / Introduction Breast milk feeding improve developmental quotient, transfers immunoprotective factors and prevent complications such as necrotizing enterocolitis and sepsis. New born (NB) whose their own mother’s milk is not available, donated human milk (DHM) is the best choice. For this reason, the opening human milk banks (HMB) continue to expand. Actually in these HMB there are no protocols for drug screening in DHM. Most of substances consumed or inhaled by mothers partially pass into the breast milk. It is important having DHM safe and in optimal conditions to be provided to NB. The objectives of this work are: (1) Evaluate toxicological safety of DHM; (2) analyze drugs concentration found in DHM and its possible clinical effect in neonates; and (3) justify a drug screening during the donor selection and milk processing. Methodology: In a first stage (February to July 2009), 63 lactating women donors from HMB Hospital 12 de Octubre in Madrid were included in the study. 400 pasteurized milk samples and 34 samples before and after the pasteurization were analyzed. In a second period (August 2010 to February 2012), 36 women who participated in drugs of abuse screening in hair and milk were included. In the same period, 54 urine samples were collected during donor selection process and were analyzed. We compared results obtained from substance abuse determination in 3 different biological matrices with data collected in lifestyle questionnaires. Milk from 3 women cannabis, cocaine and methadone respectively consumers were analyzed as control. Biochemical analysis of milk and urine samples was performed by liquid chromatography tandem mass (LC-MS/MS), a validated method for quantifying simultaneously 18 substances in a single sample. Hair determinations were performed by gas chromatography. Results: Caffeine (CAF) was found in 45,3% of donor milk samples, with a mean concentration of 496 ng/ml. Milk and hair analysis from 36 women indicated: CAF (272,76 ng/ml) in 50% of human milk; CAF (1.769 ng/mg) in 77% of hair samples and nicotine (NIC) (1.805 ng/ml) and its metabolite cotinine (COT)(0,148 ng/mg) were found in 33,33% of the total hair samples. Urinalysis revealed CAF (343,70 ng/ml) in 57,4% of samples; COT (0,52 ng/ml) and 3-OH-COT (2,01 ng/ml) in 35,12% of samples. No illegal drugs were identified in any of 3 biological matrices. Pasteurization had no effect on caffeine concentration measured in milk samples. Discussion: Actually there are no guidelines in HMB which a target DHM target method of screening substances of abuse is included. Information reported in lifestyle questionnaire is considered as valid. HMB donors are sensitized to prevent substance abuse consumption; by this reason results can not be extrapolated to the rest of the population. Conclusion: Lifestyle questionnaire used during donor selection and pasteurization process does not constitute a measure of toxicological safety. However CAF levels found in DHM are not high enough to produce /cause symptoms in the NB. Nowadays necessary technology exists for toxic screening in HMB. Including urine and hair analysis for donor selection and screening drugs in milk pool before pasteurization and aliquoted process may guarantee dispensing breast milk without drugs.

OBJETIVOS: El objetivo principal fue determinar la prevalencia de hiperparatiroidismo secundario (sHPT) y de déficit de vitamina D (DVD) en pacientes con insuficiencia cardíaca (IC) estable. Otros objetivos fueron: analizar la relación entre sHPT/DVD y los tratamientos de la IC, evaluar su relación con datos ecocardiográficos, clínicos y analíticos, la relación con otros biomarcadores y con la morbimortalidad, así como determinar los pacientes candidatos a realizar tratamiento del sHPT. MATERIAL Y MÉTODOS: Estudio prospectivo y observacional de una cohorte de pacientes con IC estable en el Hospital Vall d'Hebrón. Se incluyeron pacientes diagnosticados de IC en fase estable, sin diagnóstico previo de hiperparatiroidismo primario, con filtrado glomerular (eGFR) >30mL/min/1,73m² y que no estuvieran bajo tratamiento con suplementos de VD o quelantes del fósforo. Se evaluaron las características demográficas, clínicas, analíticas, ecocardiográficas y el tratamiento administrado. Se analizaron la mortalidad, ingresos y visitas a urgencias tras un año de seguimiento. Se ha realizado un análisis descriptivo de los resultados.Para comparar la media de una variable entre 2 o más grupos, se utilizaron respectivamente el test t de Student o el test ANOVA. Para las variables categóricas se utilizó el test chi-cuadrado o el exacto de Fisher. Con las variables significativas se realizó un modelo de regresión lineal múltiple. Se consideró significativo un valor de p<0,05. RESULTADOS: Entre febrero 2013 y marzo 2014 se incluyeron 260 pacientes. El 71% fueron varones con una edad media de 71±11 años. La etiología más frecuente de IC fue la isquémica (40%). Las comorbilidades más frecuentes fueron: dislipemia (78%), hipertensión (62,7%) y obesidad (41%). El 82% tomaba betabloqueantes, el 95% inhibidores del enzima conversor de la angiotensina o antagonistas del receptor de la angiotensina II y el 47% antagonistas de la aldosterona. El 61% tenía una función sistólica reducida. La prevalencia de sHPT fue del 69,2% y los niveles de VD fueron subóptimos en el 97,7%. Calcemia y fosfatemia fueron normales. El sHPT se relacionó con los niveles de NT-proBNP (p<0,005), sin encontrar relación entre sPTH con los valores de renina y aldosterona, aunque sí con el tratamiento a dosis bajas de antialdosterónicos. Las dosis elevadas de diuréticos y el eGFR se relacionaron con el sHPT (p<0,005). La supervivencia fue del 95%. La causa de la muerte fue cardiovascular en el 86%. El 89,2% de los pacientes no eran tributarios de tratamiento sustitutivo. DISCUSIÓN: La prevalencia de sHPT fue superior a la esperada. La activación de la PTH en respuesta a la hipocalcemia secundaria al déficit de calcitriol y al tratamiento farmacológico logró mantener la calcemia y la fosfatemia dentro de la normalidad. La hipovitaminosis D fue casi universal a pesar de vivir en un país soleado, debido probablemente a un insuficiente aporte vitamínico en la dieta y a la inmovilización en el contexto de la IC. Hemos hallado una relación entre la insuficiencia renal y marcadores pronósticos como el NT-proBNP en aquellos pacientes con PTH elevada, destacando la asociación con la elevada dosis de diuréticos y baja de antialdosterónicos. La elevada supervivencia no permite identificar la presencia de factores pronósticos a medio plazo en esta población. CONCLUSIONES: 1.- La prevalencia de sHPT y déficit de VD en pacientes con IC estable es superior a la descrita en otras poblaciones. 2.- Existe una relación entre sHPT con dosis altas de diuréticos y bajas de antialdosterónicos. 3.- No existe una relación entre sHPT ni hipovitaminosis con el pronóstico. 4. Sólo un 10% de los pacientes precisarían tratamiento sustitutivo. / AIMS: The aim of the study was to determine the prevalence of secondary hyperparathyroidism (sHPT) and vitamin D deficiency (VDD) in patients with stable chronic heart failure (HF). Secondary endpoints included the relationship between both sHPT and VDD with clinical, analytical and echocardiographic features, their association to other biomarkers, survival rate, and the identification of at-risk individuals who would benefit from sHPT treatment. METHODS: Prospective cohort study of consecutive patients diagnosed with stable heart failure between February 2013 and March 2014 from the Heart Failure Unit at Vall d’Hebron Hospital (Barcelona). Only patients with a glomerular filtration rate (eGFR) >30mL/min/1,73m² who were not under treatment with VD supplements, calcium or phosphate binders, and with no previous history of primary hyperparathyroidism, were included. Baseline data (demographic, clinical, echocardiographic and laboratory, as well as current treatment) were recorded in case report forms upon recruitment. After a 12-month period of follow-up, survival, number of hospital admissions and Emergency Department visit requirements were collected. Statistical analysis: For categorical variables the chi-square test or the Fischer test when appropriate, were used. The mean of variables for 2 or more groups were compared using the Student's t-test or the ANOVA test. A multiple linear regression model was built. The level of significance was established at p<0.05 RESULTS: 260 patients were included. 71% were men and their mean age was 71 (SD 11) years-old. Coronary artery disease was the most common cause of heart failure (40%). Reduced systolic function was detected in 61%. Associated comorbidities included dyslipidemia (78%), hypertension (62.7%) and obesity (41%). Treatment regimens comprised β-Blockers (82%), ACE-inhibitors or angiotensin receptor blockers (95%) and aldosterone antagonists (47%). The prevalence of sHPT and VDD was 69.2 and 97.7% respectively, with normal levels of calcium and phosphate in almost all patients. sHPT was directly related to NT-proBNP levels (p<0.005). No significant association was found between sHPT and renin-aldosterone levels but there existed an association between sHPT and the subgroup of patients under low doses of aldosterone antagonists and high-doses of diuretics. eGFR was also related to sHPT (p<0.005). Only 10% of the cohort would benefit from substitutive treatment. After one year follow-up, there was a 95% survival rate, 84% of death due to cardiovascular disease. DISCUSSION: Prevalence of sHPT was higher than expected. The activation of parathyroid hormone (PTH) in response to secondary hypocalcemia, due to calcitriol-deficiency together with therapeutic drugs, could keep calcemia and phosphatemia under normal values. Despite living in a sunny environment, hypovitaminosis D was nearly universal, probably because of both insufficient intake and HF-related immobilisation. A direct relation between renal impairment and prognostic markers like NT-proBNP was found in patients with high PTH levels, particularly in those taking high-dose diuretics and low-dose aldosterone antagonists. The high survival rate precluded the identification of prognostic factors in the medium-term. CONCLUSIONS: 1. The prevalence of sHPT and VDD in patients with stable HF is higher than in other populations. 2. There is a direct relation between sHPT and high-dose diuretics and low-dose aldosterone antagonists. 3. No relation between sHPT/VDD and prognosis has been found. 4. Only 10% of patients would require substitutive treatment.

La hernia incisional (HI) es una complicación potencialmente grave y de elevada incidencia de las laparotomías. Se caracteriza por la pérdida de estructura y de función miofasciales y la eventual ruptura del tejido. De progresión lenta, conlleva una morbilidad importante y su reparación quirúrgica usando biomateriales es un desafío. Aunque se han identificado diversos factores de riesgo (dependientes de la técnica o del paciente), la etiología fundamental de la HI es desconocida. Por el momento, no hay modelos experimentales apropiados para el estudio los mecanismos iniciales de la enfermedad y así extrapolar hallazgos preclínicos. Debido a su coste socio-sanitario, y la posible mejora en su prevención, diagnóstico y tratamiento, tiene sentido desarrollar estudios descriptivos con los que poder generar nuevas hipótesis y habilitar una nueva medicina de precisión más basada en la evidencia. En esta tesis nos hemos centrado en el estudio del nicho conectivo local, comparando muestras humanas peroperatorias derivadas de tejidos de la pared abdominal (fascia y músculo esquelético) de pacientes con historia de HI y pacientes control. El objetivo primordial del trabajo es avanzar en la comprensión de los mecanismos efectores (celulares y moleculares) implicados, y así poder contribuir a reducir el rango de variables candidatas asociadas al desarrollo y/o la predicción de la HI. Planteamos una investigación de tipo clínico orientada al paciente. De manera específica nos hemos centrado en tres ejes principales: 1.- Caracterización tisular, 2.- Caracterización de fibroblastos primarios in vitro; y, 3.- Evaluación de la respuesta fenotípica y funcional de dichos fibroblastos en sustratos de cultivo con propiedades mecánicas (rigidez) reguladas, con la intención de modelar in vitro las condiciones mecánicas del tejido humano afectado. En el análisis de la caracterización tisular, demostramos alteraciones al nivel de la matriz extracelular (MEC) local (específicas de tejido), que en última instancia podrían ser las responsables de la pérdida tisular y de la eventual ruptura. Destaca la presencia de señales diversas de muerte celular en los tejidos de pacientes con HI, que probablemente podrían provocar la destrucción progresiva de los fibroblastos locales. Bajo un segundo objetivo hemos caracterizado fibroblastos primarios in vitro, identificando un funcionamiento alterado en los derivados de HI (HIFs). Demostramos que los HIFs adquieren in vivo un fenotipo diferencial que se mantiene en cultivo a lo largo de diferentes pases, y que afecta a procesos celulares fundamentales. Finalmente, sobre la base de este progreso y para obtener una imagen más completa de estos eventos, sabiendo que la patología local puede generar también cambios en las tensiones mecánicas de los tejidos, bajo un tercer objetivo y utilizando sustratos de propiedades mecánicas reguladas, hemos podido demostrar que cambios en la rigidez del sustrato son suficientes para provocar alteraciones fenotípicas y funcionales en los fibroblastos primarios estudiados. La respuesta difiere entre fibroblastos primarios derivados de pacientes con HI y fibroblastos derivados de pacientes control. Estos resultados confirman que la modulación de la rigidez del sustrato permite una aproximación in vitro al estudio de determinados aspectos de la fisiología de fibroblasto en respuesta a cambios de rigidez del tejido conectivo (i.e., presentes en la HI). En conclusión, nuestros datos aportan evidencias indirectas pero convincentes de que el microentorno de la MEC local puede influenciar el desarrollo de la HI, actuando de una manera específica para cada tejido. Estos hallazgos pueden ser relevantes ayudando a identificar nuevos actores en el proceso HI, tal y como sucederían in vivo, y aportan información para poder definir nuevas dianas que podrían ayudar a diferentes niveles: a) mejor identificación y estratificación de paciente, b) mejora de resultados clínicos, c) contribuir a adaptar el diseño de biomateriales para mejorar el rendimiento clínico. / The incisional hernia (IH) is a potentially serious and high incidence complication of laparotomy. It is characterized by the loss of structure and function and eventual myofascial tissue breakdown. HI is a slowly progressive pathology, leading to significant morbidity, and surgical repair using biomaterials is a challenge. Even thought it has described several risks factors (technique or patient dependent), the primary aetiology for IH is unknown. At this time, no appropriate experimental models have been developed to study the initial mechanisms of disease, and then extrapolate the preclinical findings. Due to its social and health costs, and the possibility to improve the prevention, diagnosis and treatment of HI, it would be reasonable to develop descriptive studies to generate new hypotheses and enable a new precision medicine based on evidence. In this thesis, we focused on the study of the local connective niche, comparing perioperative human samples derived from the abdominal wall tissue (fascia and skeletal muscle) in patients with a history of HI and control patients. The primary objective of this work is to advance the understanding of the effector mechanisms (cellular and molecular) involved in HI, and thus contributing to efforts to narrow the range of candidate variables associated with the development and / or prediction of the HI. We propose a patient-oriented clinical type research. Particularly, we have focused on three main subjects: 1. - Tissue characterization, 2. - Characterization of primary fibroblasts in vitro; and 3. - evaluation of phenotypic and functional response of before mentioned fibroblasts in culture substrates with mechanical regulated properties (stiffness), with the aim of modelling the in vitro mechanical conditions of the human tissue affected. In the analysis of tissue characterization we showed alterations at local extracellular matrix (ECM) level, (tissue-specific), which ultimately may lead to tissue loss and eventual rupture. The presence of various signs of cell death in tissues of patients with IH is particularly noteworthy, which could probably lead to progressive destruction of local fibroblasts. We achieved the second goal of characterizing primary fibroblasts in vitro, identifying an altered function in IH derived fibroblast (IHFs). We show that IHFs acquire a differential phenotype in vivo which is maintained in culture over different passages, affecting fundamental cellular processes. Finally, based on these evidences and to obtain a more complete picture of these events, knowing that local pathology can also cause changes in the mechanical tension of tissue, and using substrates with mechanical properties regulated, we have demonstrate that changes in substrate stiffness are sufficient to cause phenotypic and functional alterations in primary fibroblasts studied. The response differs between primary fibroblasts derived from patients with IH and fibroblasts derived from control patients. These results confirm that modulating substrate stiffness allows an in vitro approach to the study of certain aspects of fibroblast physiology in response to changes in rigidity of the connective tissue (i.e, present in HI). In conclusion, our data provide indirect but convincing evidence that local ECM microenvironment can influence in the development of IH, acting in a specific manner for each tissue. These findings may be relevant by helping to identify new agents in the IH process, as would occurs in vivo, providing information to define new targets that could help different aspects of pathology: a) better identification and stratification of patients, b) improvement of clinical outcomes, c) contribute to adapt the design of the biomaterials to improve clinical performance.