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Uveal melanoma (UM) is an intraocular cancer with propensity for liver metastases. The median overall survival (OS) for metastatic UM (MUM) is 1.07 years, with a reported range of 0.84–1.34. In primary UM, high cysteinyl leukotriene receptor 1 (CysLT1) expression associates with poor outcomes. CysLT1 antagonists, quininib and 1,4-dihydroxy quininib, alter cancer hallmarks of primary and metastatic UM cell lines in vitro. Here, the clinical relevance of CysLT receptors and therapeutic potential of quininib analogs is elaborated in UM using preclinical in vivo orthotopic xenograft models and ex vivo patient samples. Immunohistochemical staining of an independent cohort (n = 64) of primary UM patients confirmed high CysLT1 expression significantly associates with death from metastatic disease (p = 0.02; HR 2.28; 95% CI 1.08–4.78), solidifying the disease relevance of CysLT1 in UM. In primary UM samples (n = 11) cultured as ex vivo explants, 1,4-dihydroxy quininib significantly alters the secretion of IL-13, IL-2, and TNF-α. In an orthotopic, cell line-derived xenograft model of MUM, 1,4-dihydroxy quininib administered intraperitoneally at 25 mg/kg significantly decreases ATP5B expression (p = 0.03), a marker of oxidative phosphorylation. In UM, high ATP5F1B is a poor prognostic indicator, whereas low ATP5F1B, in combination with disomy 3, correlates with an absence of metastatic disease in the TCGA-UM dataset. These preclinical data highlight the diagnostic potential of CysLT1 and ATP5F1B in UM, and the therapeutic potential of 1,4-dihydroxy quininib with ATP5F1B as a companion diagnostic to treat MUM.

For breast cancer diagnosis, the computation of the KI-67 score is a useful metric to define patient?s treatment. For a proper computation of this score, tumour and stroma areas have to be distinguished in KI-67 images. However, this is not an easy task due to the fact that in KI-67 images, tumour and stroma regions have a similar appearance. The objective of this research is to develop a semantic segmentation model capable of separating these two regions in KI-67 images. As for training this segmentation model there is the need of ground truth masks, a large part of the project has been focused on the generation of these masks. Regarding the ground truth generation, in this research we present a way to generate KI-67 ground truth masks based on translating KI-67 images into the CK-19 domain and segment them using simple morphological operators and manual corrections. The image-to-image translation model used for the translation purpose is a FASTCUT model and the semantic segmentation model we trained is a U-Net. Moreover, we present a comparative study of the performance of the U-Net for three different fields of view scope. From this research, quite good results are obtained for both ground truth generation and semantic segmentation processes.

Objective Ki67 is a prognostic and predictive marker in breast cancer (BC). However, manual scoring (MS) by visual assessment suffers from high inter-observer variability which limits its clinical use. Here, we developed a new digital image analysis (DIA) workflow, named KiQuant for automated scoring of Ki67 and investigated its equivalence with standard pathologist's assessment. Methods Sequential immunohistochemistry of Ki67 and cytokeratin, for precise tumor cell recognition, were performed in the same section of 5 tissue microarrays containing 329 tumor cores from different breast cancer subtypes. Slides were digitalized and subjected to DIA and MS for Ki67 assessment. The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate inter-observer reproducibility. The Kaplan-Meier analysis was used to determine the prognostic potential. Results KiQuant showed an excellent correlation with MS (ICC:0.905,95%CI:0.878–0.926) with satisfactory inter-run (ICC:0.917,95%CI:0.884–0.942) and inter-antibody reproducibilities (ICC:0.886,95%CI:0.820–0.929). The distance between KiQuant and MS increased with the magnitude of Ki67 measurement and positively correlated with analyzed tumor area and breast cancer subtype. Agreement rates between KiQuant and MS within the clinically relevant 14% and 30% cut-off points ranged from 33% to 44% with modest interobserver reproducibility below the 20% cut-off (0.606, 95%CI:0.467–0.727). High Ki67 by KiQuant correlated with worse outcome in all BC and in the luminal subtype (P = 0.028 and P = 0.043, respectively). For MS, the association with survival was significant only in 1 out of 3 observers. Conclusions KiQuant represents an easy and accurate methodology for Ki67 measurement providing a step toward utilizing Ki67 in the clinical setting., Highlights • Automated Ki67 scoring workflow improved reproducibility. • Sequential immunohistochemistry in the same section for precise cell recognition. • Use of a tumor mask for automatic tumor region selection. • Outperform pathologist-based Ki67 scoring in prognostic prediction.

Sigma-1 receptor (σ1R) ligands have been shown to be effective at relieving neuropathic and inflammatory pain, but have not yet been tested in experimental models of fibromyalgia. The objective of this study was to evaluate the effect of a σ1R antagonist (BD1063) compared to pregabalin. ICR-CD1 female mice were subjected to either six repeated injections of reserpine, to cause reserpine-induced myalgia (RIM6), or acidified saline intramuscular injections (ASI). In these two models, we evaluated the effect of BD1063 and pregabalin on thermal hypersensitivity, anxiety-like and depression-like behaviors, and on spinal cord gliosis. BD1063 exerted an antinociceptive effect on both reflexive (thermal hyperalgesia) and nonreflexive (anxiety- and depression-like) pain behaviors, and reduced spinal astroglial and microglial reactivity, following repeated treatment for 2 weeks. Interestingly, the effects of BD1063 were long-term, lasting several weeks after treatment discontinuation in both fibromyalgia-like models. Similar results were obtained with pregabalin, but the effects on pain behaviors lasted for a shorter length of time, and pregabalin did not significantly modulate spinal glial reactivity. The inhibitory and long-lasting effect of pharmacological blockade of σ1Rs on both sensory and affective dimensions of nociplastic-like pain and spinal cord gliosis in two experimental models of fibromyalgia support the application of this therapeutic strategy to treat fibromyalgia This research was funded by ESTEVE Pharmaceuticals (Grant number 066/13 23/01/2017) and by the Vice-Chancellorship of Research of the University of Girona (Grant number MPCUdG2016/087)

El carcinoma ductal in situ (CDIS) és un ampli espectre de lesions de caràcter neoplàsic, amb alteracions cel·lulars similars al càncer ductal invasiu però confinades al sistema ductolobulillar, amb indemnitat de la membrana basal. En el càncer de mama invasor, la identificació dels diferents subtipus moleculars i la seva aproximació mitjançant immunohistoquímica determinen un comportament diferencial i, per tant, la indicació terapèutica en cada cas. Tot i que en el CDIS també s’han descrit subtipus moleculars similars, el seu valor pronòstic real encara es desconeix. El pronòstic del CDIS es considera excel·lent, i existeix preocupació creixent quan el seu posible sobrediagnòstic i sobretractament. La nostra hipòtesi de treball és que les característiques moleculars del carcinoma in situ de mama condicionen el seu comportament a nivell local i la seva evolució, i mitjançant la seva aproximació per immunohistoquímica, s’han analitzat les dades de 282 casos de CDIS que van ser tractats a l’Hospital de la Mar de Barcelona des de l’any 2000 al 2014, amb seguiment en el centre fins a l’any 2020. Factors anatomo-patològics relacionats habitualment amb el mal pronòstic s’han associat en el nostre treball a lesions de CDIS més extenses, destacant la negativitat dels receptors hormonals i la positivitat de Her-2. A més, la positivitat de Her-2 s’ha associat a la presència de multifocalitat/multicentricitat. Les proves d’imatge utilitzades per a l’estudi prequirúrgic del CDIS han mostrat limitacions en l’estimació de la grandària, amb risc de infraestimar les lesions de pitjor pronòstic. L’estimació incorrecta de l’extensió del CDIS per mamografia s’ha associat a més a major risc de recurrència infiltrant, i el inmunofenotipus triple negatiu s’ha associat amb major risc de recurrència homolateral. El término carcinoma ductal in situ (CDIS) se refiere a un amplio espectro de lesiones de carácter neoplásico, con alteraciones celulares similares al cáncer ductal invasivo pero confinadas al sistema ductolobulillar, con indemnidad de la membrana basal. En el cáncer de mama invasor, la identificación de los distintos subtipos moleculares y su aproximación mediante inmunohistoquímica determinan un comportamiento diferencial y, por lo tanto, la indicación terapéutica en cada caso. Aunque en el CDIS también se han descrito subtipos moleculares similares, su valor pronóstico real en esta entidad aún se desconoce. El pronóstico del CDIS se considera excelente, y existe preocupación creciente en cuanto su posible sobrediagnóstico y sobretratamiento. Nuestra hipótesis de trabajo es que las características moleculares del carcinoma in situ de mama condicionan su comportamiento a nivel local y su evolución, y mediante su aproximación por inmunohistoquímica, se han analizado los datos de 282 casos de CDIS que fueron tratados en el Hospital del Mar de Barcelona desde el año 2000 al 2014, con seguimiento en el centro hasta el año 2020. Factores anatomo-patológicos asociados habitualmente al mal pronóstico se han asociado en nuestro trabajo a lesiones de CDIS más extensas, destacando la negatividad de los receptores hormonales y la positividad de Her-2. Además, la positividad de Her-2 se ha asociado a la presencia de multifocalidad/multicentricidad. Las pruebas de imagen utilizadas para el estudio prequirúrgico del CDIS han mostrado limitaciones en la estimación del tamaño, con riesgo de infraestimar las lesiones de peor pronóstico. La estimación incorrecta de las extensión del CDIS por mamografía se ha asociado además a mayor riesgo de recidiva infiltrante, y el inmunofenotipo triple negativo se ha asociado con mayor riesgo de recidiva homolateral. Ductal carcinoma in situ (DCIS) is a broad spectrum of neoplastic lesions, with cellular alterations similar to invasive ductal cancer but confined to the ductolobular system, with spare basement membrane. In invasive breast cancer, the identification of the different molecular subtypes and their approximation by immunohistochemistry determine a differential behavior and, therefore, the therapeutic indication in each case. Although similar molecular subtypes have also been described in DCIS, their actual prognostic value in this entity is still unknown. The prognosis of DCIS is considered excellent, and there is growing concern regarding possible overdiagnosis and overtreatment. Our working hypothesis is that the molecular characteristics of carcinoma in situ of the breast determine its behavior at the local level and its evolution, and through its immunohistochemical approach, data from 282 cases of DCIS treated at Hospital del Mar have been analyzed from 2000 to 2014, with follow-up at the center until 2020. Pathological and anatomical factors usually associated with poor prognosis have been associated in our work with more extensive DCIS lesions, highlighting the negativity of hormone receptors and the positivity of Her-2. In addition, Her-2 positivity has been associated with the presence of multifocality/multicentricity. The imaging tests used for the preoperative study of DCIS have shown limitations in estimating the size, with the risk of underestimating the lesions with the worst prognosis. The incorrect estimation of the DCIS extent by mammography has been associated in our work with a higher risk of infiltrative recurrence, and the triple negative immunophenotype has been associated with a higher risk of ipsilateral recurrence. Universitat Autònoma de Barcelona. Programa de Doctorat en Pediatria, Obstetrícia i Ginecologia

El melanoma de cèl·lules globoses (Balloon cell malignant melanoma - BCMM) és una estranya presentació histològica de les neoplàsies melanocítiques que, tot i ser un tipus de tumor poc comú en petits animals, sempre s’hauria de considerar en el diagnòstic diferencial d’una neoplàsia cutània que estigui composta d’elements cel·lulars amb citoplasma vacuolitzat i/o de tinció clara. El present estudi té com a objectius identificar i descriure les característiques morfològiques i immunohistoquímiques clau que faciliten el diagnòstic d’aquesta variant de BCMM en gats i gossos. Per acomplir-los, s’ha realitzat una recerca bibliogràfica àmplia i s’han estudiat histomorfològicament, histoquímicament, i immunohistoquímicament 26 casos reals. Els resultats de la revisió bibliogràfica demostren que el comportament pagetoide, l’activitat d’unió, un patró de creixement difús i sòlid o formant lòbuls i nius, i una proliferació cel·lular composta per més del 50% de cèl·lules grans, de rodones a poligonals, amb un citoplasma abundant de clar i vacuolitzat a lleugerament eosinòfil, són característiques microscòpiques altament compatibles amb BCMM. Tot i això, es requereix un panel immunohistoquímic que contingui anticossos contra Melan-A, S-100, HMB-45, PNL-2, NSE, tirosinasa o vimentina pel diagnòstic definitiu en gats i gossos. En l’estudi experimental, tot i que la majoria dels casos van presentar les característiques morfològiques i histoquímiques típiques de cèl·lules globoses i d’origen melanocític, només a través de la immunohistoquímica es va concloure el diagnòstic final. La tinció especial Fontana-Masson per si sola no va ser una tècnica fiable per establir el diagnòstic definitiu, en canvi, la tinció PAS (àcid periòdic Schiff) va ser molt específica per descartar un origen melanocític. L’expressió de Melan-A, S-100, i HMB-45, i la falta d’expressió de CKAE1/AE3 van confirmar el diagnòstic de BCMM. Aquest estudi es considera preliminar i es requereixen noves línies d’investigació per estudiar possibles marcadors descrits en la literatura que augmentin la fiabilitat diagnòstic del BCMM.

To evaluate the effects of feeding frequency (FF) and dietary protein/carbohydrate (P/CH) ratios on appetite regulation of gilthead seabream, two practical diets were formulated to include high protein and low carbohydrate (P50/CH10 diet) or low protein and high carbohydrate (P40/CH20 diet) content and each diet was fed to triplicate groups of fish until visual satiation each meal at a FF of 1, 2, or 3 meals per day. Feed intake and feed conversion ratio were higher in fish fed 2 or 3 meals than 1 meal per day and in fish fed the P40/CH20 than the P50/CH10 diet. The specific growth rate was only affected by FF, being higher in fish fed 2 or 3 meals per day than 1 meal per day. Expression of the cocaine-amphetamine-related transcript, corticotropin-releasing hormone, ghrelin receptor-a (ghsr-a), leptin, and neuropeptide y in the brain, cholecystokinin (cck) in the intestine, and leptin and ghrelin in the stomach was not affected by FF or dietary P/CH ratio. This is the first time that ghrelin cells were immune-located in the stomach of gilthead seabream. Fish fed 3 meals per day presented lower cck expression in the brain than those fed twice per day and higher hepatic ghsr-b expression than those fed once per day. Fish fed P40/CH20 diet presented higher hepatic leptin expression than those fed P50/CH10 diet. In conclusion, present results indicate that feeding a P40/CH20 diet at 3 meals a day seems to decrease the satiation feeling of gilthead seabream compared to fish fed higher P/CH ratio diets or fed 1 or 2 meals a day.

BACKGROUND AND OBJECTIVES: Cutaneous malignant melanoma arises from transformed melanocytes de novo or from congenital or acquired melanocytic nevi. We have recently reported that T-type Ca2+ channels (TTCs) are upregulated in human melanoma and play an important role on cell proliferation. The aim of this study was to describe for the first time in formalin-fixed-paraffin-embedded tissue the immunoexpression of TT-Cs in biopsies of normal skin, acquired melanocytic nevi and melanoma, in order to evaluate their role in melanomagenesis and/or tumor progression, their utility as prognostic markers and their possible use in targeted therapies. METHODS: Tissue samples from normal skin, melanocytic nevi and melanoma were subjected to immunohistochemistry for two TT-Cs (Cav3.1, Cav3.2), markers of proliferation (Ki67), cell cycle (Cyclin D1), hypoxia (Glut1), vascularization (CD31) and autophagy (LC3), V600E/BRAF mutation (VE-1) and PTEN. Immunostaining was evaluated by histoscore. In silico analysis was used to assess the prognostic value of TT-Cs over-expression. RESULTS: TT-Cs immunoexpression increased gradually from normal skin to common nevi, dysplastic nevi and melanoma samples, but with differences in distribution of both isoforms. Particularly, Cav3.2 expression was significantly higher in metastatic melanoma than in primary melanoma. Statistical correlation showed a lineal interaction between PTEN-loss/ V600E-BRAF/ Cav3.1/ LC3/ Ki67/ Cyclin D1/ Cav3.2 /Glut1. Disease-free survival (DFS) and global survival (OS) correlated inversely with over-expression of Cav3.2. DFS also correlated inversely with over-expresion of Cav3.1. DISCUSSION: TT-Cs immunoexpression on melanocytic neoplasms 1) is consistent with our previous in vitro studies, 2) appears related to tumor progression, and 3) TT-Cs upregulation can be considered as a prognostic marker using TCGA database. The high expression of Cav3.2 in metastatic melanoma encourages the investigation of the use of TT-Cs blockers in targeted therapies. This article is protected by copyright. All rights reserved. This study was supported by grants from ISCIII (FIS-PI1200260 to R.M.M., FIS-PI1301980 to J.H. and RETICS-RD12/0036/0013 to X.M.G.); from Fundació la Marató de TV3 (FMTV 201331-31 to R.M.M.) and from Generalitat de Catalunya (2014/SGR138 to X.M.-G.) and was cofinanced by FEDER ‘Una manera de hacer Europa’. O.M. and C.B. hold predoctoral fellowships from the University of Lleida and S.M. a predoctoral fellowship from IRBLleida/Diputació de Lleida. Tumour samples were obtained with the support of Xarxa de Bancs de Tumors de Catalunya, sponsored by Pla Director d'Oncología de Catalunya (XBTC), IRBLleida Biobank (B.0000682) and PLATAFORMA BIOBANCOS (PT13/0010/0014)

The use of epithelial cell adhesion molecule (EPCAM) immunohistochemistry (IHC) is not included in the colorectal cancer (CRC) screening algorithm to detect Lynch syndrome (LS) patients. The aim of the present study was to demonstrate that EPCAM IHC is a useful tool to guide the LS germ-line analysis when a loss of MSH2 expression was present. We retrospectively studied MSH2 and EPCAM IHC in a large series of 190 lesions composed of malignant neoplasms (102), precursor lesions of gastrointestinal (71) and extra-gastrointestinal origin (9), and benign neoplasms (8) from different organs of 71 patients suspicious of being LS due to MSH2 alterations. LS was confirmed in 68 patients, 53 with MSH2 mutations and 15 with EPCAM 3&prime, end deletions. Tissue microarrays were constructed with human normal tissues and their malignant counterparts to assist in the evaluation of EPCAM staining. Among 154 MSH2-negative lesions, 17 were EPCAM-negative, including 10 CRC and 7 colorectal polyps, and 5 of them showed only isolated negative glands. All lesions showing a lack of EPCAM expression belonged to patients with EPCAM 3&prime, end deletions. EPCAM IHC is a useful screening tool, with 100% specificity to identify LS patients due to EPCAM 3&prime, end deletions in MSH2-negative CRC and MSH2-negative colorectal polyps.

Gap junctions; Immunohistochemistry; Lung cancer Unions comunicants; Immunohistoquímica; Càncer de pulmó Uniones comunicantes; Inmunohistoquímica; Cáncer de pulmón Direct intercellular communication, mediated by gap junctions formed by the connexin transmembrane protein family, is frequently dysregulated in cancer. Connexins have been described as tumour suppressors, but emerging evidence suggests that they can also act as tumour promoters. This feature is connexin- and tissue-specific and may be mediated by complex signalling pathways through gap junctions or hemichannels or by completely junction-independent events. Lung cancer is the number one cancer in terms of mortality worldwide, and novel biomarkers and therapeutic targets are urgently needed. Our objective was to gain a better understanding of connexins in this setting. We used several in silico tools to analyse TCGA data in order to compare connexin mRNA expression between healthy lung tissue and lung tumours and correlated these results with gene methylation patterns. Using Kaplan-Meier plotter tools, we analysed a microarray dataset and an RNA-seq dataset of non-small cell lung tumours in order to correlate connexin expression with patient prognosis. We found that connexin mRNA expression is frequently either upregulated or downregulated in lung tumours. This correlated with both good and poor prognosis (overall survival) in a clear connexin isoform-dependent manner. These associations were strongly influenced by the histological subtype (adenocarcinoma versus squamous cell carcinoma). We present an overview of all connexins but particularly focus on four isoforms implicated in lung cancer: Cx26, Cx30.3, Cx32 and Cx43. We further analysed the protein expression and localization of Cx43 in a series of 73 human lung tumours. We identified a subset of tumours that exhibited a unique strong nuclear Cx43 expression pattern that predicted worse overall survival (p = 0.014). Upon sub-stratification, the prognostic value remained highly significant in the adenocarcinoma subtype (p = 0.002) but not in the squamous carcinoma subtype (p = 0.578). This finding highlights the importance of analysis of connexin expression at the protein level, particularly the subcellular localization. Elucidation of the underlying pathways regulating Cx43 localization may provide for novel therapeutic opportunities.

Background: Venezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela. Results: Thirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose; ears; ears and nose; and nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie. Conclusions: FeL should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum.

La Immunohistoquímica (IHC) i els tissue microarrays (TMAs) formen part de la clínica assistencial en els serveis d’anatomia patològica i oncologia. Aquesta tesi maximitza l’ús d’aquestes dues tècniques en diferents àmbits i tumors de la recerca mèdica. S'han utilitzat la IHC i els TMA per a l’optimització d’un protocol que permet la detecció de PTEN en un ampli rang de condicions analítiques i preanalítiques; per validar un algoritme que permeti identificar el tumor primari d'origen desconegut en metàstasis peritoneals i ovàriques, així com comparar la seva utilitzat amb tècniques d’expressió de microarrays; per avaluar el paper en la progressió tumoral dels canals de calci de tipus T (TT-CCs) en el melanoma i nevus, a més de correlacionar els TT-CCs amb marcadors d’hipoxia, proliferació, autofàgia i la mutació de BRAF V600E; per últim, per establir una relació en biòpsies parafinadas entre snail1 nuclear i melanomes metastàtics amb la mutació BRAF V600E., Immunohistochemistry (IHC) and tissue microarrays (TMAS) take part of clinical in oncology and pathology services. This thesis maximizes the use of these two techniques and tumors in different areas of medical research. We used the IHC and TMA to optimize a protocol that allows the detection of PTEN in a wide range of analytical and preanalytical conditions; to validate an algorithm to identify the primary tumor of unknown origin in ovarian and peritoneal metastases, as well as compare their techniques used expression microarrays; to evaluate the role in tumor progression of T-type calcium channels (TT-CCS) in melanoma and nevi in addition to correlate the TT-CCS with markers of hypoxia, proliferation, autophagy and BRAF V600E mutation; Finally, to establish a relationship between FFPE biopsies snail1 nuclear and metastatic melanoma with the BRAF V600E mutation., La Inmunohistoquímica (IHC) y los tissue microarrays (TMAS) forman parte de la clínica asistencial en los servicios de anatomía patológica y oncología. Esta tesis maximiza el uso de estas dos técnicas en diferentes ámbitos y tumores de la investigación médica. Se han utilizado la IHC y los TMA para la optimización de un protocolo que permite la detección de PTEN en un amplio rango de condiciones analíticas y preanalíticas; para validar un algoritmo que permita identificar el tumor primario de origen desconocido en metástasis peritoneales y ováricas, así como comparar su utilidad con técnicas de expresión de microarrays; para evaluar el papel en la progresión tumoral de los canales de calcio de tipo T (TT-CCs) en el melanoma y nevus, además de correlacionar los TT-CCs con marcadores de hipoxia, proliferación, autofagia y la mutación de BRAF V600E; por último, para establecer una relación en biopsias parafinadas entre snail1 nuclear y melanomas metastásicos con la mutación BRAF V600E.

Mammary carcinomas are relatively common ailments among female canines aged around 10 years old, presenting an important morbidity with an average survival of five years. The cytoinclusion technique is frequently employed in human medicine as the investigative method of choice as it quickly provides resources for the determination of the correct therapeutic response, however, the effectiveness of the technique in canines remains understudied in veterinary medicine. This study aims at evaluating the degree of correlation with immunohistochemical marking for cytokeratin 5 (CK5), oestrogen and progesterone receptors (ER and PR) between the cytoinclusion and the histopathology technique in mammary carcinomas. Twenty-five samples of mammary carcinoma, both for the cytoinclusion and histopathological techniques were submitted for histological processing; microscope slides were created for hematoxylin-eosin (HE) staining and the immunohistochemical technique (IHC) was assessed for the ER, PR and CK5 receptors. Through the HE staining, we reached a concordance rate of 100% between the cytoinclusion and the histopathological analysis in the diagnosis of carcinomas. The immunohistochemical assay presented sensitivity of 85.71%, 95.45% and 100% and Cohen’s kappa of 0.78, 0.84 and 0.95 for ER, PR and CK5, respectively, as well as 100% specificity and P

Màster en Lideratge i Gestió d'Infermeria, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, curs: 2016-2017, Director: Tomàs Santalucía Albi, Objetivo principal: Definir, mediante un estudio experimental, los tiempos mínimo y máximo de fijación para las muestras que garanticen una determinación correcta de la expresión de PDL1. Ámbito del estudio: Se trata de un estudio metodológico orientado a definir los criterios de calidad en los laboratorios de anatomía patológica para aquellos procedimientos inmunohistoquímicos de marcadores relacionados con la terapia antineoplásica dirigida. Metodología: Estudio experimental con fragmentos de tejido amigdalar fresco, se estudiarán N=16 piezas obtenidas de 4 especímenes diferentes de forma paralela en condiciones idénticas y controladas con distintos tiempos de fijación (12, 24, 48 y 72 horas; N=16 para cada tiempo de exposición). Las muestras se marcarán con dos anticuerpos distintos (SP142 y SP263) en un diseño mixto 4x2 (muestras repetidas y grupos independientes) y los resultados serán valorados de forma ciega dicotómicamente (muestra correcta / incorrecta) según criterios predefinidos. El estudio estadístico será no paramétrico (tests de test de Friedman y McNemar. Implicaciones para la práctica: Los resultados del experimento ayudarán a definir los circuitos adecuados de procesamiento de este tipo de muestras en el futuro.

ObjectiveTo report the clinical and oncologic associations of antibodies against Kelch-like protein 11 (KLHL11-ab), recently suggested as biomarkers of a paraneoplastic brainstem cerebellar syndrome associated with testicular seminoma, and to determine the value of immunohistochemistry as a screening technique.MethodsStudies included 432 sera or CSF from 329 patients with paraneoplastic (157) or autoimmune neurologic syndromes (172); 63 with neurologic symptoms and benign teratomas; 28 with small-cell lung cancer, and 12 healthy subjects. KLHL11-abs were examined using a cell-based assay (CBA) with HEK293 cells transfected with a human KLHL11 clone. The CBA specificity was confirmed by immunoprecipitation. All positive samples were examined by immunohistochemistry on rat brain sections.ResultsKLHL11-abs were detected in 32 patients by CBA, and patients' antibodies immunoprecipitated KLHL11. Using rat brain immunohistochemistry, only 7 samples (22%) were positive. Patients' median age was 28 years (range 9–76 years), and 16 (50%) were women. Tumors were identified in 23/32 (72%) patients, including 14 teratomas and 7 seminomas or mixed germ cell tumors. Thirteen (41%) patients had cerebellar ataxia (7) or encephalitis with brainstem cerebellar symptoms (6), 7 (22%) anti-NMDA receptor (NMDAR) encephalitis (5 with ovarian teratoma), 5 (16%) opsoclonus-myoclonus, 3 (9%) limbic encephalitis, and 4 (12%) diverse neurologic symptoms (3 with benign teratomas). Concurrent autoantibodies occurred in 14 (44%) patients (7 anti-NMDAR, 6 Ma2, and 1 Hu).ConclusionsKLHL11-abs associate with a spectrum of syndromes and tumors wider than those previously reported; 44% of patients have concurrent neuronal antibodies, some of them (anti-NMDAR) pathogenically relevant. Brain immunostaining is not useful for routine screening of KLHL11-abs.

NANOG is a master regulator of embryonic stem cell pluripotency, found to be frequently aberrantly expressed in a variety of cancers, including laryngeal carcinomas. This study investigates for the first time the role of NANOG expression in early stages of laryngeal tumourigenesis and its potential utility as cancer risk marker. NANOG protein expression was evaluated by immunohistochemistry using two large independent cohorts of patients with laryngeal precancerous lesions, and correlated with clinicopathological parameters and laryngeal cancer risk. NANOG expression was detected by immunohistochemistry in 49 (60%) of 82 laryngeal dysplasias, whereas expression was negligible in patient-matched normal epithelia. Strong NANOG expression was found in 22 (27%) lesions and was established as cut-off point, showing the most robust association with laryngeal cancer risk (P = 0.003) superior to the histological classification (P = 0.320) the current gold standard in the clinical practice. Similar trends were obtained using a multicenter validation cohort of 86 patients with laryngeal dysplasia. Our findings uncover a novel role for NANOG expression in laryngeal tumourigenesis, and its unprecedented application as biomarker for cancer risk assessment.

El cáncer gástrico es el resultado de sucesivos cambios histológicos en la mucosa gástrica conocidos como lesiones preneoplásicas y cuya sucesión es conocida como la Cascada de Correa, constituyendo el mecanismo de carcinogénesis gástrica establecido en la actualidad. Entre estas lesiones preneoplásicas, la metaplasia intestinal es crucial debido a su alta tasa de progresión a cáncer gástrico y su preciso diagnóstico histológico. Los principales factores de riesgo de la carcinogénesis gástrica son tanto ambientales como genéticos. Entre los cuales se encuentran factores de susceptibilidad genética constituidos por polimorfismos de nucleótido simple (SNP), y la infección por la bacteria Helicobacter pylori, la cual induce cambios en la expresión génica y la activación de múltiples vías de señalización celular de naturaleza proliferativa y oncogénica. Esta tesis esta formada por cuatro estudios relacionados en cuanto a la exploración de la susceptibilidad del paciente al cáncer gástrico; pero usando diferentes muestras y metodología técnica en cada estudio. Hay dos estudios de susceptibilidad genética con el cáncer gástrico y sus lesiones preneoplásicas, los que revelan asociaciones estadísticas de los genes CD14, NOD2 y MUC2. Un estudio de microarrays de expresión identificó nuevos genes diferencialmente expresados (TRIM, TMEM, SNORDs116) y procesos moleculares (efecto Warburg, glicosilación mucina aberrante, tiroides desregulación hormonal y la degradación de melatonina) característicos de la metaplasia intestinal. Además, se observó un mayor número de vías de señalización desreguladas (activación del ciclo y la proliferación celular, oncogenes, supresores de tumores) en la metaplasia intestinal incompleta con respecto a la metaplasia intestinal completa, lo que apoya su mayor riesgo de progresión a cáncer gástrico observado en estudios epidemiológicos. Además, en un enfoque traslacional con el objetivo de validar en humanos los resultados de un modelo de ratón de metaplasia intestinal. Se observó que la expresión génica de SLFN5 es inducida por IFNalfa en lineas humanas de células T y monocitos, y la proteína SLFN5 es expresada por estos mismos tipos celulares en la mucosa gástrica humana. De manera relevante, se observa que la proteína se SLFN5 se sobre-expresa en la metaplasia intestinal que progresa a cáncer gástrico respecto a la misma lesión que no experimenta progresión tumoral en el transcurso del tiempo. El aumento de su expresión inmunohistoquímica incrementa de manera significativa la probabilidad de identificar a pacientes afectados por esta lesión que progresan a cáncer gástrico a lo largo del tiempo. El contenido de la tesis se estructura de acuerdo con el esquema de introducción, hipótesis, objetivos, materiales y métodos, resultados, discusión, conclusiones y referencias. Finalmente, el apéndice incluye la publicación obtenido como resultado directo de este trabajo.

Background/Aim: Invasive lobular breast carcinoma is the second most common type of breast cancer after invasive ductal carcinoma. According to the American Cancer Society, more than 180,000 women in the United States find out they have invasive breast cancer each year. Personal history of breast cancer and certain changes in the breast are correlated with an increased breast cancer risk. The aim of this work was to analyze breastfeeding in patients with infil- trating lobular breast carcinoma, in relation with: 1) clinicopathological parameters, 2) hor- monal receptors and 3) tissue-based tumor markers. Materials and Methods: The study included 80 women with ILC, 46 of which had breastfeed their children. Analyzed parameters were: age, tumor size, axillary lymph node (N), distant metastasis (M), histologi- cal grade (HG), estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), Ki-67, p53 and BCL2. Results : ILC of non-lactating women showed a larger (p = 0.009), lymph node involvement (p = 0.051) and distant metastasis (p = 0.060). They were also more proliferative tumors mea- sured by Ki-67 (p = 0.053). Breastfeeding history did not influence the subsequent behavior of the tumor regardless of histological subtype. Conclusion: Lactation seems to influence the biological characteristics of ILC defining a subgroup with more tumor size, axillary lymph node involvement, distant metastasis and higher prolifera- tion measured by ki-67 expression.

Aunque los pacientes con cáncer de pulmón a células no pequeñas en estadios tempranos (NSCLC) tienen buen pronóstico, el 20-30% recae, siendo relevante la identificación de biomarcadores pronósticos. Los retinoides regulan crecimiento y diferenciación, y pueden antagonizar la progresión tumoral. Su efecto depende del transporte citosólico mediado por moléculas como CRBP1, y de la unión a receptores específicos (RARβ). Alteraciones en esta vía se asociaron con cáncer. Nuestro objetivo fue estudiar la expresión, mediante inmunohistoquímica, de RARβ y CRBP1 en el tejido tumoral de 49 pacientes NSCLC Estadio I/II, obtenido durante la cirugía. La supervivencia se analizó mediante los test Log Rank y multivariado de Cox. El 44.9% de los tumores fueron positivos para RARβ con expresión a nivel citoplasmático, mientras que el 34.7% lo expresó a nivel nuclear. La tinción para CRBP1 se observó en el 61.2% de los tumores. No se encontró asociación entre la expresión de ambas moléculas y las características clinicopatológicas (sexo, tamaño tumoral, nódulos línfáticos comprometidos, histopatología y p53). Tampoco se encontró asociación con el hábito de fu-mar. La presencia de células tumorales en el lavado pleural se asoció significativamente con la expresión de CRBP1. Por otro lado, se demostró asociación entre la expresión elevada de RARβ citoplasmático y menor supervivencia global (LR 4.17, p=0.0412). El análisis multivariado no mostró asociación con otras variables de pronóstico en NSCLC. En conclusión, en este grupo de pacientes NSCLC Estadio I/II, RARβ pareciera predecir la supervivencia global en forma independiente.Although early-stage non-small-cell lung carcinoma (NSCLC) patients have a relative by favorable prognosis, the risk of a bad outcome remains substantial. Identification of reliable prognostic markers for disease recurrence and death has meaningful clinical application. Retinoids are involved in cell growth and differentiation and may antagonize cancer progression. Their effects are mediated through nuclear receptors called Retinoic Acid Receptor (RAR) and regulated by molecules such as Cellular Retinol-Binding Protein 1 (CRBP1) that function in retinol storage. The aim of this work was to analyze by immunohistochemistry the expression patterns of RARβ and CRBP1, involved in retinoid-mediated signaling, in the tumoral tissue of a cohort of stage I/II NSCLC patients (n=49) who underwent a successful surgical resection. Prognostic evaluation was performed with the multivariate Cox proportional hazard model: 44.9% of tumors were positive for RARβ staining at cytoplasmic level, while 34.7% showed nuclear staining. CRBP1 staining was observed in 61.2% of the lung tumors. No relationship was found between the number of cells expressing the studied molecules and clinical pathological features, including sex, T and N (stage), histopathology and p53 expression. Univariate analysis showed a significant association between positive cytoplasmatic expression of RARβ with shorter overall survival (Log-rank test 4.17, p=0.0412). Multivariate studies indicated that RARβ expression was not influenced by other clinical pathological parameters. In conclusion, in this cohort of stage I and II NSCLC, only the expression of RARβ at cytoplasmatic level is a significant independent unfavorable prognostic factor.

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re- specification of non-sensory areas into sensory epithelia, and the loss of Reissner’s membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror- image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea., This work was supported by the Spanish MinEco [BFU2010-15477, BFU2011-24057, BFU2013-40944]; Fundació La Maratód e TV3; TerCel [RD06/0010/0000]; and Red de Terapia Célular de la Junta de Castilla y León.

Generalment el Càncer d’Endometri (EC) s’associa a bon pronòstic, no obstant el 20% dels EC associats a bon pronòstic recidiven. El propòsit d’aquesta tesi ha estat l’estudi de les principals diferències en l’expressió de gens implicats en el desenvolupament i progressió del CE i que estan relacionats amb la hipòxia, l’apoptosi i l’adaptació a la radioteràpia mitjançant la Immunohistoquímica i els Arrays matricials de teixit en CE primaris i recidives post radioteràpia. Les recidives presenten valors més elevats de p53, HIF-1α, FLIP, Ki67, p50, c-REL, Rel B i β-catenina. La hipòxia activa tant la via NF-κB canònica com l’alternativa i tant la cinasa IKKα com IKKβ són necessàries per l’acumulació de RelA (p65) i p100, mentre que el processament de p52 depèn de IKKα. La hipòxia també indueix l’expressió nuclear de HIF-1α, FLIP, β-catenina i l’augment de l’activitat de via NF-κB, en canvi això no s’observa quan la línia cel•lular Ishikawa es sotmet a radiació. Per la seva banda, ANXA2 juga un paper en promoure el procés de metàstasi en el EC i és un marcador predictiu de recidiva també en els EC de risc baix-intermedi. La classificació histològica del EC pot ser difícil en alguns carcinomes d’alt grau o mixtes. Per aquest motiu també hem determinat una firma de 9 biomarcadors que ajuda a predir el tipus histològic en el EC., Generalmente el Cáncer de Endometrio (EC) se asocia a buen pronóstico, sin embargo el 20% de los EC asociados a buen pronóstico recidivan. El propósito esta tesis doctoral ha sido el estudio de las principales diferencias en la expresión de genes implicados en el EC y que están relacionados con la hipoxia, la apoptosis i la adaptación a la radioterapia mediante la Inmunohistoquímica y los Arrays matriciales de tejido en EC primarios y recidivas post radioterapia. Las recidivas presentan valores más elevados de p53, HIF-1α, FLIP, ki 67, p50, c-REL, Rel B y β-catenina. La hipoxia activa tanto la vía NF-κB canónica como la alternativa y tanto la cinasa IKKα como IKKβ son necesarias para la acumulación de RelA (p65) y p100, mientras que el procesamiento de p52 depende de IKKα. Además, la hipoxia induce la expresión nuclear de HIF-1α, FLIP, β-catenina y el aumento de la actividad de NF-κB aunque dichos cambios no se observan cuando la línea celular Ishikawa se somete a radiación. Por otro lado ANXA2 juega un papel en la promoción del proceso de metástasis en el EC y es un marcador predictivo de recidiva incluso en los EC de riesgo bajo-intermedio. La clasificación histológica del EC puede ser difícil en algunos carcinomas de alto grado o mixtos. Por ese motivo también hemos querido determinar una firma de biomarcadores que ayude a predecir el tipo histológico en el EC., Endometrial Cancer (EC) is usually associated with good prognosis. However, 20% of the EC associated with good prognosis have recurrent disease. The aim of this work was the study of the expression of relevant genes involved in development, progression and recurrence of EC and those involved in resistance to apoptosis, hypoxia and adaptation to radiation, using Immunohistochemistry and Tissue Micro Arrays in primary EC and post radiation recurrences. Post radiation recurrences showed higher p53, HIF-1α, FLIP, Ki67, p50, c-REL, Rel B and β-catenin expression values. Hypoxia activates not only canonical NF-ΚB molecular pathway but also the alternative and both IKKα and IKKβ are necessary to RelA (p65) and p100 accumulation whereas p52 processing is IKKα dependent. Additionally, hypoxia induced nuclear expression of HIF-1α, FLIP, β-catenin and increased NF-κB activity. However, these changes were not observed when Ishikawa cell line was subjected to radiation. ANXA2 plays a role in promoting metastasis in EC and is a predictive biomarker of recurrent disease also among low-intermediate risk EC. Histological typing is difficult in some high grade or mixed endometrioid-non endometrioid EC. For this reason we have assessed an internally and externally 9-biomarker signature to predict histological type in EC.

[cat] La present tesi doctoral està centrada en l'estudi de l'expressió dels receptors d'entrada del virus de l'hepatitis C (VHC) i la detecció dels antígens virals en mostres de fetge parafinades de pacients sotmesos a un trasplantament hepàtic (TH). La primera part de l'estudi està basada en la caracterització i anàlisi, mitjançant tècniques d'immunofluorescència i microscòpia confocal, de l'expressió dels receptors d'entrada claudina-1 (CLDN1), ocludina (OCLN) i SRB1. També s'ha dut a terme l'estudi de la cinètica viral precoç amb mostres de sèrum obtingudes abans i després del TH. Els resultats obtinguts mostren que la localització de les proteïnes CLDN1 i OCLN resta inalterable al pol apical de la cèl.lula, on colocalitzen fortament independentment de la fase de la infecció. Paral.lelament s'observa una correlació significativa entre: 1. els nivells d'SRB1 en el moment de la reperfusió del nou òrgan i la disminució de la càrrega viral (CV) a les 24h després del TH, el que suggereix l'entrada massiva del virus a l'empelt i 2. els nivells de CLDN1 i OCLN i l'increment de la CV a la setmana després del TH, el que és indicatiu de l'elevada capacitat d'adaptació i replicació del VHC. En la segona part del treball s'analitza l'expressió dels antigens virals core i NS5A i es determina la colocalització entre abdues proteïnes i la seva distribució relativa versus les estructures cel.lulars denominades gotes lipídiques. Els resultats mostren que en un 75% de les biòpsies de fase aguda analitzades és possible detectar l'antigen core, l'expressió del qual es limita als hepatòcits. El marcatge és citoplasmàtic i granular, amb diferents graus d'intensitat. El patró de distribució de la tinció és difús, amb certa tendència a concentrar-se a les zones periportals. Existeix una correlació significativa entre el percentatge de cèl.lules core positives i: 1. l'activitat inflamatòria al lobulet i 2. els nivells de transaminasas ALT i AST. La detecció de l'expressió de la proteïna core s'associa de forma significativa amb la CV i existeix colocalització entre NS5A, core i adipofil.lina (proteïna constitutiva de les gotes lipídiques). Aquesta localització es dóna en forma d'estructures circulars, essent NS5A la proteïna que es situa majoritàriament a la part més perifèrica. Els resultats demostren per primera vegada in vivo la importància de les gotes lipídiques en la replicació i l'assamblatge del VHC., [eng] Liver transplantation (LT) is a unique model to study the mechanisms of hepatitis C virus (HCV) entry into hepatocytes. The aims of the first study of this thesis were: 1) to characterize the expression patterns of SBR1, claudin-1 and occludin in grafts from LT recipients; 2) to explore their potential changes after infection. Our results showed that SRB1 expression was particularly abundant in the sinusoidal domain. Claudin-1 and occludin expression was restricted to the apical zone. There was a significant correlation between the amount of SRB1 at the time of reperfusion and the HCV-RNA decay during the first 24 hours following LT. Similarly, there was significant correlation between the levels of claudin-1 and occludin and the HCV-RNA increase during the first week after LT. Overall, SRB1 levels remained stable after LT, whereas claudin-1 and occludin expression increased significantly 12 months after LT, both in patients with mild and severe HCV recurrence. Despite the increase in claudin-1 and occludin levels, their expression pattern remained unchanged and restricted to the apical membrane, where claudin-1 and occludin colocalized strongly. The aim of the second study was to detect HCV antigens in liver biopsies of these patients. All reperfusion biopsies were negative for either core or NS5A staining. NS5A and core were detected in 75% of liver biopsies obtained during the acute phase of HCV infection and in 33% of biopsies belonging to patients with active infection. Importantly, HCV antigens were not detected in any of the 11 samples from patients who cleared HCV after antiviral treatment. Single infected hepatocytes were found throughout the liver sections, with propensity to localize in the periportal areas. Immunostaining was mainly hepatocellular with a granular cytoplasmic pattern and a wide spectrum of intensity. There was strong colocalization of both proteins and in some cells the staining revealed ring-like structures, supporting the localization of core and NS5A around lipid droplets. A detailed 3D analysis showed the relative position of each protein in these structures, with core localized inside and NS5a in the periphery.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions. This work was supported by the 7th Framework Programme of European Union [grant number HEALTH-F2-2008-223713, REPROBESITY], Ministerio de Ciencia e Innovación [grant numbers SAF2010-19087, SAF 2010-20521], Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, UE-ERDF [grant number CP12/03109], Red de Trastornos Adictivos [grant numbers RD12/0028/0001, RD12/0028/0009], CIBERobn, Plan Nacional Sobre Drogas, Ministerio de Sanidad y Consumo [grant number PNSD2010/143], Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE/ERDF [grant number CTS-433, P-11-CVI-07637], Consejería de Salud, Junta de Andalucía [grant numbers PI0232/2008, PI0029/2008, SAS111224], and Fundació La Marató de TV3 [grant number 386/C/2011]. Juan Suárez is recipient of a “Miguel Servet” research contract from the National System of Health (Instituto de Salud Carlos III, grant number CP12/03109).

Array-CGH represents a comprehensive tool to discover genomic disease alterations that could potentially be applied to body fluids. In this report, we aimed at applying array-CGH to urinary samples to characterize bladder cancer. Methods: Urinary DNA from bladder cancer patients and controls were hybridized on 44K oligonucleotide arrays. Validation analyses of identified regions and candidates included fluorescent in situ hybridization (FISH) and immunohistochemistry in an independent set of bladder tumors spotted on custom-made tissue arrays (n = 181). Results: Quality control of array-CGH provided high reproducibility in dilution experiments and when comparing reference pools. The most frequent genomic alterations (minimal recurrent regions) among bladder cancer urinary specimens included gains at 1q and 5p, and losses at 10p and 11p. Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002). The amplification and overexpression of Prefoldin (PFND2), a selected candidate mapping to 1q23.3-q24.1, correlated to increasing stage and tumor grade by means of custom-designed and optimized FISH (p = 0.013 and p = 0.023, respectively), and immunohistochemistry (p ≤0.0005 and p = 0.011, respectively), in an independent set of bladder tumors included in tissue arrays. Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005). PFND2 was amplified and overexpressed in bladder tumors belonging to patients providing urinary specimens where 1q23.3q24.1 amplification was detected by array-CGH. Conclusions: Genomic profiles of urinary DNA mirrowed bladder tumors. Molecular profiling of urinary DNA using array-CGH contributed to further characterize genomic alterations involved in bladder cancer progression. PFND2 was identified as a tumor stratification and clinical outcome prognostic biomarker for bladder cancer patients, Supported by grants (SAF2009-13035 and SAF2012-40206) from the Spanish Ministry of Education and Culture (to Dr Sánchez-Carbayo). Virginia López is recipient of a predoctoral award from the Spanish Ministry of Education and Culture

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus., This work was supported by grants BIO2008-05493-C02-02 and BIO2009-11818 from Spanish Ministerio de Ciencia e Innovacion to A. Ferrando. B. Belda-Palazon is a recipient of a VALi+d predoctoral contract of Generalitat Valenciana ACIF2010/085. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Con el fin de determinar la presencia del virus de influenza en piaras de producción porcina intensivas, de mediana y gran escala, se llevaron a cabo tres estudios independientes. Se realizó un estudio serológico retrospectivo en un grupo de muestras provenientes de 17 establecimientos. Se determinó la presencia de anticuerpos contra influenza serotipos H1N1 y N3N2 a través de las pruebas de inhibición de la hemaglutinación (IHA) y la prueba de ELISA. La prueba de IHA utilizó antígenos contra los serotipos circulantes en la población humana durante el año 2002 y la prueba de ELISA antígenos de subtipos porcinos circulantes en Norte America. Los sueros evaluados a través de la técnica de IHA mostraron un alto porcentaje de establecimientos y animales seropositivos a H3N2 y H1N1. A pesar del bajo porcentaje de sueros positivos detectados con la prueba de ELISA, esta detectó por lo menos un animal positivo por granja. Muestras de pulmón, obtenidas en plantas de faena con lesiones compatibles con bronconeumonia necróticas fueron evaluadas por IHQ y microscopia electrónica. Se observó inmuno marcación en macrófagos alveolares y epitelio respiratorio en cortes por congelación. En los mismos tejidos se pudo observar partículas virales intracitoplasmáticas compatibles con virus de influenza. Se realizó un estudio longitudinal prospectivo en 10 establecimientos de producción intensiva. Se evalúo seroconversion así como la eliminación del agente y la presencia de lesiones pulmonares compatibles con la infección por el virus de influenza durante un ciclo completo de producción. No se detectaron anticuerpos y no se observó la circulación del virus de influenza durante el periodo en estudio. Sumado a la evaluación de influenza se evalúo la presencia de múltiples agentes que afectan al aparato respiratorio y factores de riesgo que pueden afectar parámetros productivos. La seroconversión contra Mycoplasma hyopneumoniae durante la ultima etapa de producción mostró tener una alta correlación con una alta prevalencia de lesiones pulmonares durante la faena. Sumado a esto se observo que la presencia de estas lesiones tiene un impacto negativo en la ganancia diaria de peso durante la etapa de desarrollo. Se determinaron numerosos factores de riesgo asociados a la presencia de animales seropositivos a PCV-2. Finalmente se realizó un estudio de brote en una piara que presentó un cuadro respiratorio agudo con alta morbilidad y baja mortalidad. A través de estudios anatomopatológicos se observaron lesiones características en patrón de tablero de ajedrez. La marcación con anticuerpos antinucleoproteína de influenza mostraron tinción positiva en células epiteliales y macrófagos alveolares. Finalmente las muestras procesadas por rRT-PCR mostraron señal positiva con marcadores universales contra influenza. La eliminación viral fue determinada por la presencia de efecto citopatico e infección en embriones de pollo con muestras obtenidas por hisopados traqueales y muestras pulmón de cerdos clínicamente afectados. Se realizó la secuenciación viral observándose que el brote fue debido la circulación e infección de una cepa H3N2 humana no contemporánea., Facultad de Ciencias Veterinarias

Diet-induced obesity produces changes in endocannabinoid signaling (ECS), influencing the regulation of energy homeostasis. Recently, we demonstrated that, in high-fat-diet-fed rats, blockade of CB1 receptor by AM251 not only reduced body weight but also increased adult neurogenesis in the hippocampus, suggesting an influence of diet on hippocampal cannabinoid function. To further explore the role of hippocampal ECS in high-fat-diet-induced obesity, we investigated whether the immunohistochemical expression of the enzymes that produce (diacylglycerol lipase alpha and N-acyl phosphatidylethanolamine phospholipase D) and degrade (monoacylglycerol lipase and fatty acid amino hydrolase) endocannabinoids may be altered in the hippocampus of AM251 (3 mg/kg)-treated rats fed three different diets: standard diet (normal chow), high-carbohydrate diet (70% carbohydrate) and high-fat diet (60% fat). Results indicated that AM251 reduced caloric intake and body weight gain, and induced a modulation of the expression of ECS-related proteins in the hippocampus of animals exposed to hypercaloric diets. These effects were differentially restricted to either the 2-arachinodoyl glycerol or anandamide signaling pathways, in a diet-dependent manner. AM251-treated rats fed the high-carbohydrate diet showed a reduction of the diacylglycerol lipase alpha : monoacylglycerol lipase ratio, whereas AM251-treated rats fed the high-fat diet showed a decrease of the N-acyl phosphatidylethanolamine phospholipase D : fatty acid amino hydrolase ratio. These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high-fat-diet-fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS-related proteins in the rat hippocampus in a diet-specific manner. Overall, these results suggest that the hippocampal ECS participates in the physiological adaptations to different caloric diets.

El càncer de mama és el més freqüent entre les dones de les nostres contrades, fins ara per classificar-lo s’ha utilitzat una classificació histològica però amb les noves tecnologies es vol realitzar una classificació molecular. Nosaltres valorem la utilitat com a factors pronòstics dels nous grups de classificació molecular, dels factors clàssics histològics i d’algunes variables immunohistoquímiques. S’inclouen 311 pacients diagnosticades de carcinoma ductal infiltrant NOS, es realitzen tissue-arrays i tincions immunohistoquímiques. Dels resultats obtinguts s’ha de destacar el paper pronòstic, ja reconegut, dels factors clàssics (mida tumoral, grau histològic, nombre de ganglis axil· lars metastàtics), també cal destacar l’efecte protector dels receptors d’andrògens (augmenta la supervivència i disminueix la recidiva), i referent a la recidiva cal destacar el mal pronòstic conferit per la CK 5/6 i el bon pronòstic de la CK 34 E12. En quant a la classificació molecular el grup triple negatiu, sobretot el subgrup basal, és el que presenta pitjor pronòstic., El cáncer de mama es el más frecuente entre las mujeres de nuestra zona, hasta ahora se ha usado una clasificación histológica pero con las nuevas tecnologías se está planteando realizar una clasificación molecular. Queremos estudiar la utilidad como factores pronósticos de los nuevos grupos de clasificación molecular, de los factores histológicos clásicos y de algunos parámetros inmunohistoquímicos. Incluimos 311 pacientes diagnosticadas de carcinoma ductal NOS, se realizan tissue-arrays i tinciones inmunohistoquímicas. Entre los resultados obtenidos destaca el papel pronóstico, ja conocido, de los factores clásicos (tamaño tumoral, grado histológico, número de ganglios axilares metastáticos), también destacar el efecto protector de los receptores de andrógenos (aumentando la supervivencia y disminuyendo la recidiva), y referido a la recidiva destacar el mal pronóstico de la expresión de CK 5/6 y el bueno de la CK 34 E12. Por lo referido a la clasificación molecular el grupo triple negativo, sobretodo el subgrupo basal, es el de peor pronóstico. Abstract Breast cancer is more common among women in our region, so far it has been used, Breast cancer is more common among women in our region, so far it has been used to classify a histological classification but with new technologies is to make a molecular classification. We value the utility as prognostic factors of new molecular classification groups, the classic histopathologic factors and immunohistochemical variables. Included 311 patients diagnosed with infiltrating ductal carcinoma NOS, we performed tissue-arrays and immunohistochemical staining. The results obtained have highlighted the role of expectations, as recognized classics of the factors (tumour size, histological grade, number of metastatic axillary nodes), also include the protective effect of androgen receptors (increasing survival and reduces relapse) and relapse include reference to the poor prognosis conferred by CK 5 / 6, and CK 34 E12 good prognosis. As for the molecular classification triple negative group, especially the basal subgroup, presents the worse prognosis.

Introducción: la localización y diagnóstico de los tumores neuroendocrinos (TNE) es difícil. La ultrasonografía endoscópica (USE) tiene un papel significativo en la detección de TNE sospechados por la clínica u otras técnicas de imagen, así como en la localización exacta y confirmación citológica mediante USEPAAF. Objetivo: valorar la utilidad y precisión de la PAAF-USE en el diagnóstico diferencial y de confirmación de los TNE, en una revisión retrospectiva de la experiencia de nuestro grupo. Pacientes y métodos: de un total de 55 enfermos con sospecha de TNE a los que se le practicó USE radial o sectorial, se detectaron 42 tumores en 40 casos. En 16 casos con sospecha de TNE funcionantes (trastornos hormonales: 6 casos) y no funcionantes (10 casos), se les practicó USE-PAAF con 22 G. En todos se efectuó Ki 67 o inmunocitoquímica (ICQ). Hubo confirmación quirúrgica en 9 casos (5 M y 4 V), con una media de edad de 51 años (rango: 41-81 años). Los tumores se localizaban todos en el páncreas, excepto uno en el mediastino y uno en el recto, con un tamaño medio de 19 mm (rango: 10-40 mm). Resultados: no hubo complicaciones atribuibles a la PAAF. La sensibilidad fue del 100% (8/8), y la precisión y el VPP fue del 89% (8/9), ya que hubo un falso positivo que en el estudio cito - lógico sugirió el diagnóstico de TNE pero que su resección quirúrgica confirmó el diagnóstico de tumor sólido seudopapilar del páncreas. Conclusiones: la USE-PAAF con 22 G de los TNE posee una alta sensibilidad y VPP en la confirmación citológica de estos pacientes, con muy escasas complicaciones.

Background: The DNA repair protein O-6-Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. Methods: A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Results: Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Conclusions: Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably., This study was supported by grants from the Ministerio de Sanidad y Consumo (Fondo de Investigacion Sanitaria 08/1085; Instituto de Salud Carlos III-RETIC RD06/0020/0097) and the Fundacion Medica Mutua Madrilena, 2007.

It has been postulated that the nasal mucosa, like other human tissues, is affected by a complex interactive network of neuropeptides, cytokines, allergic and inflammatory mediators and hormones such as estrogen, in which associations between symptoms (e.g. nasal stuffiness and coryza) and hormonal variations deriving from pregnancy, use of hormonal contraceptives and menstrual cycle phases are observed. The objective is evaluating the presence of specific estrogen receptors (types alpha and beta) in inferior turbinate mucosa in healthy subjects without nasal symptoms. Samples of nasal inferior turbinate were removed from patients undergoing aesthetic nasal surgery, and analyzed using hematoxylin-eosin staining, followed by immunohistochemical preparations on paraffin-embedded sections from the material sample, to detect estrogen receptors alpha and beta. Positive immunohistochemical reactions for both beta and alpha receptors were found in various regions of the inferior nasal turbinate. In conclusion both alpha and beta receptors were found, though the expression of beta was greater and more intense in the anterior portion of the inferior turbinate. No difference was found between male and female patients regarding the intensity of expression of receptors in the inferior turbinate. Se ha postulado que la mucosa nasal, al igual que otros tejidos humanos, se ve afectada por una compleja red interactiva de neuropéptidos, citoquinas, mediadores alérgicos e inflamatorios, y hormonas como el estrógeno, en el que las asociaciones entre los síntomas (por ejemplo, congestión nasal y catarro) y hormonales las variaciones derivadas del embarazo, se observó el uso de anticonceptivos hormonales y las fases del ciclo menstrual. El objetivo es evaluar la presencia de receptores de estrógenos específicos (tipos de alfa y beta) en la mucosa de la concha nasal inferior en sujetos sanos sin síntomas nasales. Las muestras de la concha nasal inferior fueron retirados de los pacientes sometidos a cirugía nasal estética y analizados mediante hematoxilina-eosina, seguidos de cortes de preparados de inmunohistoquímica incluídos en parafina de la muestra de material, para detectar los receptores de estrógenos alfa y beta. Las reacciones de inmunohistoquímica fueron positiva para ambos receptores alfa y beta, éstas se encuentran en diversas regiones del cornete nasal inferior. En conclusión, tanto los receptores alfa y beta se encuentran, aunque la expresión de la beta fue mayor y más intensa en la porción anterior de la concha nasal inferior. No se encontraron diferencias entre pacientes hombres y mujeres en relación con la intensidad de la expresión de los receptores en el concha nasal inferior.

6 páginas, 2 figuras, 3 tablas., Introduction While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of “carcinoma NOS (not otherwise specified)” in a prospective series of small tumor samples. Methods With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC. Results The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p, This work was partially funded by grants from Fundacion Mutua Madrileña to EC, FLR, and LPA; CIBER Respiratory Disease to ALE (ISCIII-CB06/06); and Red Temática de Investigacion Cooperativa en Cancer (RTICC) to MSC (RD06/0020/0062). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

L'adenocarcinoma ductal pancreàtic (PDAC) és un dels càncers més letals. Per tal de millorar el diagnòstic precoç, s'estan investigant les etapes inicials de la formació del càncer, com és el cas de les lesions preneoplàstiques, i es vol desxifrar l'origen cel·lular de la malaltia. Les proteïnes Polycomb constitueixen una família de silenciadors epigenètics que es troben en una varietat de tumors sòlids. La hipòtesi principal és que Polycomb pot estar participant en els processos preneoplàstics del pàncreas i en l'aparició i progressió del tumor. La expressió de Bmi1 i Ring1B fou analitzada durant el desenvolupament del pàncreas, en teixit pancreàtic de diferents models murins de la malaltia i en mostres humans de teixit pancreàtic. Es va dur a terme l'anàlisi del mecanisme de Bmi1 mitjançant models in vitro i induint la depleció de Bmi1. Bmi1 i Ring1B s'expressaren en precursors pancreàtics durant etapes primerenques del desenvolupament i en cèl·lules ductals i dels illots,però no en els acins, en el pàncrees adult. Bmi1 s'induí en cèl·lules acinars durant lesió aguda, en lesions metaplàstiques acinoductals, en neoplàsies intraepitelials pancreàtiques (PanIN) i en PDAC. Ring1B s'incrementà significativament en PanINs de grau alt i en PDAC. La disminució dels nivells de Bmi1 en la línia cel·lular acinar canvià l'expressió dels enzims digestius pancreàtics. Aquests resultats suggereixen que Bmi1 i Ring1B podrien estar contribuint de diferent manera en la progressió tumoral., Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. To improve early diagnosis, research efforts are focused in characterising early events of cancer formation like preneoplastic lesions and deciphering the cell origin of the malignancy. Polycomb proteins constitute a family of epigenetic silencers found in a variety of solid tumours. The main hypothesis is that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development and progression. The expression of Bmi1 and RingB was analysed during pancreatic development, in pancreatic tissue from mouse models of disease and in human pancreatic tissue samples. Mechanistic insights of Bmi1 were performed using in vitro models and with induced Bmi1 depletion. Bmi1 and Ring1B were expressed in pancreatic exocrine precursors during early development and in ductal and islet cells, but not in acinar cells, in the adult pancreas. Bmi1 was induced in acinar cells during acute injury, in acinar-ductal metaplastic lesions, in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B was significantly increased in high-grade PanINs and in PDAC. Bmi1 knockdown in acinar cell line changed the expression of pancreatic digestive enzymes. These results suggest that Bmi1 and Ring1B could contribute differently to tumour development.

La 3,4-metilendioximetamfetamina (MDMA, èxtasi) i el cannabis són dues drogues les quals es consumeixen conjuntament de manera habitual. Malgrat que tots dos compostos presenten propietats reforçant i potencial addictiu, també tenen propietats farmacològiques oposades. La MDMA es una droga psicoestimulant, la qual causa hiperlocomoció, hipertèrmia, resposted de tipus asiogènic i neurotoxicitat. Per altra banda el Δ9-tetrahydrocannabinol (THC), principal compost psicoactiu del cannabis, posseeix efectes relaxants, hipolocomotors, hipotèrmics i neuroprotectors. Els efectes de la MDMA i el THC al sistema nerviós central es troben mediats per dos mecanismes notablement diferents. La MDMA augmenta els nivells extracel·lulars de dopamina i serotonina, mentre que el THC produeix l'activació del receptor cannabinoide CB1. Cal destacar a més que les interaccions entre els sistemes monoaminèrgic i endocannabinoide s'observa de manera freqüent en l'organisme.En el present estudi hem explorat la implicació del sistema endocannabinoide i la MDMA en diversos aspectes. Per una banda el receptor cannabinoide CB1 juga un important paper en els efectes hiperlocomotors i hipertèrmics, i en les respostes de tipus ansiogènic produïdes per la MDMA. Curiosament, encara que el receptor CB1 no participa en els efectes recompensants primaris de la MDMA, és imprescindible per que tinguin lloc els seus efectes reforçants. Així mateix, l'alliberació de serotonina per part de la MDMA redueix de manera dosi-depenent la simptomatologia física causada pel síndrome d'abstinència a cannabinoides precipitada per un antagonista del receptor CB1. Finalment, el tractament amb THC era capaç de prevenir la hipertèrmia, activació glial, estrès oxidatiu i pèrdua de terminals causada per la MDMA. Com a conseqüència el THC exerceix un efecte neuroprotector contra la neurotoxicitat induïda per la MDMA., 3,4-methylenedioximethamphetamine (MDMA, ecstasy) and cannabis are two drugs frequently consumed in combination. Despite both compounds have rewarding properties and abuse liability, they show opposite pharmacological properties. On the one hand, MDMA is a psychostimulant drug with hyperlocomotor, hyperthermic, anxiogenic-like and neurotoxic effects. On the other hand, Δ9-tetrahydrocannabinol (THC), the main psychoactive compound of cannabis, has relaxant, hypolocomotor, hypothermic and neuroprotective properties. The effects of MDMA and THC in the central nervous system are mediated by two different mechanisms. MDMA enhances the extracellular levels of dopamine and serotonin, whereas THC activates the CB1 cannabinoid receptor. Likewise, interactions between the monoaminergic and the endogenous cannabinoid system have been frequently observed.In the current study, we explored the involvement of CB1 cannabinoid receptor on the hyperlocomotor, hyperthermic, anxiogenic-like, rewarding and reinforcing effects of MDMA. We also studied the effect of acute and chronic administration of MDMA on rimonabant-precipitated THC withdrawal syndrome. Furthermore, we explored the neuroprotective effects of THC on MDMA-induced neurotoxicity.As a result of this study we may conclude that endocannabinoid system and MDMA interact in a wide variety of aspects. CB1 receptor plays an important role on the hyperlocomotor, hyperthermic, and anxiogenic-like effects of MDMA. Interestingly, CB1 receptor is essential for the reinforcing but not the primary rewarding properties of MDMA. In addition, the release of serotonin by MDMA dose-dependently reduced the severity of THC withdrawal syndrome triggered by a CB1 antagonist. Finally, pretreatment with THC prevented the hyperthermia, glial activation, oxidative stress and terminal loss caused by MDMA. Consequently, THC exerts a neuroprotective effect against MDMA-induced neurotoxicity.

Brain damage caused by an acute injury depends on the initial severity of the injury and the time elapsed after the injury. To determine whether these two variables activate common mechanisms, we compared the response of the rat medial septum to insult with a graded series of concentrations of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) with the time-course effects of a low dose of AMPA. For this purpose we conducted a dose-response study at concentrations of AMPA between 0.27 and 10.8 nmol to measure atrophy of the septal area, losses of cholinergic and GABAergic neurons, astroglial and microglial reactions, and calcification. Cholinergic neurons, whose loss paralleled the degree of septal atrophy produced by AMPA, are more sensitive than GABAergic neurons to the injury produced by AMPA. At doses of AMPA above 2.7 nmol, calcification and the degree of microglial reaction increased only in the GABAergic region of the septal area, whereas atrophy and neuronal loss reached a plateau. We chose the 2.7-nmol dose of AMPA to determine how these parameters were modified between 4 days and 6 months after injection. We found that atrophy and neuronal loss increased progressively through the 6-month study period, whereas astrogliosis ceased to be observed after 1 month, and calcium precipitates were never detected. We conclude that septal damage does not increase with the intensity of an excitotoxic insult. Rather, it progresses continuously after the insult. Because these two situations involve different mechanisms, short-term paradigms are inappropriate for interpreting the pathogenic mechanisms responsible for long-term neurodegenerative processes.

La membrana sinovial, és el lloc on s'inicia i es perpetua el procés inflamatori que caracteritza a les diferents artropaties inflamatòries. A diferència d'altres òrgans no té una estructura definida, motiu pel qual el context on es produeix la investigació és un teixit poc diferenciat que dificulta trobar diferències rellevants.Els treballs presentats en aquesta tesi, intenten aportar algunes claus per aprofundir en la patògenia i el pronòstic dels diferents tipus d'artritis.L'objectiu de la tesi seria demostrar si existeixen diferències macroscòpiques vasculars (patrons vasculars valorats per artroscòpia) així com d'expressió cel·lular i de certes mol.lècules entre els diferents tipus d'artritis i la seva correlació amb els mecanismes fisiopatogènics subjacents i amb el pronòstic.La hipòtesi de treball seria que aquestes diferències ens poden orientar sobre el tipus de sinovitis i ens ajuden a trobar marcadors sinovials d'utilitat diagnòstica i pronòstica.L'artroscòpia ha demostrat ser el millor mètode per l'estudi directe de la membrana sinovial i l'obtenció de mostres dirigides. La introducció d'aquesta tècnica en algunes unitats d'artritis ha permès un estudi sistemàtic dels patrons vasculars i la seva correlació amb la clínica. Per altra banda, la anàlisi immunohistoquímica de l'expressió de diferents tipus cel·lulars i mol.leculars, ha estat àmpliament validada en l'estudi microscòpic del teixit sinovial.Els patrons sinovials són útils per diferenciar entre entitats clíniques i entre subtipus d'una mateixa malaltia. Existeixen diferències d'expressió cel·lular i de la proteïna p53 entre els pacients amb artritis reumatoide (AR) i artritis psoriàsica (APso), i aquestes diferències tant a nivell macroscòpic com microscòpic es corresponen amb una realitat clínica i pronòstica dels pacients.Les aportacions d'aquesta tesi apunten a utilitzar aquestes troballes com a ajuda en el coneixement fisiopatògenic d'aquestes malalties i com a via per establir factors pronòstics i de resposta al tractament., "CLASSIFICATION OF SYNOVITIS BY ARTHROSCOPIC AND IMMUNOHISTOQUIMIC METHODS. DIAGNÒSTIC AND PROGNOSTIC VALUE"TEXT:Inflammation of synovial tissue (synovitis) is a common final pathway in different inflammatory arthritides. Synovium, has not a well defined structure compared with other organs, so investigation in this field makes difficult to find relevant differences. The studies conforming this Doctoral Thesis, try to clarify some of the aspects of pathogenesis and prognostic of different types of arthritis. Our objective would be to assess if there are macroscopic vascular differences (vascular patterns by arthroscopy) either different molecular and cellular expression among different types of arthritis. We also try to correlate these features, with subjacent pathogenic mechanisms and prognosis.Our hypothesis would be that these differences may give us information about the type of synovitis and aid us to find synovial markers with diagnostic and prognostic utility.Arthroscopy has been proved as the best method to provide a direct study of synovial membrane and to obtain specific samples. Introduction of this technique in arthritis units has provide insight into the systematic study of vascular patterns and their correlation with clinical. Furthermore, immunohistochemical analysis of different cellular and molecular expression has been widely validated in the microscopic study of synovial tissue. Synovial vascular patterns may be of interest to discriminate different types of arthritis and different subgroups of patients with the same disease. There is a differential cellular expression and of the protein p53 between patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA). These differences correlate with a clinical and prognostic reality. The contribution of this thesis aim to use these findings as a pathway in the pathogenic knowledge of inflammatory diseases and alights a way to establish prognostic factors and evaluate the efficacy of treatments.

INTRODUCCIÓ: Des de 1989, un ampli cos de literatura s'ha ocupat dels aspectes biològics i alteracions de p53, romanent encara un cert grau de controvèrsia pel que fa al valor com a factor pronòstic en càncer colorectal, de l'estudi d'aquesta proteïna. P21 i p27, inhibidors de CDKs, han estat també objecte de diverses publicacions referents al seu valor com a factors pronòstics al llarg de la passada dècada, amb conclusions igualment diverses. Ciclina E, cofactor de CDK2 durant la fase G1 del cicle, es troba expressada a uns nivells molt superiors als fisiològics en molts tumors, però la seva expressió en còlon s'ha associat més aviat a estats d'infiltració incipient que no pas a estadis avançats. El seu valor com a factor pronòstic és, novament, motiu de controvèrsia.HIPÒTESI: L'estudi d'algunes proteïnes reguladores del cicle cel.lular en càncer colorectal, ha d'aportar informació pronòstica complementària a l'estadi de Dukes.OBJECTIUS: Identificació de perfils fenotípics en l'adenocarcinoma colorectal que puguin aportar informació pronòstica suplementària a l'estudi anàtomo-patològic habitual, mitjançant l'anàlisi integrat de mutacions de p53, del polimorfisme AA 72 Arg/Pro de p53, i d'expressió de p53, p21, p27 i ciclina E. MATERIAL I MÈTODES: Sèrie consecutiva de 185 adenocarcinomes colorectals ressecats electivament. Anàlisi mutacional del exons 5-8 de p53 mitjançant PCR-SSCP i seqüenciació, i del polimorfisme Arg/Pro 72 de p53 mitjançant PCR- SSCP. Anàlisi mitjançant immunohistoquímica (IHQ) sobre seccions parafinades, de sobreexpressió de p53 i d'expressió de p21, p27 i ciclina E. Repetició de la IHQ de p21 i p27 en un array tissular.RESULTATS. Major freqüència de mutacions de p53 en els tumors GG (arg72) que en els CC (pro72) o GC. Les mutacions de p53 localitzades en L3 i LSH es van associar a pitjor supervivència global(p = 0.04; ajustada per Dukes < 0.01). La sobreexpressió IHQ de p53 va correlacionar amb l'estadi de Dukes, però no amb supervivència. Els tumors amb mutacions de p53 inhibidores de p73 van mostrar en l'anàlisi multivariant una pitjor supervivència global que els tumors amb altres mutacions (p= 0.04). En el grup de tumors GG, els carcinomes amb aquestes mutacions van tendir a una pitjor supervivència global (p= 0.08). Dins del grup de tumors GG, aquells amb mutació en L3 i LSH van mostrar al ajustar per Dukes, pitjor supervivència global que els tumors amb p53 salvatge o amb altres mutacions (p= 0.005). Aquesta correlació no es va observar dins el grup GC-CC. L'expressió IHQ de p21 es va associar a millor supervivència. També es va associar a millor supervivència lliure de malaltia en el grup de pacients que havien rebut tractament amb quimioteràpia. Els carcinomes amb mutació hot spot en L3 i LSH de p53 i pèrdua d'expressió p21 van mostrar pitjor supervivència global, fins i tot ajustant per Dukes. L'expressió de p27 va correlacionar amb millor supervivència global però de manera dependent a l'estadi de Dukes.Els carcinomes amb alt percentatge d'expressió de ciclina E van mostrar inesperadament, millor supervivència global. A més, els carcinomes amb expressió dèbil de ciclina E en més d'un 12% de nuclis van mostrar millor supervivència que aquells amb expressió nul.la o molt intensa d'aquesta proteïna.Finalment, en una sèrie de 146 adenocarcinomes curativament ressecats, l'anàlisi dels codons 245, 248, 273 i 282 de p53, i l'anàlisi IHQ de p21, es van mostrar com uns bons marcadors de supervivència global.CONCLUSIONS: En càncer colorectal, l'anàlisi dels codons 245, 248, 273 i 282 de p53, aporta informació pronòstica sobre supervivència global independent de l'estadi de Dukes.L'estudi de p21 mitjançant IHQ en carcinomes colorectals completament ressecats, és un mètode d'anàlisi senzill i econòmic, amb valor pronòstic independent de l'estadi de Dukes, i podria ser utilitzat en la pràctica assistencial., ASSESSMENT OF THE VALUE AS PROGNOSTIC MARKERS IN COLORECTAL CANCER OF P53, P21, P27 AND CYCLIN E.ABSTRACTINTRODUCTION: The value as prognostic markers in colorectal cancer of p53, p21, p27 and cyclin E still remains controversial.HIPOTHESIS: The study of some proteins that regulate the cell cycle should give prognostic information, complementary to the Dukes' stage.AIMS: To identify phenotypical profiles in the colorectal adenocarcinoma which could give prognostic information complementary to the usual pathological study, by means of an integral analysis of p53, with assessment of mutations, protein overexpression and the AA 72 Arg/Pro polymorphism of p53, and expression analysis of p21, p27 and cyclin E. MATERIAL AND METHODS: A consecutive series of 185 colorectal cancers electively extirpated. Mutational analysis of the exons 5-8 of p53 by means of PCR-SSCP and sequencing and of the 72 Arg/Pro polymorphism by PCR-SSCP. Expression analysis by means of immunohistochemistry (IHC) on whole paraffin sections of p53, p21, p27 and cyclin E. Repetition of the IHC analysis of p21 and p27 on tissue-array sections.RESULTS: It was observed a greater frequency of p53 mutations in GG tumours (Arg 72) than in CC or GC tumours. The mutations of p53 located at the L3 loop and the LS -helix motif was associated to a clear worse overall survival (pP53 overexpression assessed by means of IHC correlated with Dukes'stage but not with survival. The tumours with mutations of p53 which inhibit p73 showed, at the multivariant analysis, a worse overall survival than the tumours with other p53 mutations (p= 0.04). Among the GG tumours, those with these mutations of p53 tended to a worse overall survival (p= 0.08). Also, in the group of GG tumours, those with p53 mutations at L3 and LSH showed, adjusting by Dukes, a worse overall survival than the tumours with other p53 mutations or wild type protein.P27 expression correlated with a better overall survival but in a depending mode with the Dukes'stage.Finally, in a series of 146 curatively extirpated tumours, the analysis of the codons 245, 248, 273 and 282, and the analysis of p21 expression by means of IHC showed to be good prognostic markers for overall survival.CONCLUSIONS: In colorectal cancer, the study of the codons 245, 248, 273 and 282 of p53, and the analysis of p21 expression, give prognostic information about overall survival independently of the Dukes'stage.

O Syzygium cumini é uma planta medicinal que tem sido utilizada popularmente para o tratamento da diabetes melito insulino dependente (DMID). Este estudo verificou o efeito do extrato da casca de Syzygium cumini sobre a regeneração de células insulino-positivas, a partir do ducto pancreático, no pâncreas de ratos normais e diabéticos. Os animais foram divididos em grupo controle (C), controle tratado (CT), diabético controle (DC) e diabético tratado (DT). Nos grupos tratados foi realizada a administração oral do extrato aquoso da casca de Syzygium cumini, na dose de 1g/kg de peso vivo. Após um período de 30 dias, os animais foram submetidos à eutanásia e o pâncreas retirado para análise imunohistoquímica. Neste estudo, foram visualizadas células insulino-positivas no ducto pancreático e no tecido conjuntivo próximo a ele, no pâncreas dos animais dos grupos DT e CT. Estes resultados indicam que o tratamento com o extrato da casca de Syzygium cumini na dose de 1g/kg estimula a formação de células insulino-positivas a partir das células epiteliais do ducto pancreático. Syzygium cumini is a plant that has been used in popular medicine for the treatment of insulin dependent diabetes mellitus (DMID). This study verified the effect of Syzygium cumini upon the regeneration of insulin producing cells in the pancreatic duct wall. The animals were divided into four groups, control (C), treated control (TC), diabetic control (DC) and treated diabetic (TD). An aqueous extract from Syzygium cumini bark was given by gavage in a daily dose of 1g/kg of body weight. After a thirty day period the animals were euthanized and the pancreas taken to immunohistochemical analysis. In this study, it was observed the positive staining for insulin on cells of the pancreatic duct and connective tissue in the pancreas of TD and TC animals. These results indicate that Syzygium cumini bark extract stimulates development of insulin positive cells from the pancreatic duct epithelial cells.

Syzygium cumini is a plant that has been used in popular medicine for the treatment of insulin dependent diabetes mellitus (DMID). This study verified the effect of Syzygium cumini upon the regeneration of insulin producing cells in the pancreatic duct wall. The animals were divided into four groups, control (C), treated control (TC), diabetic control (DC) and treated diabetic (TD). An aqueous extract from Syzygium cumini bark was given by gavage in a daily dose of 1g/kg of body weight. After a thirty day period the animals were euthanized and the pancreas taken to immunohistochemical analysis. In this study, it was observed the positive staining for insulin on cells of the pancreatic duct and connective tissue in the pancreas of TD and TC animals. These results indicate that Syzygium cumini bark extract stimulates development of insulin positive cells from the pancreatic duct epithelial cells. O Syzygium cumini é uma planta medicinal que tem sido utilizada popularmente para o tratamento da diabetes melito insulino dependente (DMID). Este estudo verificou o efeito do extrato da casca de Syzygium cumini sobre a regeneração de células insulino-positivas, a partir do ducto pancreático, no pâncreas de ratos normais e diabéticos. Os animais foram divididos em grupo controle (C), controle tratado (CT), diabético controle (DC) e diabético tratado (DT). Nos grupos tratados foi realizada a administração oral do extrato aquoso da casca de Syzygium cumini, na dose de 1g/kg de peso vivo. Após um período de 30 dias, os animais foram submetidos à eutanásia e o pâncreas retirado para análise imunohistoquímica. Neste estudo, foram visualizadas células insulino-positivas no ducto pancreático e no tecido conjuntivo próximo a ele, no pâncreas dos animais dos grupos DT e CT. Estes resultados indicam que o tratamento com o extrato da casca de Syzygium cumini na dose de 1g/kg estimula a formação de células insulino-positivas a partir das células epiteliais do ducto pancreático.