Univ., Diss., 2010--Kassel.

Regensburg, Univ., Diss., 2009.

Fibronectin (Fn) is an extracellular matrix (ECM) protein involved in embryonic development, wound healing and tumorigenesis. Structurally Fn is mainly composed of three repeated modules: FI, FII and FIII, together with an alternatively spliced type III connecting segment (IIICS). The IIICS has no sequence homology to these repeated modules and contains integrin, proteoglycan and zinc binding sites. These sites facilitate adherence and spreading of leukocytes, peripheral neurons and melanoma cells, which can lead to disease states such as inflammation, autoimmunity and cancer metastasis. Therefore, there is the potential to develop therapeutic agents based on the IIICS structure. In this study, nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the structure and dynamics of both the IIICS and its adjacent FIII15 module, two of the few Fn regions for which a structure has not been elucidated. An ensemble of solution state NMR structures calculated for the isolated FIII15 module showed that FIII15 forms a rare six-stranded FIII fold, homologous to a typical seven-stranded FIII fold, with a disordered N-terminal linker sequence. NMR relaxation data and chemical shift analysis showed that the IIICS is an intrinsically disordered region with no areas of well-defined secondary structure. A structure was also calculated for FIII15 within a construct containing the IIICS, which showed that contrary to a previous hypothesis, the IIICS does not contribute to the FIII15 structure. In addition, structural comparisons between IIICS splice variants suggested that alternative splicing confers no stable structural features to the IIICS. Furthermore, ligand binding studies showed that, under conditions tested, neither zinc nor the proteoglycan heparin, induced the formation of any secondary structure to this region. Zinc binding did, however, induce oligomerisation of a IIICS containing construct and appeared to enhance the binding of heparin to the IIICS. Data was obtained to suggest that FIII15 forms a transient interaction with an adjacent module, which is likely to be FIII14. It is hoped that the work presented will contribute to further studies into this important area of Fn and may aid in the future development of novel therapeutics.

Bacteroides fragilis is a member of the normal microbiota that resides in the human lower gastrointestinal tract. This bacterium is of clinical significance because it is the most frequently isolated Gram-negative obligate anaerobe from peritoneal abscesses and bloodstream infections. Human fibrinogen is a hexameric-glycoprotein that is important for fibrin-mediated abscess formation and limiting the spread of infection. B. fragilis can bind and degrade fibrinogen which may aid in its escape from abscesses into the bloodstream, thereby promoting bacteraemia. In addition to fibrinogen, binding of B. fragilis to fibronectin, a component of the extracellular matrix, found in association with fibrinogen at wound sites, has also been reported. An outer membrane protein, BF1705, expressed by B. fragilis was found to share homology with BspA from Tannerella forsythia which is known to bind fibrinogen. The gene encoding BF1705 was deleted from the B. fragilis NCTC 9343 genome in the present work using a markerless gene deletion technology. Proteins derived from the outer membranes of wild-type B. fragilis were able to bind fibronectin and fibrinogen in far-western blots. Similar protein extracts from the ΔBF1705 strain did not bind fibrinogen and fibronectin, which confirms the role of BF1705 in adhesive interactions with proteins of the host extracellular matrix. The possible involvement of BF1705 in fibrinogen degradation was ruled out because the ΔBF1705 strain still degraded fibrinogen. To identify the proteases involved in degradation of fibrinogen, four genes encoding putative extracellular metallo- and serine proteases in the size range 45-50 kDa were deleted from the NCTC 9343 genome. All of the single and multiple mutants defective in these selected proteases were still capable of degrading fibrinogen as determined by zymography. Expression of eight B. fragilis proteases in E. coli did not lead to detectable degradation of fibrinogen. These observations suggest that these proteases alone cannot degrade fibrinogen and either that an unidentified protease is responsible for degradation or that there is redundancy in the proteases involved. Under conditions of nitrogen limitation bacteria resort to scavenging nitrogen from the environment to replenish the depleting intracellular nitrogen content. By examining the differential regulation of the B. fragilis transcriptome under nitrogen replete and depleting conditions, a potential role for BF1705 and secreted proteases in nutrient binding and assimilation were studied. Growth on conventional glucose defined medium with ammonia as the nitrogen source was compared to growth in defined medium with glutamine as nitrogen source. A reduced doubling time and diauxic growth in the medium containing glutamine indicated nitrogen limitation. Comparison of the transcriptome derived from cultures of B. fragilis grown on either ammonia or glutamine by RNA-Seq did not reveal a significant upregulation of BF1705 in response to nitrogen limitation. This observation in conjunction with its inability to degrade fibrinogen suggests that the primary role of BF1705 might be as an adhesin and does not act directly in nutrient binding and degradation. Nevertheless, nitrogen limitation was found to induce the expression of four protease-encoding genes by over a 2-fold (adjusted p value < 0.05). The molecular weight of three of these proteases were identified to be within the size range of 45-55 kDa which corresponded to the lysis bands detected by fibrinogen zymography with wild-type B. fragilis protein extracts. Therefore the possible involvement of these three proteases in fibrinogen degradation could be assessed. A 155-fold upregulation (adjusted p value < 0.05) in asnB, encoding a homologue of asparagine synthetase B, under conditions of nitrogen limitation suggest a previously uncharacterised aspartate metabolism pathway for ammonia generation via arginine catabolism in B. fragilis. Ammonia thus formed might aid in sustaining B. fragilis growth under nitrogen deprived conditions. In addition to nitrogen assimilation, significant upregulation was observed in the expression of genes involved in regulation of oxidative stress and metronidazole resistance. The observed changes in the transcriptome will add to our understanding of the B. fragilis metabolism and potential assist with unravelling the mechanisms of infection mediated by this important opportunistic pathogen.

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-25Bitstream added on 2014-06-13T18:55:57Z : No. of bitstreams: 1 oliveira_hc_me_arafcf.pdf: 1420769 bytes, checksum: b06f79633d6d6736be281777e90904a5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Paracoccidioides brasiliensis (Pb) é o agente etiológico da paracoccidioidomicose, micose sistêmica de grande importância no Brasil, país que possui a maior concentração de áreas endêmicas para essa doença no mundo. Uma das estratégias possivelmente utilizadas pelo patógeno seria a expressão de genes envolvendo adaptação às condições do hospedeiro, que pode também estar relacionadas à captação de micronutrientes. A matriz extracelular (MEC) desempenha um papel importante na regulação da adesão celular, diferenciação, migração e proliferação das células. Este estudo propõe uma análise de transcritos e de proteínas expressas em condições de depleção de cobre, na presença de quatro matrizes extracelulares – laminina, fibronectina e colágeno I e IV, mimetizando as condições de infecção por P. brasiliensis por meio das técnicas de RDA (Representational Difference Analysis) e eletroforese bidimensional. Para isso, o isolado Pb01 foi cultivado por 3 horas no meio quimicamente definido (MVM), depletado de cobre (Cu) e a seguir colocado em contato com os quatro diferentes componentes da MEC e a adesão foi avaliada por citometria de fluxo. Um aumento significativo (p ≤ 0,05) na adesão frente a todos os componentes da MEC foi observado quando o fungo foi cultivado sem Cu. Então, o RNA e os extratos protéicos foram obtidos de Pb sem Cu e Pb sem Cu em contato com os diferentes componentes da MEC. Ensaios de RDA foram realizados para demonstrar os genes envolvidos neste processo. Duas hibridizações foram realizadas nas proporções de 1:10 e 1:100 de tester e driver, respectivamente, com um excesso de driver para remover as seqüências comuns em ambas condições. Os produtos diferencialmente expressos foram amplificados, resultando em padrões distintos que foram seqüenciados... / Paracoccidioides brasiliensis (Pb) is the etiologic agent of paracoccidioidomycosis, a systemic mycosis of great importance in Brazil, which has the highest concentration of endemic areas for this disease in the world. One of the strategies used by the pathogen may be the expression of proteins related to the adaptation to the host conditions, which may also be related to the uptake of micronutrients. Extracellular matrix (ECM) plays an important role in the regulation of cell adhesion, differentiation, migration and proliferation of cells. This study proposes an analysis of transcripts and proteins expressed in condition of copper depletion in the presence of four components of extracellular matrix - laminin, fibronectin and collagen I and IV, mimicking the conditions of infection by P. brasiliensis, using techniques of RDA (Representational Difference Analysis) and two-dimensional electrophoresis. For this, we cultured the Pb 01 strain at 3 hours in a chemically defined media (MVM) with depletion copper (Cu). After, the fungus was placed at contact with the four differents ECM components and the adhesion was evaluated by flow cytometry. A significant increase of binding (p ≤ 0,05) to all ECM components was observed when the fungal was cultured without Cu. So RNA and protein extracts were obtained of Pb without Cu, and Pb without Cu in contact with the differents ECM components. RDA assay was developed to demonstrate the genes involved in this process. Two hybridizations were performed in the proportions of 1:10 and 1:100 of tester and driver respectively, with an excess of driver to remove the common sequences in both conditions. The differentially expressed products were amplified, resulting in distinct patterns that were sequenced revealing genes involved in differents process like virulence (25%), protein synthesis... (Complete abstract click electronic access below)

Resumo: Objetivo: Estudar a expressão da Galectina-3 e a distribuição das proteínas de matriz, laminina, fibronectina e colágeno IV, em 30 amostras teciduais de carcinoma de nasofaringe (CNF) e correlacionar com as características clínicopatológicas, agressividade tumoral e sobrevida dos indivíduos. Forma de estudo: clínico retrospectivo. Casuística e Material: Foram estudadas por método imunohistoquímico 30 amostras teciduais de 26 pacientes com diagnóstico de Carcinoma de Nasofaringe (CNF), cujos blocos parafinados estavam arquivados no Departamento de Patologia da Faculdade de Medicina de Botucatu, UNESP. As lâminas foram revisadas e reclassificadas de acordo com a classificação de 2005 da WHO. As proteínas de matriz e a galectina-3 foram analisadas por método imunohistoquímico. Resultado: A análise mostrou que a media etária foi de 48anos, com o pico de prevalência entre 60 a 69 anos, e predominância do sexo masculino de 2:1. O Carcinoma Escamoso Não Ceratinizante Indiferenciado (CENCI) foi mais comuns em 23 amostras (76,7%), o Carcinoma Escamoso Não Ceratinizante Diferenciado (CENCD) em 4 amostras (13.3%) e Carcinoma Escamoso Ceratinizante (CEC) em 3 amostras (10,0%). A expressão da laminina que normalmente é restrita à parede dos vasos e na lâmina própria, estava muito aumentada na matriz das células neoplásicas em 23 amostras (76,7%); a fibronectina foi positiva em 13 amostras (43%) e a galectina-3 foi positiva em 21 amostras (70%). Tivemos correlação positiva da laminina, fibronectina e galectina-3 em 7 amostras (23,3%) e entre laminina e galectina-3 em 11amostras (36,6%). Tivemos um caso de CEC na recidiva de um CENCD. Conclusão: A expressão positiva da Galectina-3 e da laminina não apresentaram significância estatística quanto a agressividade tumoral e a sobrevida dos pacientes. A presença da fibronectina parece reduzir a chance de recidiva do tumor e também sobrevida maior ao contrário da laminina / Abstract: Objectives: Analyze the expression of galectin-3 and distribution of matrix proteins, laminin, fibronectin and collagen IV, in 30 paraffinated samples of nasopharynx carcinoma(NFC); and correlate the to clinicopathological characteristics, as tumor aggressiveness and survival of individuals. Study design: retrospective clinical study. Methods: The analyses were performed by immunohistochemistry in 30 tissue samples of 26 patients diagnosed with NPC were archived in the Pathology Department of Botucatu of Medical School, São Paulo State University, UNESP, Brazil. The slides were reviewed and reclassified according to the WHO classification of 2005. The matrix proteins and galectin-3 analyses were performed by immunohistochemistry. Results: The analysis showed that the patients mean age was 48 years-old, with the age peak prevalence was from 60 to 69 years-old, with male predominance of 2:1. The undifferentiated non-keratinized squamous cell carcinoma (UNKSCC) was predominant in 23 samples (79%), differentiated non-keratinized squamous cell carcinoma (DNKCC) was found in 4 samples (13.3%) and keratinized squamous cell carcinoma (KCC) was found in 3 samples (10%). The laminin expression, which is normally restricted to the vessel walls and lamina propria, was increased in the neoplasic cell matrix in 23 samples (76.7%). Fibronectin was positive in 23 samples (43%). Galectin-3 was observed in 21 samples (70%). Seven cases showed positive correlation between all three proteins. Eleven cases presented positive correlation of laminin and galectin-3 immunoexpression. There was one case of KCC in recurrence of DNKCC. Conclusions: The statistical analysis was not conclusive, maybe because of the sample size. Nevertheless, fibronectin presence seems to reduce the chance of tumor recurrence and also, increase the survival rate, differently from laminin and galectin-3 / Orientador: Jair Cortez Montovani / Coorientador: José Vicente Tagliarini / Coorientador: Maria A. Custódio Domingues / Banca: José Victor Maniglia / Banca: Onivaldo Cervantes / Banca: Emanuel Celice Castilho / Banca: Odair Carlito Michelin / Doutor

WNT lipoglycoproteins (WNTs) modulate a plethora of cellular functions through the activation of the family of frizzled receptors (FZDs). Deregulation in components of the WNT signalling pathways is often observed in human cancers and associated with uncontrolled proliferation and metastasis. Frizzled receptor 6 (Fzd6), one of the ten human FZDs, is frequently overexpressed in cancer, but its role in tumorigenesis is still unclear. In this study we investigated the role Fzd6 in breast cancer. We found that expression of Fzd6 predicts distant relapse in patients with localised breast cancers, particularly in those bearing the triple negative subtype. Using a loss of function approach, we demonstrated that Fzd6 is important to regulate motility and invasion of breast cancer cells in vitro and in vivo. Indeed, Fzd6 regulates the tropism of breast cancer cells the bone, liver and heart of mice. Mechanistically, we found that Fzd6 signalling activates the small GTPase Rho and is important in the organisation of the fibronectin matrix. Both Rho and fibronectin have been previously implicated in the development of metastasis in different systems. All together, these results demonstrate that Fzd6 is an important driver of metastatic spread and a predictive marker of metastatic relapse in breast cancer patients. Fzd6 could therefore be used as a biomarker and target in metastatic breast cancer.

Abstract Type XIII collagen is an integral membrane protein in type II orientation. In cells and tissues type XIII collagen has been located in various adhesive structures, like focal adhesions. Due to this, its biological role has been implicated in cell adhesion. This collagen also exists as a soluble protein due to the release of the ectodomain from the plasma membrane. In this thesis, ectodomain shedding, i.e. enzymatic release of the extracellular domain, was studied in detail, focusing on the phenomenon as it occurs in mammalian cells. It was found that the ectodomain is released by members of the mammalian proprotein convertase family, e.g. furin. Shedding was shown to take place at the cell surface, but based on additional observations, this cleavage may also take place intracellularly in the Golgi apparatus. Various intracellular mechanisms, depending on cell type, were found to be involved in the regulation of ectodomain shedding. Apparently, due to the liberation of the ectodomain, the level of type XIII collagen on the plasma membrane is maintained at a relatively even amount. The released ectodomain was shown to retain biological activity. It showed distinct matrix-specificity so that on vitronectin its influence on cell functions was anti-adhesive, anti-migratory, anti-proliferative and non-supportive of cell spreading. It was also demonstrated to affect the fibronectin matrix assembly in a manner that resulted in reduced amounts of the fibrillar fibronectin matrix. A large collection of human epithelial and mesenchymal cancer samples were screened for type XIII collagen mRNA expression and compared to the expression levels of pre-malignant and normal samples. It was discovered that malignant transformation upregulates the expression of type XIII collagen in mesenchymal cancers and particularly in the stroma of epithelial cancers, more so than in cancer epithelia. TGF-β1 was demonstrated as one factor contributing to the stimulation of expression. Based on cell culture experiments in this study, it was also deduced that the upregulated expression of type XIII collagen and the concomitant shedding of the ectodomain can remodel the tumour stroma, making it inauspicious for adhesion-dependent cell functions, particularly in vitronectin-rich milieu.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21013号 / 医博第4359号 / 新制||医||1028(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 長船 健二, 教授 瀬原 淳子, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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