Introdução: Agregação familial é observada em cerca de 10% dos casos de câncer e 1 a 3% é hereditário. O tipo difuso pode estar relacionado à agregação familial e a alterações genéticas no gene CDH1, que codifica a proteína E-caderina. Alterações na imunoexpressão de Beta-catenina e p53 também são observadas. Objetivos: Analisar a imunoexpressão da E-caderina, Beta-catenina e TP53 em adenocarcinomas gástricos de pacientes com câncer gástrico familial e comparar com os dados clinicopatológicos, além dos achados das alterações genéticas destes pacientes, estudadas previamente nesta Instituição. Casuística e Métodos: Vinte e seis casos de adenocarcinoma gástrico em blocos de parafina de pacientes do HC-FMUSP foram submetidos ao estudo imunoistoquímico para detecção e análise do padrão de imunoexpressão da E-caderina, Beta-catenina e TP53 através do método da streptavidina-biotina-peroxidase. A análise da imunoexpressão dos marcadores foi classificada segundo escala de intensidade e distribuição e os testes estatísticos utilizados foram o Teste t de Student e Exato de Fisher. Resultados: A localização predominante do tumor foi no antro (61,5%). 11 (42,3%) casos alterados para a imunoexpressão da E-caderina, sendo todos do tipo difuso; 15 (57,7%) casos normais, sendo 9 do tipo difuso e 6 do tipo intestinal (p=0,02). Em estudo prévio realizado nesta instituição, uma mutação missense no exon 12 do gene CDH1, códon 617, nucleotídeo 1849 G>A foi encontrada no mesmo caso em que foi observada ausência de imunorreatividade da E-caderina. 11 (42,3%) casos alterados para a imunoexpressão de Beta-catenina e 46,2% de imunorreatividade nuclear positiva para TP53. Conclusões: 1) O tipo difuso de Laurén está associado à alteração da imunoexpressão da E-caderina no Câncer Gástrico Familial; 2) Não houve associação entre a imunoexpressão da E-caderina, idade, gênero e localização do tumor; tampouco houve associação entre a imunoexpressão da Beta-catenina e os dados clínico-patológicos; houve associação inversa entre a imunoexpressão da E-caderina e TP53; 3) Nos casos em que foram detectadas alterações na imunoexpressão, parece haver duas rotas distintas de carcinogênese envolvidas no CGF. / Introduction: Familial clustering is observed in about 10% of the gastric cancer cases and 1-3% is hereditary. Diffuse type gastric cancer is related to genetic alterations in CDH1 gene, which translates the E-cadherin protein. The abnormal expression of E-cadherin is characterized by low expression of cytoplasmatic staining, or loss of membranous immunoreactivity. Aim: to analyze the immunoexpression of E-cadherin, Beta-catenin and TP53 in gastric adenocarcinomas in patients with Familial Gastric Cancer and compare with clinical-pathologic data, including the genetic alterations of these patients, found previously on this institution. Methods: 26 cases of paraffin-embedded gastric adenocarcinoma tissue of patients of Hospital das Clinicas - School of Medicine of University of Sao Paulo underwent immunostaining to detect the presence and to analyze the pattern of immunoexpression of E-cadherin, Beta-catenin and TP53 using Streptavidine-Biotine-Peroxidade technique. The immunoexpression evaluation was performed utilizing a semiquantitative scale for intensity and distribution. The statistical analysis was done through Students t test and Fishers Exact test. Results: E-cadherin immunoexpression was negative in 11 cases (42.3%), and all of them were diffuse type of Laurén. 15 cases (57.7%) were positive for E-cadherin, from which 9 were of the diffuse type and 6 of intestinal type (p=0.02). In previous study performed on this institution, one missense mutation in exon 12 of CDH1 gene, codon 617, nucleotide 1849 G>A was found on the same case that absence of E-cadherin immunostaining was observed. 61.5% of the tumors were located in the antrum. Beta-catenin immunoexpression was altered in 43.2% and TP53 nuclear immunoreactivity was positive in 46.2% of the tumors. TP53 was solely detected in 12 (46.2%) of the tumors, while E-cadherin was altered in 10/26 (38.5%) negative TP53 tumors, p=0.01. Conclusions: 1) Diffuse type of Laurén is associated to E-cadherin immunoexpression alteration in Familial Gastric Cancer; 2) There was no association between E-cadherin immunoexpression and age, gender or tumor location, as well as there was no association between Beta-catenin and the clinical-pathologic data; there was an inverse association between immunoexpression of TP53 and E-cadherin; 3) There may be two distinct carcinogenesis pathways on familial gastric cancer cases that imunoexpression alterations were detected.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

Les ADAMs forment une sous-famille d’enzymes appelées “métalloprotéases”. Elles sont impliquées dans de nombreux processus aussi bien physiologiques que pathologiques de par leur capacité à cliver un certain nombre de substrats tels que des facteurs de croissance, des cytokines ou des protéines d’adhérence. Malgré de nombreuses études sur l’activité des ADAMs, on ne connaît que très peu d’éléments de leur régulation.Les tétraspanines constituent une super-famille de protéines membranaires ayant en commun une structure particulière. Elles sont impliquées dans un grand nombre de processus biologiques fondamentaux tels que la migration, les interactions intercellulaires, la réponse immunitaire, la fusion des gamètes… Les tétraspanines interagissent non seulement entre elles mais aussi avec un certain nombre de partenaires protéiques à la membrane plasmique, formant ainsi un réseau multi-moléculaire appelé « réseau de tétraspanines » ou « tetraspanin web ». Les travaux précédents de notre laboratoire ont montré que la métalloprotéase ADAM10 est associée au réseau de tétraspanines. Cependant, la tétraspanine en association directe avec ADAM10 permettant à cette dernière d’être incluse dans le réseau n’avait jusqu’ici pas été identifiée.Tout d’abord, afin d’établir un modèle permettant une mesure de la modulation de l’activité d’ADAM10 par les tétraspanines, nous avons démontré que l’engagement des tétraspanines par des anticorps monoclonaux augmente le clivage d’E-cadhérine par ADAM10. De plus, l’activation d’un récepteur muscarinique à l’acétylcholine permet aussi une augmentation du clivage d’E-cadhérine mais de manière ADAM17-dépendante cette fois. La transactivation de l’EGFR n’est pas impliquée dans la régulation muscarinique du clivage de l’E-cadhérine alors que l’activation directe de l’EGFR par un de ses ligands conduit, elle, à une stimulation de ce clivage.Revenant à notre quête initiale des conséquences de l’interaction entre ADAM10 et les tétraspanines, nous avons démontré que la métalloprotéase ADAM10 interagit avec l’ensemble des membres de la sous-famille de tétraspanines à 8 cystéines « TspanC8 » regroupant Tspan5, Tspan17, Tspan14, Tspan15, Tspan10 et Tspan33. Ces interactions ainsi que l’expression relative de chacun des membres des TspanC8s influent sur la sortie d’ADAM10 du réticulum endoplasmique. ADAM10 et son interaction avec les TspanC8s sont conservées dans l’Evolution et jouent un rôle dans la régulation de la voie de signalisation Notch. Lorsque nous avons examiné plus en détail l’interaction entre la tétraspanine Tspan5 et ADAM10, nous avons découvert qu’elle avait un effet négatif sur les expressions membranaires et totales d’ADAM10. De plus, cette interaction semble impliquée dans la prolifération de la lignée cellulaire PC3 dérivée de cancer de la prostate. En effet, la surexpression de Tspan5 cause une croissance diminuée de cette lignée. Cette inhibition semble due à un ou plusieurs facteurs solubles qui pourraient être sécrétés moins efficacement par les cellules surexprimant Tspan5 que par leurs homologues sauvages. Egalement, de manière inattendue, les cellules PC3 surexprimant Tspan5 sont totalement insensibles aux drogues ciblant le récepteur à tyrosine-kinase EGFR alors que la croissance des PC3 sauvages est très réduite après de tels traitements. Ceci impliquant donc que la croissance de ces dernières est au moins partiellement dépendante de la signalisation EGFR. Enfin, nous montrons qu’un autre récepteur à tyrosine-kinase appelé EphA2 pourrait avoir un rôle important dans la régulation de la dépendance à la signalisation EGFR des cellules PC3. / ADAMs are a sub-family of enzymes called “metalloproteases” which are implicated in a variety of physiological as well as pathological processes through their ability to cleave a number of substrates including growth factors, cytokines or adhesion proteins. Despite numerous studies on ADAM activity, very little is known about their regulation.Tetraspanins form a super-family of membrane proteins with a common conserved structure. They are implicated in numerous biological processes including migration, intercellular interactions, immune response, gamete fusion… Tetraspanins are known to interact with one another and with a restricted number of protein partners at the cell surface, thus forming a multi-molecular network referred to as « the tetraspanin web ». Previous studies in our laboratory have shown that the metalloprotease ADAM10 is associated to the tetraspanin web. Nevertheless, the tetraspanin in direct interaction with ADAM10 enabling it to be part of the web was not identified at the time. To begin with, in order to establish a model providing a read-out for a modulation of ADAM10 activity by tetraspanins, we demonstrate that tetraspanin engagement by monoclonal antibodies enhances E-cadherin shedding by ADAM10. Furthermore, muscarinic receptor activation also augments E-cadherin shedding but this time in an ADAM17-dependent manner. This occurs without the intervention of EGFR transactivation whereas a direct EGFR activation is able to stimulate E-cadherin shedding. Refocusing on the initial subject of the consequences of an interaction between ADAM10 and the tetraspanins, we conclusively show that the metalloprotease ADAM10 interacts with members of the conserved TspanC8 subfamily consisting of tetraspanins Tspan5, Tspan17, Tspan14, Tspan15, Tspan10 and Tspan33. These interactions and the relative expression of each of the TspanC8 members play a role in ADAM10 trafficking. ADAM10 and TspanC8 interactions are conserved throughout the Evolution and play a role in Notch signaling pathway regulation. When we examined in more details the particular interaction between the tetraspanin Tspan5 and ADAM10, we discovered that it had a negative effect on ADAM10 membrane as well as total expression. Moreover, this interaction seems to have implications on prostate cancer PC3 cell proliferation as Tspan5 overexpression causes a diminished growth rate. This inhibition could be caused by one or more soluble factors which could be less secreted by cells overexpressing Tspan5 than wild-type counterparts. Furthermore, oddly enough, PC3 cells overexpressing Tspan5 were completely unaffected by drugs targeted against the tyrosine-kinase receptor EGFR whereas this type of treatment impaired PC3 WT cell growth which therefore seems at least partly dependent on EGFR signalling. Finally, we reveal that another tyrosine-kinase receptor called EphA2 could play the proeminent role of regulating EGFR signalling-dependence in PC3 cells.

Infertility is one of the major problems of obesity. Studies have shown that administration of leptin reversed obesity-induced infertility in rats and mice. Coenzyme Q10 (CoQ10) is an antioxidant and also supplies the energy needed for ovulation and embryo development. We hypothesized that leptin when combined with CoQ10 could greatly improve obesity-induced infertility. The results showed a significant decrease in food intake, body weight, and the regular estrous cycle was restored after treatment with leptin+CoQ10. There was a significant increase (p10 significantly (p10 can improve fertility in obese infertile female rats. This study could provide a novel therapeutic strategy for the treatment of infertility and formulation of new drugs for the treatment of obesity-induced infertility in females.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences The University of Alcalá, Faculty of Medicine, Department of biomedicine and biotechnology Student: Zuzana Chvojanová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: María Isabel Arenas Jimenéz Title of the diploma thesis: Study of the variations in the expression of different adhesion and cytoskeletal proteins of podocytes (E-Cadherin, Podocin, Vimentin) due to Bisphenol A Bisphenol A (BPA) is one of the most widespread compounds in the world, producing over 6 billion metric tons per year. It is widely used as part of polycarbonate plastics and epoxy resins, from which reusable plastic bottles, food boxes and some medical equipment are made. It is also used to coat the inner layer of the cans. Previous studies have shown that BPA contributes to many chronic diseases in the human body, such as kidney disease - diabetic nephropathy. Podocytes - terminally differentiated cells of the Bowman's capsule in glomerulus - are an integral part of the filtration barrier, where they play an important role in preventing the plasmatic proteins from penetrating to the urine. Therefore, in this study, we looked at the effect of BPA on these cells and their particular proteins, using both in vivo and...

La métaplasie épithéliale est un phénomène d'adaptation tissulaire apparaissant à la suite d'une agression chronique. Caractérisées par le remplacement d'un épithélium par un autre, ces transformations métaplasiques peuvent apparaître dans différentes régions anatomiques (bronches, sophage, col de l'utérus, estomac et vessie). Bien que mieux adapté à résister à un agent irritatif, l'épithélium métaplasique présente cependant une susceptibilité accrue au développement cancéreux. Ainsi, la grande majorité (87%) des lésions (pré)néoplasiques cervicales se développe au sein du microenvironnement métaplasique de la zone de transformation du col utérin. Les travaux réalisés dans le cadre de cette thèse ont contribué à une meilleure compréhension des mécanismes responsables de la métaplasie épidermoïde cervicale et ont démontré, pour la première fois, une implication du TGF-β1 et de la PGE2 dans limmunodéficience locale observée dans les foyers de métaplasie mature et immature. Ainsi, en réduisant lexpression de E-cadhérine, le TGF-β1 altère indirectement la rétention épithéliale des cellules présentatrices dantigène en empêchant leurs interactions avec les cellules épithéliales. Quant à la PGE2, elle affecte la migration et le phénotype de ces cellules immunitaires, les rendant tolérogènes et, par conséquent, incapables dactiver les lymphocytes T naïfs. Par ailleurs, il est possible que la réduction de ΔNp63 par les facteurs de transcription Snail et Slug participe à la mise en place des épithélia métaplasiques ainsi quà leur transformation maligne.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

El mal pronòstic en una neoplàsia epitelial està associada a l'adquisició de característiques mòbils o invasives per part de les cèl·lules canceroses. Aquesta transformació morfològica es denomina transició epiteli-mesènquima (TEM). Snail és un factor de transcripció implicat en aquest procés, responsable de reprimir l'expressió de l'E-cadherina. Aquest treball demostra que Snail té la capacitat de reprimir l'expressió de MUC1 i VDR a través de la seva unió directa a caixes de reconeixement situades en els diferents promotors proximals. A més, la sobreexpressió d'Snail en diverses línies cel·lulars provoca un augment dels nivells d'ARNm de ZEB1 i un increment de l'activitat del seu promotor. L'activitat del promotor mínim d'Snail i els seus nivells d'ARNm depenen de la senyalització d'ERK. Finalment, hem demostrat que Snail i WT1, un regulador positiu de l'expressió de l'E-cadherina, competeixen per unir-se al promotor de l'E-cadherina i regular la seva transcripció. / The poor prognosis in epithelial neoplasia is associated with the acquisition of motile or invasive properties by the cancerous cells. This morphological transformation is often referred to as epithelial to mesenchymal transition (EMT). The Snail transcription factor is involved in this process by repressing the expression of E-cadherin. In this study we demonstrate the capacity of Snail to repress both MUC1 and VDR transcription by direct binding to specific sequences within their proximal promoter. Moreover, Snail overexpression in several cell lines induces ZEB1 mRNA and increases its promoter activity. The activity of the Snail minimal promoter is dependent on the ERK signaling pathway. Finally, we have demonstrated that Snail and WT1, a positive regulator of E-cadherin expression, compete for the binding to the E-cadherin promoter in order to regulate its transcription.

Colorectal carcinoma is the second most common type of cancer in both men and women in Sweden. Cancer of the colon and rectum are often considered together and their ten year survival rate is approximately 50 – 60 % depending on sex and location. Different histopathological characteristics of such cancers, including the complexity of growth, are of importance for prognosis. This thesis has compared different morphometric methods in order to achieve a quantitative and objective measurement of the invasive front of colon carcinoma. Since the growth pattern is dependent on the cell adhesiveness of different proteins we studied the distribution and localization of E-cadherin, Beta-catenin, Claudin 1,2,7 and Occludin as well as screened the genes for mutations. We found a perturbed protein expression of E-cadherin, Beta-catenin, Claudin 1,2,7 and Occludin in tumor sections compared to normal mucosa, but no relation to tumor volume or growth pattern could be seen. The tumor volume was found to be correlated to the growth pattern but not responsible to the perturbed protein expression. In the mutation screening we found a SNP in exon 13 the E-cadherin gene in the tumor, as well as in exon 2 of Claudin 1 and exon 4 of Claudin 7 in both tumor and normal mucosa. No correlation between mutations and growth pattern or tumor volume was found. In conclusion, this thesis shows that the computer image analysis with estimation of fractal dimension and number of free tumor cell clusters is superior to the semi quantitative visual grading of tumor invasive complexity. The aberrant expression of cell adhesion proteins in the tumor compared to normal mucosa as well as polymorphisms in the cell adhesion genes CLDN1 and CLDN7 in both tumor and normal mucosa can suggest that these aberrations are important in the tumorigenesis of colon carcinoma.