Made available in DSpace on 2014-07-29T15:16:33Z (GMT). No. of bitstreams: 1 dissertacao GNC primeira parte.pdf: 75754 bytes, checksum: 7caa36c31bb043ddbf91465bd48e4bbb (MD5) Previous issue date: 2008-07-31 / Breast cancer is the second most common tumor in the world and the first among women. In the past two decades, several breast cancer studies focused on the expression and on the possible routes of cellular signaling for the epithelial cell adhesion molecule (EpCAM). This molecule was found to be overexpressed in various malignant tissues of epithelial origin, including breast cancer. The importance of EpCAM protein in tumor cells is still controversial. First, because it is believed that the cell adhesion role of EpCAM reduces the ability of metastasis. On the other hand, replacing the strong cell adhesion performed by cadherins instead of weak homophilic adhesion of EpCAM may be associated with the promotion of invasion and tumor metastasis. The aim of our study was to analyze the implications of EpCAM in breast ductal carcinoma infiltration. By using immunohistochemistry methods, we noticed the absence of EpCAM expression in 10 case (10,2%), low EpCAM levels expression in 45 cases (45,9%) and overexpression in 43 cases (43,9%). Unlike other published studies, the overexpression of the EpCAM protein did not correlate with the five-year overall survival of breast cancer patients, even those with axillary lymph node involvement. Similarly, there was no correlation between EpCAM s protein expression and classical prognostic factors, including the size of the tumor, axillary lymph node involvement, hormone receptors, c-erbB-2 overexpression and p53 immunodetection. The presence of distant metastasis , reported in 26 cases (26,5%), did not correlate significantly with the overexpression of EpCAM / O câncer de mama é o segundo tipo de câncer mais freqüente no mundo e o primeiro entre as mulheres. Há cerca de duas décadas, diversos estudos sobre câncer de mama enfocaram a expressão e as possíveis rotas de sinalização celular da molécula de adesão celular epitelial (EpCAM). Esta molécula foi encontrada hiperexpressa em vários tecidos neoplásicos de origem epitelial, incluindo o câncer de mama. A importância da proteína EpCAM no suporte celular de tumores é controversa. Por um lado, acredita-se que a função de adesão celular de EpCAM reduza a capacidade de metastatização, mas por outro lado, a substituição da forte adesão celular de caderinas pela fraca adesão homofílica de EpCAM estaria associada à promoção de invasão tumoral e metástase. Nosso estudo consiste na análise das implicações clínicopatológicas da proteína EpCAM no carcinoma ductal infiltrante de mama. Utilizando técnicas de imuno-histoquímica, observamos a ausência da expressão de EpCAM em 10 casos (10,2%), baixa expressão em 45 casos (45,9%) e superexpressão em 43 casos (43,9%). Diferentemente de outros estudos, a superexpressão da proteína EpCAM não se correlacionou com a sobrevida global das pacientes em cinco anos, nem mesmo naquelas com comprometimento de linfonodos axilares. Da mesma forma, não houve correlação da expressão da proteína EpCAM com nenhum dos fatores prognósticos analisados, entre eles tamanho do tumor, linfonodos axilares, receptores hormonais, cerbB-2 e p53. A presença de metástase à distância, relatada em 26 casos (26,5%) não correlacionou-se significativamente com a superexpressão de EpCAM

Une approche prometteuse contre le cancer est le ciblage d’antigènes associés aux tumeurs par des ligands spécifiques couplés à des nanoparticules transportant des agents thérapeutiques ou d’imagerie. Les anticorps sont les molécules de ciblage les plus utilisées, mais présentent des limitations en termes de coûts de production élevés, de complexité structurale et de stabilité limitée. Les Affitins sont des protéines d'affinité hautement stables, dérivées à l'origine de Sac7d, un polypeptide d’archée de la famille de liaison à l'ADN de 7 kDa (Sul7d). Les Affitins montrent une affinité et une spécificité comparables à celles des anticorps, tout en étant thermiquement et chimiquement plus stable, moins coûteuses à produire, plus faciles à remodeler avec une taille 20 fois plus petite. Les nanocapsules lipidiques (LNC) possèdent une grande stabilité et une efficacité élevée pour l'encapsulation des médicaments lipophiles et les protègent de la dégradation rapide. Le ciblage de cellules cancéreuses par des LNC peut réduire de plus la concentration de médicament dans les tissus normaux et réduire la toxicité. Le but du projet est de combiner les avantages des Affitins et des LNC pour amener les médicaments anticancéreux vers les cellules cancéreuses. Le premier objectif a été d'identifier et de caractériser un membre plus court, mais toujours très stable, de la famille Sul7d pour encore améliorer la base moléculaire des Affitins, puis de générer des Affitins qui reconnaissant le biomarqueur EpCAM associé aux cellules tumorales. Le deuxième objectif a été d'attacher les nouvelles Affitins comme ligands d'affinité aux LNC et d'évaluer le ciblage de la tumeur par ces complexes. / A progressive strategy against cancer is the targeting of tumour-associated antigens by specific ligands coupled to nanoparticles, carrying therapeutic or imaging agents. Antibodies are the most widely used targeting molecules, but they possess limitations as high production costs, complex structure and limited stability. Affitins are highly stable engineered affinity proteins, derived originally from Sac7d, an archaeal polypeptide from the 7 kDa DNA-binding family (also known as Sul7d family). These binders show comparable affinity and specificity to those of antibodies, while being thermally and chemically more stable, cheaper to produce, easier to engineer and present a simpler structure and 20-fold smaller size. Lipid nanocapsules (LNCs), prepared by solvent free process, possess great stability and high efficiency for lipophilic drugs encapsulation and protect the drug from rapid degradation. Targeting drug-LNC to cancer cells can further decrease drug concentration in normal tissues and lower the toxicity. The aim of the project is to combine the advantages of Affitins as targeting agents and LNCs as carriers in order to create vehicles for delivering payloads to cancer cells. The first goal of this work was to identify and characterize a shorter member, but still very stable, of the Sul7d family in order to further improve the affinity scaffold, and then to use it for the generation of Affitins, recognizing the tumour-associated Epithelial Cell Adhesion Molecule. The last goal was to attach the new binders as affinity moieties to LNCs and to assess the tumour targeting of these complexes.

abstract: Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors. This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g. serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2019

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21000号 / 医博第4346号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 中川 一路, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<p>Based on the cancer stem cell (CSC) model, tumors develop as a hierarchy, much like normal tissue. At the apex, CSCs are capable of self-renewal and differentiating into non-tumorigenic cells that form the bulk of the tumor. CSCs have been isolated for many types of cancer based on cell surface marker expression. For instance in human breast carcinoma, the CD44+ CD24-/low EpCAM+ population are enriched in CSCs. These cells are more resistant to traditional chemo and radiation therapy relative to the bulk of the tumor. As such, many believe CSCs to be responsible for relapse and metastasis events. Hence, a more targeted therapy towards breast CSCs can prove to be very effective. EpCAM has been shown to be a reliable, albeit not exclusive, marker of breast CSCs. A more thorough understanding of EpCAM’s function can provide new angles for designing therapeutic agents. Originally thought to be a mere adhesion molecule, EpCAM is now known to derive a signalling pathway that promotes transcription in its target genes. We aimed to provide further insight into this novel pathway, by manipulating EpCAM’s expression and function in MCF7 breast cancer cell line. We designed a dominant negative allele of the EpCAM protein to hinder the activity of the endogenous EpCAM. However, this transgene offered no significant effect on our cell line. Furthermore, we used RNA interference technology to reduce EpCAM expression. The introduction of the EpCAM short hairpin RNA constructs into our cell line had inhibitory effects on transcription reporter expression, cellular growth, adherent colony and unattached sphere formation. The inhibition of EpCAM in MCF7 spheres was followed by signs of cell death, differentiation and an epithelial mesenchymal transition process. Sphere cultures are used because they are enriched in cancer stem cells. The adverse effects of EpCAM inhibition in MCF7 spheres provides further evidence as to the role of this protein in cancer stem cells.</p> / Master of Science (MSc)

Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). The virally encoded X antigen, HBx, contributes importantly to the development of HCC through its trans-activating role in various signal transduction pathways. Pathways implicated in stem cell self-renewal also contribute to carcinogenesis. Thus, experiments were designed to test if HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HBx expressing (HepG2X) and control (HepG2CAT) human cell lines were assayed for phenotypic and molecular characteristics of "stemness." Western blotting of protein extracts from HepG2X and HepG2CAT cells as well as immunohistochemical staining of HCC and adjacent liver tissue sections from HBV infected patients showed up-regulation of "stemness"-associated (EpCAM and beta-catenin) and "stemness" (Oct-4, Nanog, Klf-4) markers by HBx. Moreover, HBx stimulated cell migration and spheroid formation. HBx expression was also associated with depressed levels of E-cadherin and subsequent activation of beta-catenin and EpCAM. Results from ChIP-chip data performed previously in this lab suggest an associative link between HBx and the expression of epigenetic co-repressor, mSin3A, which is known to repress E-cadherin when complexed with histone deacetylases. Thus, experiments were also designed to test if HBx represses the E-cadherin gene (CDH1) through histone deacetylation by the mSin3A/HDAC complex. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was showed by immunofluorescent staining. Chromatin immunoprecipitation revealed that HBx mediated the recruitment of the mSin3A/HDAC complex to the CDH1 promoter. HDAC inhibition by Trichostatin A treatment restored E-cadherin expression. Thus, HBx-associated epigenetic repression of E-cadherin and up-regulated expression of multiple "stemness" markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristic of CSCs. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. / Biology

No description available.

Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell-specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold

Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell- specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold