The Reelin gene located on the human chromosome region 7q22, encodes an extracellular matrix glycoprotein, a ligand for ApoER2 and low-density lipoprotein receptors (LDL) Receptor, is required for mediating the correct positioning of neurons during embryonic brain development1. In the current study, first we applied RT-PCR and immunohistochemistry analysis (IHC) analysis on tissue microarrays (TMA) to verify the Reelin expression patterns in a variety of adult tissues, suggesting additional roles for Reelin in stabling the cyto-architecture and controlling the remodeling of many organs during development. Second, we report the Reelin expression status in tumorigenesis. We discover that the loss of Reelin expression is associated with multiple types of cancers, including more than 80% of both breast and colorectal cancers. Interestingly, our study also found suspension small cell lung cancer (SCLC) cell lines that grow as large aggregates retained high Reelin expression, whereas attached non small cell lung cancer cultures do not. That may imply the Reelin expression may be also associated with cell culture morphology and growth characteristics in the in vitro culture system for lung cancers. Our results here also demonstrated that epigenetic silencing of Reelin expression by DNA hypermethylation in tumors directly correlates with loss of Reelin expression in many cancers. Reelinmethylation was reversed and expression restored by treating tumor cell lines with the demethylating agent 5-aza-2-deoxycytidine. In conclusion, from the molecular basis of Reelingene inactivation in human cancer here, we propose that the Reelinvariation in more than 80% of breast and colorectal cancers makes it a significant novel tumor marker.

Das Kolorektale Karzinom (KRK) ist eines der häufigsten malignen Erkrankungen, an der in Deutschland jährlich 26000 Menschen versterben. Auf der Suche nach einem neuen Biomarker für dieses Malignom wurde in dieser Arbeit L1-CAM, ein neuronales Zell-Adhäsionsmolekül, untersucht. Es ist, exprimiert an der Zelloberfläche, assoziiert mit einem signifikant schlechteren Outcome bedingt durch eine raschere lokale Tumorausbreitung und Metastasierung. Es zeigte sich anhand der Untersuchung von 62 Tumorpräparate und 39 präoperativ gewonnenen Seren, dass L1-CAM sowohl immunhistologisch nachgewiesen auf der Tumoroberfläche als auch mittels ELISA bestimmt im Serum der Patienten nachweisbar ist. Patienten mit L1-CAM positiven Tumoren waren im Mittel deutlich jünger als Patienten ohne L1-CAM Expression (60 vs. 69 Jahre). Zudem zeigte sich, dass Patienten mit schwach L1-CAM positiven Tumoren im Mittel einen signifikant höheren BMI aufwiesen (Kruskal Wallis Test p=0,0354). Die L1-CAM Expression hatte in unserem Patientengut keinen signifikanten Einfluss auf die Tumorausbreitung, wenngleich wir eine häufigere Metastasierung in die Leber (44%) bei L1-CAM positiven Tumoren gegenüber Patienten ohne L1-CAM Expression im Tumor (29%) beobachteten. Gleiches gilt für die Infiltration der Perineuralscheiden durch Tumorzellen. Bei der Untersuchung der L1-CAM Serumkonzentrationen zeigte sich im Mittel kein signifikanter Unterschied zu einer gesunden Vergleichsgruppe, sodass L1-CAM als Serum-Tumormarker ungeeignet ist.

Os tumores de glândula mamária, à semelhança do que ocorre na espécie humana, são as neoplasias que mais comumente acometem as fêmeas caninas, sendo os cães apontados como importante modelo de estudo desta doença. Com cerca da metade dos tumores mamários caninos considerados malignos, um problema crucial no manejo deste tipo de neoplasia é a busca por fatores prognósticos que auxiliem na escolha do tratamento adjuvante e na predição do curso clínico da doença, uma vez que neoplasias de baixo grau de malignidade , podem apresentar curso agressivo, contrariando os indicadores prognósticos clínicos usuais. Este trabalho teve por objetivo estudar alterações genéticas e epigenéticas em neoplasias mamárias de fêmeas caninas, buscando também avaliar seu valor prognóstico de sobrevida. Para tanto, neoplasias mamárias espontâneas de cães foram colhidas e classificadas de acordo com o tipo e grau histológico, além do imunofenótipo, obtido da marcação de ERα, PR, HER2 e Ki67. O padrão de metilação global do DNA e o perfil de expressão gênica dos marcadores tumorais BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 e GATA3 foram correlacionados com aqueles parâmetros. Para o estudo do padrão de metilação global do DNA foram utilizados 109 fragmentos de tecido mamário neoplásico, conservados em parafina, oriundos do arquivo do Hospital Veterinário da Universidade Metropolitana de Santos. Para imunofenotipagem e estudo imunoistoquímico da expressão de BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 e GATA3 foram colhidos em estudo prospectivo 78 fragmentos de tecido mamário neoplásico e não neoplásico, provenientes de 70 fêmeas da espécie canina com diagnóstico clínico de neoplasia mamária e que foram submetidas a tratamento cirúrgico. O material assim obtido foi conservado em nitrogênio líquido, para os estudos de expressão de RNAm por qPCR e em blocos de parafina, para os estudos histopatológicos e imunoistoquímicos. O estadiamento clínico mostrou-se fator prognóstico importante, assim como diâmetro tumoral, grau histológico do tumor, uso de quimioterapia adjuvante e recorrência. Os estudos epigenéticos demonstraram que o padrão de metilação do DNA independe do tipo histológico do tumor e de seu grau histológico, guardando correlação apenas com o grau de malignidade dos tumores; no entanto, este marcador demonstrou-se interessante previsor de recidiva tumoral, com diferença estatística significativa entre os animais que apresentaram recidiva e aqueles que não a apresentaram. Nos estudos de imunofenotipagem, foi possível observar por meio da técnica de imunoistoquímica que, tanto a expressão de ERα quanto de PR nas fêmeas caninas guardam, à semelhança da mulher, relação com o estado reprodutivo e hormonal das pacientes. Também foi observado que os marcadores ERα, PR e HER2 em sua maioria se correlacionam com o tipo e grau histológico quando transformados em variável categórica; porém, não apresentam valor prognóstico de sobrevida. No entanto, Ki67 demonstrou forte correlação com tipo e grau histológico dos tumores, com a presença de linfonodos positivos e metástase pulmonar, apresentando-se como fator prognóstico independente nas neoplasias mamárias caninas. Na busca por candidatos a marcadores tumorais, observou-se que, em sua maioria, os marcadores BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 e GATA3 não apresentaram correlação com tipo e grau histológico do tumor; porém, FGFR2 mostrou-se fator prognóstico de sobrevida para as fêmeas caninas portadoras de neoplasia. A finalidade de estudos integrados que correlacionem aspectos clínicos, morfológicos e moleculares das neoplasias é avançar em seu conhecimento e obter novas perspectivas para o sucesso no tratamento destas doenças. Esperamos que, com este trabalho, tenhamos contribuído para atingir estes objetivos. / Tumors of the mammary gland, similar to what occurs in the human species, are the most common cancers that affect the female dogs, being this species pointed out as an important model for the study of this disease. With about half of canine mammary tumors considered malignant, a crucial issue in the management of this type of cancer is the search for prognostic factors that assist in the choice of adjuvant therapy and in predicting the clinical course of the disease, since low-grade neoplasms malignancy can present aggressive course, contrary to the usual clinical prognostic indicators. This work aimed to study genetic and epigenetic changes in mammary tumors in female dogs, seeking also to evaluate its prognostic value for survival. For this purpose , spontaneous mammary tumors of dogs were collected and classified according to the histological type and grade, plus the immunophenotype obtained by evaluating ERα, PR , HER2 and Ki67 . The pattern of global DNA methylation and gene expression of tumor markers BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 and GATA3 profile were correlated with those parameters . Pattern of DNA global methylation was studied in 109 formalin-fixed paraffin-embedded neoplastic mammary tissue, obtained from the archives of the Veterinary School Hospital of the Universidade Metropolitana de Santos. Immunophenotyping and immunohistochemical study of the expression of BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 and GATA3 were performed in a prospective study with 78 fragments of neoplastic and non-neoplastic mammary tissue from 70 female dogs, with clinical diagnosis of mammary cancer and that underwent surgical treatment. The material was stored in liquid nitrogen for studies of mRNA expression by qPCR and in paraffin for histopathological and immunohistochemical studies. Clinical staging was an important prognostic factor, as well as tumor size, histological and tumor grade, use of adjuvant chemotherapy and recurrence. Epigenetic studies have shown that the pattern of DNA methylation is independent of the histological and tumor grade, keeping, however, correlation with the degree of malignancy of the tumors and interesting predictor value for tumor recurrence. Immunophenotyping studies showed that both ERα and PR expression, like in women, presented correlation with reproductive and hormonal status of the patients. It was also observed that ERα, PR and HER2 tumor markers mostly correlate with histological type and grade when considered a categorical variable, but they have not shown any prognostic survival value. However Ki67 showed strong correlation with histological type and grade of the tumors, the presence of positive lymph nodes and lung metastasis, and can be considered an independent prognostic factor in canine mammary tumors. In search for candidates for tumor markers, we found that, mostly BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 and GATA3 markers showed no correlation with histological type and grade of the tumor, although FGFR2 showed prognostic survival value for female dogs affected by breast cancer. The purpose of integrated studies correlating clinical, morphological and molecular aspects of cancer is to gain further knowledge and to glimpse new perspectives for success in treating these diseases. We hope that with this work, we have contributed to achieve these goals.

Os tumores de glândula mamária, à semelhança do que ocorre na espécie humana, são as neoplasias que mais comumente acometem as fêmeas caninas, sendo os cães apontados como importante modelo de estudo desta doença. Com cerca da metade dos tumores mamários caninos considerados malignos, um problema crucial no manejo deste tipo de neoplasia é a busca por fatores prognósticos que auxiliem na escolha do tratamento adjuvante e na predição do curso clínico da doença, uma vez que neoplasias de baixo grau de malignidade , podem apresentar curso agressivo, contrariando os indicadores prognósticos clínicos usuais. Este trabalho teve por objetivo estudar alterações genéticas e epigenéticas em neoplasias mamárias de fêmeas caninas, buscando também avaliar seu valor prognóstico de sobrevida. Para tanto, neoplasias mamárias espontâneas de cães foram colhidas e classificadas de acordo com o tipo e grau histológico, além do imunofenótipo, obtido da marcação de ERα, PR, HER2 e Ki67. O padrão de metilação global do DNA e o perfil de expressão gênica dos marcadores tumorais BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 e GATA3 foram correlacionados com aqueles parâmetros. Para o estudo do padrão de metilação global do DNA foram utilizados 109 fragmentos de tecido mamário neoplásico, conservados em parafina, oriundos do arquivo do Hospital Veterinário da Universidade Metropolitana de Santos. Para imunofenotipagem e estudo imunoistoquímico da expressão de BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 e GATA3 foram colhidos em estudo prospectivo 78 fragmentos de tecido mamário neoplásico e não neoplásico, provenientes de 70 fêmeas da espécie canina com diagnóstico clínico de neoplasia mamária e que foram submetidas a tratamento cirúrgico. O material assim obtido foi conservado em nitrogênio líquido, para os estudos de expressão de RNAm por qPCR e em blocos de parafina, para os estudos histopatológicos e imunoistoquímicos. O estadiamento clínico mostrou-se fator prognóstico importante, assim como diâmetro tumoral, grau histológico do tumor, uso de quimioterapia adjuvante e recorrência. Os estudos epigenéticos demonstraram que o padrão de metilação do DNA independe do tipo histológico do tumor e de seu grau histológico, guardando correlação apenas com o grau de malignidade dos tumores; no entanto, este marcador demonstrou-se interessante previsor de recidiva tumoral, com diferença estatística significativa entre os animais que apresentaram recidiva e aqueles que não a apresentaram. Nos estudos de imunofenotipagem, foi possível observar por meio da técnica de imunoistoquímica que, tanto a expressão de ERα quanto de PR nas fêmeas caninas guardam, à semelhança da mulher, relação com o estado reprodutivo e hormonal das pacientes. Também foi observado que os marcadores ERα, PR e HER2 em sua maioria se correlacionam com o tipo e grau histológico quando transformados em variável categórica; porém, não apresentam valor prognóstico de sobrevida. No entanto, Ki67 demonstrou forte correlação com tipo e grau histológico dos tumores, com a presença de linfonodos positivos e metástase pulmonar, apresentando-se como fator prognóstico independente nas neoplasias mamárias caninas. Na busca por candidatos a marcadores tumorais, observou-se que, em sua maioria, os marcadores BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 e GATA3 não apresentaram correlação com tipo e grau histológico do tumor; porém, FGFR2 mostrou-se fator prognóstico de sobrevida para as fêmeas caninas portadoras de neoplasia. A finalidade de estudos integrados que correlacionem aspectos clínicos, morfológicos e moleculares das neoplasias é avançar em seu conhecimento e obter novas perspectivas para o sucesso no tratamento destas doenças. Esperamos que, com este trabalho, tenhamos contribuído para atingir estes objetivos. / Tumors of the mammary gland, similar to what occurs in the human species, are the most common cancers that affect the female dogs, being this species pointed out as an important model for the study of this disease. With about half of canine mammary tumors considered malignant, a crucial issue in the management of this type of cancer is the search for prognostic factors that assist in the choice of adjuvant therapy and in predicting the clinical course of the disease, since low-grade neoplasms malignancy can present aggressive course, contrary to the usual clinical prognostic indicators. This work aimed to study genetic and epigenetic changes in mammary tumors in female dogs, seeking also to evaluate its prognostic value for survival. For this purpose , spontaneous mammary tumors of dogs were collected and classified according to the histological type and grade, plus the immunophenotype obtained by evaluating ERα, PR , HER2 and Ki67 . The pattern of global DNA methylation and gene expression of tumor markers BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 and GATA3 profile were correlated with those parameters . Pattern of DNA global methylation was studied in 109 formalin-fixed paraffin-embedded neoplastic mammary tissue, obtained from the archives of the Veterinary School Hospital of the Universidade Metropolitana de Santos. Immunophenotyping and immunohistochemical study of the expression of BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 and GATA3 were performed in a prospective study with 78 fragments of neoplastic and non-neoplastic mammary tissue from 70 female dogs, with clinical diagnosis of mammary cancer and that underwent surgical treatment. The material was stored in liquid nitrogen for studies of mRNA expression by qPCR and in paraffin for histopathological and immunohistochemical studies. Clinical staging was an important prognostic factor, as well as tumor size, histological and tumor grade, use of adjuvant chemotherapy and recurrence. Epigenetic studies have shown that the pattern of DNA methylation is independent of the histological and tumor grade, keeping, however, correlation with the degree of malignancy of the tumors and interesting predictor value for tumor recurrence. Immunophenotyping studies showed that both ERα and PR expression, like in women, presented correlation with reproductive and hormonal status of the patients. It was also observed that ERα, PR and HER2 tumor markers mostly correlate with histological type and grade when considered a categorical variable, but they have not shown any prognostic survival value. However Ki67 showed strong correlation with histological type and grade of the tumors, the presence of positive lymph nodes and lung metastasis, and can be considered an independent prognostic factor in canine mammary tumors. In search for candidates for tumor markers, we found that, mostly BRCA1, BRCA2, ERRα, ERRβ, ERRγ, FGFR2 and GATA3 markers showed no correlation with histological type and grade of the tumor, although FGFR2 showed prognostic survival value for female dogs affected by breast cancer. The purpose of integrated studies correlating clinical, morphological and molecular aspects of cancer is to gain further knowledge and to glimpse new perspectives for success in treating these diseases. We hope that with this work, we have contributed to achieve these goals.

Un système est dit robuste s'il est possible de garantir son bon comportement Le cancer est en passe de devenir la première cause de décès dans le monde avec un nombre de cas de cancer qui a pratiquement doublé sur les trente dernières années. Le diagnostic du cancer est d’autant plus important qu’il est maintenant reconnu que, plus la prise en charge du patient est rapide, plus les traitements thérapeutiques sont efficaces. Ce diagnostic doit être précis, fiable, et établi dans les premiers stades de la maladie afin d’augmenter significativement les chances de succès du/des traitements. Les techniques conventionnelles pour le diagnostic du cancer sont essentiellement basées sur des techniques d’imagerie (radiographies, IRM…) associés à des tests cytologiques et biochimiques. Avec le développement récent des technologies de biologie moléculaire (et notamment en protéomique), de nombreux marqueurs tumoraux ont été identifiés et sont utilisés dans des tests d’immunoassay pour le diagnostic voire pronostic du cancer en oncologie clinique. Cependant, le faible taux de marqueurs tumoraux dans le sérum de patient, ainsi que leur grande diversité, sont un challenge important pour l’établissement d’un diagnostic d’autant plus que les techniques de détection souffrent souvent d’un manque de sensibilité et de sélectivité. De plus, du fait de la diversité et de la variabilité des cancers, aucun marqueur tumoral n’est suffisamment spécifique pour permettre un diagnostic précis. Aussi, afin d’augmenter la fiabilité et la précision du diagnostic, il est nécessaire d’utiliser plusieurs marqueurs tumoraux. Dans ce contexte, grâce à leur capacité d’analyse haut débit en parallèle et le faible volume d’échantillon nécessaire, les technologies de puces à protéines (protein microarray)présentent de nombreux avantages pour l’identification de marqueurs tumoraux associés à la réponse humorale. Comme les marqueurs tumoraux sont souvent présents dans les échantillons en très faible quantité (à l’échelle sub micro-molaire), il y a un besoin urgent de développer des puces à protéines avec une détection ultrasensible de marqueurs tumoraux. La spécificité du diagnostic sera fortement liée au choix des protéines que l’on veut détecter(notées protéines cibles) et par conséquent au choix des protéines sondes que l’on va immobiliser sur le support. Un des paramètres critiques dans le développement de puces à protéines sensibles est la chimie de surface qui détermine le mode d’immobilisation de la protéine sonde sur le support et influence son activité biologique et donc sa capacité à reconnaitre et interagir avec la protéine cible que l’on cherche à détecter. Comme de nombreuses études suggèrent qu’un seul biomarqueur n’est pas suffisamment spécifique et sensible, la recherche d’une combinaison pertinente de biomarqueurs est un axe important pour l’amélioration d’un tel diagnostic. L’objectif de ce travail de thèse est donc le développement d’un outil original basé sur la technologie de puces à protéines fonctionnalisées avec différentes chimies de surface pour la détection sensible et spécifique de biomarqueurs tumoraux afin d’améliorer le diagnostic du cancer. Deux types de puces à protéines seront développés pour des applications différentes. Une première puce, avec comme protéines sondes des anticorps, sera développée pour la détection de biomarqueurs tumoraux impliqués dans le cancer colorectal. Une deuxième puce, où les protéines sondes seront des antigènes, sera étudiée en vue de l’identification de réponses autoimmunes de patientes atteintes d’un cancer du sein. [...] / Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients’ and 28 healthy donors’ sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-09-27T14:29:18Z No. of bitstreams: 1 RAILDA SHELSEA TAVEIRA ROCHA DO NASCIMENTO - TESE (PPGEP) 2008.pdf: 2253809 bytes, checksum: ec04359cafb5760ae50bf56746b420d4 (MD5) / Made available in DSpace on 2018-09-27T14:29:18Z (GMT). No. of bitstreams: 1 RAILDA SHELSEA TAVEIRA ROCHA DO NASCIMENTO - TESE (PPGEP) 2008.pdf: 2253809 bytes, checksum: ec04359cafb5760ae50bf56746b420d4 (MD5) Previous issue date: 2008-11-17 / Sólidos obtidos em escala nanométrica têm demandado um grande interesse científico e tecnológico, dadas às significativas alterações nas propriedades físicas e químicas observadas em materiais com estas características, potencializando assim as aplicações tecnológicas. Neste trabalho foram sintetizados quatro nanocomplexos do tipo Ln(MI)3.3H2O onde, Ln = Er ou Eu e MI = 5(4-clorofenil)-3-fenil-1,3,4-tiodiazólio-2-tiolato (MI-1) ou 5(4-clorofenil)-3-metil-4-fenil-1,3-tioazólio-2-tiolato (MI-2). Estes complexos foram caracterizados através de técnicas físico-químicas, espectroscópicas, térmicas e estrutural. A metodologia utilizada para a síntese dos complexos partiu de uma solução etanólica de LnCl3 adicionando-se o mesoiônico, sob agitação e temperatura constante de 65oC, mantendo em refluxo por quatro horas. O resultado da análise elementar de C, H e N dos complexos Er(MI-1)3.3H2O, Eu(MI-1)3.3H2O, Er(MI-2)3.3H2O e Eu(MI-2)3.3H2O mostraram uma boa concordância entre os valores experimentais e os calculados. Os espectros de infravermelho indicam que a coordenação dos compostos mesoiônicos com os cátions Eu+3 e Er+3 acontece possivelmente através do átomo de enxofre exocíclico onde, pode-se perceber um deslocamento considerável na ligação carbono-enxofre (C-S-). Os espectros de RMN 1H dos complexos revelaram a presença de todos os sinais dos hidrogênios aromáticos, evidenciados nos compostos mesoiônicos. Através dos espectros de RMN 13C (APT), foi possível reconhecer o padrão de hidrogenação correspondente a cada átomo de carbono, possibilitando atribuir com segurança o deslocamento químico dos carbonos (C-2) e (C-5) do anel mesoiônico. Nos espectros de absorção na região do UV-visível as bandas dos complexos se deslocam quando comparadas às bandas dos compostos mesoiônicos, sendo possível atribuir a inserção dos cátions de lantanídeos à modificação das bandas de absorção, visto que os complexos apresentaram energias em comprimento inferior. Os espectros de emissão dos compostos MI-1 e MI-2 assim como os dos complexos Eu(MI-1)3.3H2O e Er(MI-2)3.3H2O apresentaram comportamento semelhante com uma banda larga de emissão entre 560 e 720 nm, não sendo possível a observação das bandas de emissão dos íons lantanídeo. As curvas TG/DTG indicaram que os compostos mesoiônicos se decompuseram em cinco etapas, diferentemente da decomposição dos complexos Er(MI-1)3.3H2O, Eu(MI-1)3.3H2O, Er(MI-2)3.3H2O e Eu(MI-2)3.3H2O que ocorreu em 5, 4, 7 e 6 etapas, respectivamente. Os complexos sintetizados com Eu apresentaram maior estabilidade térmica em detrimento dos complexos de Er, independente do mesoiônico utilizado. As curvas DSC, mostraram que os complexos estudados neste trabalho iniciam a reação de decomposição térmica ainda em estado sólido, antes da fusão. Os difratogramas de raios-x dos compostos mesoiônicos e dos complexos, apresentaram mais de uma fase, com características cristalinas, e cristalito inferior a 60nm. / The solids obtained in nanometric scale has been attracted the technological and scientific interest due to the significant modifications observed on their physical and chemical properties which increases the technological applications. In this work, four nanocomplex materials were synthesized: Ln(MI)3.3H2O where, Ln = Er or Eu and MI = 5(4-chlorophenyl)-3-phenyl-1,3,4-thiodiazole-2-tiolat (MI-1) or 5(4-chlorophenyl)-3-methyl-4-phenyl-1,3-thioazole-2-thiolate (MI-2). These complexes were characterized using physico-chemical, spectroscopic, thermal and structural techniques. To synthesize the complexes, it was used a LnCl3 ethanolic solution mixed with the mesoionic, maintained under agitation and at a constant temperature at 65oC and refluxed for a period of four hours. It was observed a good agreement between the experimental and calculated results of the C, H and N elemental analysis of Er(MI-1)3.3H2O, Eu(MI-1)3.3H2O, Er(MI-2)3.3H2O and Eu(MI-2)3.3H2O. The IR spectra indicates that the coordination of the mesoionic compounds with cations Eu+3 and Er+3, must happen probably because of the exocyclic sulfur atom and it can be perceived a considerable displacement in the carbon-sulfur (C-S-) ligation. The RMN 1H spectra suggested the presence of aromatic hydrogen evidenced in the mesoionic compounds. Through the RMN 13C (APT), it was possible to recognize the hydrogenation standard corresponding to each carbon atom. It can be safely attributed the carbon chemical displacement (C-2) and (C-5) of the mesoionic ring. In the UV-visible absorption spectra of the UV the complexes bands are displaced comparing to the mesoionic compound bands. It can be possible to attribute the insertion of the lanthanide cations to the modification of the absorption bands, once the complexes presented energies at inferior length. The MI-1 and MI-2 compounds emission spectra and the complexes Eu(MI-1)3.3H2O and Er(MI-2)3.3H2O presented a behavior similar to a large emission band between 560 and 720 nm. It was not possible to observe the emission bands of the lanthanide ions. The TG/DTG curves indicate that the mesoionic compounds were decomposed in five levels, with a different behavior of the Er(MI-1)3.3H2O, Eu(MI-1)3.3H2O, Er(MI-2)3.3H2O and Eu(MI-2)3.3H2O, which decomposition occurred in 5,4,7 and 6 steps, respectively. The complexes synthesized with Eu presented a higher thermal stability than the ones that used Er, independently of the kind of mesoionic compound that was used. The DSC curves showed that the studied complexes begin the thermal decomposition reaction before the fusion, in the solid state. The x-ray diffractograms of the complexes and mesoionic compounds present more than a level, with crystalline characteristics and crystallite size inferior to 60nm.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Lectinas sÃo proteÃnas que pertencem a um grupo heterogÃneo de molÃculas com capacidade de ligaÃÃo especÃfica e reversÃvel a carboidratos. Desde sua descoberta, as lectinas se tornaram importantes ferramentas para a investigaÃÃo de fenÃmenos como a adesÃo, a migraÃÃo e a proliferaÃÃo celular, em condiÃÃes normais e patolÃgicas. Durante o processo de diferenciaÃÃo, cÃlulas tumorais apresentam vÃrios graus de modificaÃÃo na expressÃo de glicoproteÃnas de membrana. Neste aspecto, a investigaÃÃo da estrutura da membrana tumoral pode ser compreendida como um mÃtodo sensÃvel e especÃfico de diagnÃstico. Portanto, o reconhecimento de marcadores capazes de identificar e quantificar estas caracterÃsticas, pode ser usado como ferramenta de diagnÃstico. Neste trabalho propomos um modelo experimental para detectar marcadores de membrana de tumores primÃrios do Sistema Nervoso Central usando lectinas vegetais (Con A e Con Br) acopladas à cromÃforos. Amostras de tumores foram obtidas de pacientes que tinham diagnÃstico clÃnico e radiolÃgico de tumores do Sistema Nervoso Central, apÃs cirurgias realizadas no ServiÃo de Neurologia da Santa Casa de MisericÃrdia de Sobral. As amostras foram processadas e investigadas com tÃcnicas imunohistoquÃmicas, usando lectinas vegetais acopladas à fluoresceÃna, e microscopia de fluorescÃncia. Os resultados mostraram que meningiomas e gliomas apresentaram um padrÃo diferente de interaÃÃo com Con Br-FITC e Con A-FITC quando comparados ao controle (BSA/FITC). Estes dados sugerem que as lectinas vegetais estudadas podem ser ferramentas Ãteis na identificaÃÃo de marcadores de membrana em tumores primÃrios do Sistema Nervoso Central. / Lectins are proteins that belong to a heterogeneous group of molecules capable of binding specifically and reversibly to carbohydrates. Since its discovery, lectins have become important tools for investigating phenomena such as adhesion, migration and cell proliferation in normal and pathological conditions. In the process of differentiation, tumor cells display different degrees of modification in the expression of membrane glycoproteins. In this respect, the investigation of membrane structure tumor can be understood as a sensitive and specific method of diagnosis. Therefore, the recognition of markers capable of identifying and quantifying these characteristics can be used as diagnostic tool. In this paper we propose an experimental model to detect markers of the membrane of primary tumors of the central nervous system using plant lectins (Con A and Con Br) attached to the chromophores. Tumor samples were obtained from patients who had clinical and radiological diagnosis of tumors of the central nervous system after surgeries performed at the Department of Neurology, Santa Casa de Misericordia de Sobral. The samples were processed and investigated with immunohistochemical techniques, using plant lectins coupled to fluorescein, and fluorescence microscopy. The results showed that meningiomas and gliomas showed a different pattern for interaction with Con Br-FITC and Con A-FITC compared to control (BSA / FITC). These data suggest that the studied plant lectins can be useful tools in identifying membrane markers in primary tumors of the central nervous system.

Background: Among the common malignancies, pancreatic cancer has the shortest long-term survival. The aggressive, rapid, and infiltrative growth pattern of pancreatic cancer, together with the lack of specific symptoms, often leads to late diagnosis. Metastases are frequently found at the time of diagnosis, which prevents curative surgical treatment. Good tumor markers would enable early detection, thus improving the prognosis. Unfortunately, no such markers are available in the clinic. The tumor stroma is defined as the non-malignant cells and the extracellular matrix (ECM) of a cancer. Pancreatic cancer is characterized by an abundant tumor stroma, rich in ECM proteins such as collagens, which have been shown to play important roles in tumor progression. Furthermore, pancreatic cancer cells produce large quantities of ECM proteins, especially the basement membrane (BM) protein type IV collagen. All epithelial cells are anchored to a BM, which must be degraded in order for an in situ cancer to become invasive. Matrix metalloproteinases (MMPs) are enzymes involved in BM degradation. In this thesis, the tumor stroma of pancreatic cancer is studied, focusing on the BM proteins type IV and type XVIII collagen, with the aim to clarify if the stroma could be a source of novel tumor markers for this form of cancer. Additionally, the role of type IV collagen produced by the cancer cells is studied. Methods: Expression patterns of type IV and type XVIII collagen, MMPs involved in collagen degradation, and collagen receptors (integrins) were studied by immunoflourescence in both normal and pancreatic cancer tissue, and in pancreatic cancer cell lines. Circulating plasma levels of type IV and type XVIII collagen and conventional tumor markers (TPS, Ca 19-9, CEA and Ca 125) were measured in controls and pancreatic cancer patients at the time of diagnosis and after treatment. The role of cancer cell produced type IV collagen was studied in human pancreatic cancer cell lines by functional blocking of integrin receptors (integrin a1, a2 and b1) and integrin-binding sites on type IV collagen, and by siRNA-induced down-regulation of type IV collagen synthesis. Proliferation was analyzed by a luminescence based cell viability assay, migration by time-lapse microscopy, and apoptosis by M30-neoepitope detection. Results: MMPs involved in BM degradation were upregulated in pancreatic cancer tissue. The expression of type XVIII collagen shifted from a general BM expression pattern in normal tissue, to mainly being found in the tumor vasculature in pancreatic cancer. Type IV collagen, on the other hand, remained highly expressed in the vicinity of the cancer cells. The a1, a2, and b1 integrin receptors were highly expressed at the cancer cell surface. Both down-regulation of type IV collagen synthesis and blocking the integrin/type IV collagen interaction decreased cell proliferation and migration. The proliferative capacity was rescued by the addition of exogenous type IV collagen. Furthermore, the circulating levels of both type IV and type XVIII collagen were increased in pancreatic cancer patients at the time of diagnosis compared to controls. After treatment, the levels were normalized for type XVIII collagen, whereas the levels of type IV collagen remained high after surgery. High postoperative levels of type IV collagen were associated with short overall survival. A similar association to short survival was found for preoperative type XVIII collagen levels. No such associations to survival could be detected for the conventional markers.   Conclusion: The results of this thesis show that type IV and type XVIII collagens can serve as tumor markers for pancreatic cancer with advantages compared to conventionally used markers. Additionally, evidence is provided of an autocrine loop, involving type IV collagen and its integrin receptors, with importance for retaining a proliferative and migratory phenotype in pancreatic cancer cells.

Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients' and 28 healthy donors' sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.

Das Kolorektale Karzinom (KRK) ist eines der häufigsten malignen Erkrankungen, an der in Deutschland jährlich 26000 Menschen versterben. Auf der Suche nach einem neuen Biomarker für dieses Malignom wurde in dieser Arbeit L1-CAM, ein neuronales Zell-Adhäsionsmolekül, untersucht. Es ist, exprimiert an der Zelloberfläche, assoziiert mit einem signifikant schlechteren Outcome bedingt durch eine raschere lokale Tumorausbreitung und Metastasierung. Es zeigte sich anhand der Untersuchung von 62 Tumorpräparate und 39 präoperativ gewonnenen Seren, dass L1-CAM sowohl immunhistologisch nachgewiesen auf der Tumoroberfläche als auch mittels ELISA bestimmt im Serum der Patienten nachweisbar ist. Patienten mit L1-CAM positiven Tumoren waren im Mittel deutlich jünger als Patienten ohne L1-CAM Expression (60 vs. 69 Jahre). Zudem zeigte sich, dass Patienten mit schwach L1-CAM positiven Tumoren im Mittel einen signifikant höheren BMI aufwiesen (Kruskal Wallis Test p=0,0354). Die L1-CAM Expression hatte in unserem Patientengut keinen signifikanten Einfluss auf die Tumorausbreitung, wenngleich wir eine häufigere Metastasierung in die Leber (44%) bei L1-CAM positiven Tumoren gegenüber Patienten ohne L1-CAM Expression im Tumor (29%) beobachteten. Gleiches gilt für die Infiltration der Perineuralscheiden durch Tumorzellen. Bei der Untersuchung der L1-CAM Serumkonzentrationen zeigte sich im Mittel kein signifikanter Unterschied zu einer gesunden Vergleichsgruppe, sodass L1-CAM als Serum-Tumormarker ungeeignet ist.:Bibliographische Beschreibung 2 Abkürzungsverzeichnis 3 1. Einführung 5 2. Grundlagen 7 3. Material und Methoden 12 3.1. Patientengut 12 3.2. Immunhistochemische Färbung des Primärtumors von L1-CAM und Beurteilung der Expression 13 3.3. Bestimmung der Serumkonzentrationen von L1-CAM und CEA 16 3.3.1. Bestimmung der Serum-Konzentration von L1-CAM mittels ELISA 16 3.3.2. Bestimmung der Serum-Konzentration von CEA mittels ELISA 16 3.4. Statistik 17 4. Ergebnisse 18 4.1. L1- CAM Immunhistochemie des Primärtumors 18 4.1.1. L1-CAM Expression in Bezug auf Alter und Geschlecht sowie BMI 18 4.1.2. L1-CAM Expression entsprechend der Ausdehnung des Primärtumors 20 4.1.3. L1-CAM Expression und Lymphknoten- sowie Fernmetastasierung 22 4.1.3.1 Lymphknotenmetastasierung 22 4.1.3.2. Fernmetastasierung 23 4.1.4. L1-CAM Expression entsprechend der UICC-Klassifikation des Tumors 24 4.1.5. L1-CAM Expression entsprechend der Tumordifferenzierung 25 4.1.6. Tumorlokalisation und L1-CAM Expression 27 4.2. L1-CAM im Serum 28 4.2.1. L1-CAM Konzentration im Serum der Patienten 28 4.2.2 L1-CAM im Serum einer gesunden Vergleichsgruppe 29 4.2.3. L1-CAM Serumkonzentration bezogen auf Alter und Geschlecht sowie BMI 30 4.2.4. L1-CAM Serumkonzentration bezogen auf Lymphknoten- und Lebermetastasierung 30 4.2.4.1. L1-CAM Serumkonzentration und Lymphknotenmetastasierung 30 4.2.4.2. L1-CAM Serumkonzentration und Lebermetastasierung 31 4.2.5. L1-CAM im Serum bezogen auf die Tumortherapie 33 4.3. L1-CAM histologisch und im Serum 33 5. Diskussion 35 6. Thesen 39 7. Zusammenfassung 40 8. Literaturverzeichnis 43 9. Anlagen 49