Regulation of expression of the intestinal actin-binding protein, villin, a marker of intestinal epithelial differentiation, is poorly understood. Activation of the extracellular calcium-sensing receptor (CaSR) on sub-epithelial myofibroblasts stimulated the secretion of Wnt5a, while activation of the CaSR on intestinal epithelia increased expression of Ror2, a Wnt-family co-receptor. Immunocytochemistry has localized Ror2 expression in the epithelia lining the small intestine from the crypt base to the villus tip. The aim of this study was to determine whether Wnt5a binding Ror2 in intestinal epithelia stimulated transient increases in phospho-ERK1/2 (pERK1/2) which lead to increased expression of villin transcript and protein. To examine Wnt5a-Ror2 regulation of villin expression, we transgenically overexpressed wild-type, truncated, or mutant Ror2 constructs in HT-29 adenocarcinoma cells and nontransformed fetally-derived human intestinal epithelial cells (HIECs), added conditioned media containing Wnt5a and measured changes in ERK1/2 phosphorylation, villin amplicons and protein expression by RT-PCR and Western blot techniques. Wnt5a addition caused a transient increase in pERK1/2, which was maximal at 10 min but diminished by 30 min. Transient transfection with a siRNA duplex against Ror2 diminished Ror2 amplicons and protein and reduced the extent of pERK1/2 activation. Structure-function analysis revealed that deletion of the cysteine-rich, kringle, or tyrosine kinase domain or substitution mutations of tyrosine residues in the intracellular Ser/Thr-1 region of Ror2 prevented the Wnt5a-stimulation of pERK1/2. Deletion of the intracellular proline and serine/threonine rich regions of Ror2 had no effect on Wnt5a-stimulation of pERK1/2 in HT29 cells. Western blot analysis demonstrated that villin protein was increased by over-expression of wild-type Ror2 in HT-29 cells and HIECs in the presence of Wnt5a. The increase in villin expression was blocked by pharmacological inhibition of MEK1&2 and casein kinase 1, but not by PKC and p38 inhibitors. Neither Wnt3a nor EGF addition increased villin protein. This work suggested that stromal Wnt5a will stimulate pERK1/2 via the Ror2 tyrosine kinase domain to generate increased villin protein. These findings suggested that Ror2 homeostasis and Wnt5a interaction with Ror2 are important determinants of the regulation of villin expression in the intestine. / Thesis (Master, Physiology) -- Queen's University, 2009-07-14 23:34:39.397

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-09-24T14:56:36Z No. of bitstreams: 2 Cysneiros, Maria Auxiliadora de Paula Carneiro.pdf: 1396665 bytes, checksum: f26660692713226fc47ed6704e9d53a9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2014-09-28T02:03:10Z (GMT) No. of bitstreams: 2 Cysneiros, Maria Auxiliadora de Paula Carneiro.pdf: 1396665 bytes, checksum: f26660692713226fc47ed6704e9d53a9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-28T02:03:10Z (GMT). No. of bitstreams: 2 Cysneiros, Maria Auxiliadora de Paula Carneiro.pdf: 1396665 bytes, checksum: f26660692713226fc47ed6704e9d53a9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-10-16 / Endocervical and Endometrial Adenocarcinomas are uterine neoplasms with different biological behaviors and different treatments, being all the therapeutic plan based on the origin site of these tumors. To differentiate these two neoplasms, many times the immunohistochemical study is used as an ancillary tool to assist and complement histopathological examination. Previous studies have investigated the value of various markers in the distinction of these neoplasms, with results varying and sometimes conflicting. In this study the impact of the MCM2, bcl2 and villin markers in the differential diagnosis of endocervical and endometrial adenocarcinomas, associated or not to a traditional markers panel formed by CEA, vimentin, RE, RP and p16, was evaluated. Material and methods: A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 104 hysterectomy specimens and conizations. Out of the 104, 51 samples were represented by unequivocal cases of adenocarcinomas of the endocervice and 53 represented unequivocal cases of endometrial adenocarcinomas. The sections of tissue microarray block were immunostained with the eight antibodies in study and to the antigen-antibody reaction preview it was used the polymer detection system. Scoring of immunostaining was interpreted using the German semi quantitative scoring system in considering the staining intensity and area extent of marker expression. The final Immunoreactive score was determined by multiplying the positive intensity and the positive area extent scores. The threshold for differentiating between final positive and negative immunostaining was set at 4 for interpretation (0-3 = negative; 4-12 = positive). Results: The univariate analysis showed that out of the eight markers, seven (CEA, vimentin, RE, RP, p16, MCM2 and bcl2) showed good performance in the distinction between the endocervical and endometrial origin. The multivariate analysis showed that CEA, p16 and MCM2 were the strongest predictors of site of origin. In the evaluation of six panels built by various combinations of immunomarkers , the panel with the lowest accuracy was that represented by MCM2, bcl2 and villin. The villin did not show any statistically significant difference in the differential diagnosis of these adenocarcinomas. Despite the MCM2 and bcl2 have revealed significant differences of frequency between the two sites of origin, they did not demonstrate additional benefit when added to a traditional panel. Conclusion: According to our study, the inclusion of MCM2, bcl2 and villin in the immunohistochemical assessment of uterine adenocarcinomas, added no supplementary value in the differentiation of these two neoplasms. / Adenocarcinomas endocervicais e endometriais são neoplasias uterinas com comportamentos biológicos e tratamentos distintos, sendo todo o plano terapêutico baseado no sítio de origem desses tumores. Para diferenciação dessas duas neoplasias, muitas vezes utiliza-se o estudo imuno-histoquímico como ferramenta auxiliar e complementar ao exame histopatológico. Prévios estudos têm investigado o valor de diferentes marcadores na distinção dessas neoplasias, com resultados variáveis e, por vezes, conflitantes. Neste estudo foi avaliado o desempenho dos marcadores MCM2, bcl2 e vilina no diagnóstico diferencial desses adenocarcinomas, associados ou não a um painel de marcadores tradicionais formados por CEA, vimentina, RE, RP e p16. Material e métodos: Um microarranjo de tecido (TMA) foi construído usando tecidos fixados em formol e embebidos em parafina, a partir de 104 amostras provenientes de histerectomias e conizações. Das 104 amostras, 51 eram representadas por casos inequívocos de adenocarcinomas de endocérvice e 53 representavam casos inequívocos de adenocarcinomas de endométrio. As secções do bloco de TMA foram imunomarcadas com os oito anticorpos em estudo e para vizualização da reação antígeno-anticorpo foi utilizado o sistema de detecção com polímeros. A marcação imuno-histoquímica foi graduada de acordo com o sistema semi-quantitativo alemão, levando-se em consideração a intensidade de marcação e a extensão de células marcadas. O score de imunorreatividade final foi determinado pela multiplicação da intensidade pela extensão de marcação. Um limite de corte de 4 foi determinado para diferenciação entre um resultado final positivo ou negativo (0- 3= negativo; 4- 12= positivo). Resultados: Análise univariada mostrou que dos oito marcadores avaliados, sete (CEA, vimentina, RE, RP, p16, MCM2 e bcl2) apresentaram bom desempenho na distinção entre origens endocervical e endometrial da neoplasia. Análise multivariada mostrou que CEA, p16 e MCM2 foram os mais fortes preditores do sítio anatômico. Na avaliação dos seis painéis construídos por combinações variadas desses marcadores, o painel com menor acurácia diagnóstica foi o representado por MCM2, bcl2 e vilina. A vilina não apresentou nenhuma diferença estatisticamente significativa no diagnóstico diferencial desses adenocarcinomas. Apesar do MCM2 e bcl2 terem revelado diferenças significativas de frequência entre os dois sítios de origem, eles não demonstraram valor suplementar quando adicionados a um painel tradicional. Conclusão: De acordo com este estudo, a inclusão de MCM2, bcl2 e vilina em um painel tradicional de 5 marcadores (CEA, vimentina, RE, RP, p16), não agregou nenhum benefício adicional na distinção imuno-histoquímica do sítio de origem desses adenocarcinomas uterinos.

Darmepithelzellen entwickeln eine spezielle apikale Oberfläche zur Aufnahme von Nährstoffen aus dem Darminhalt. Diese Oberfläche besteht aus F-Aktin Protrusionen und werden als Mikrovilli (MV) bezeichnet. MV regulieren die kommensalen Bakterien und schützen die inneren Gewebe gegen den Angriff pathogener Mikroorganismen. Dennoch kann das Enteropathogen Salmonella enterica (Salmonella) die MV auslöschen und zerstört durch sein Typ-3-Sekretionssystem und dessen sekretierte Virulenzsproteine die Epithelschicht. Diese Virulenzproteine werden in das Zytoplasma der Wirtzellen injiziert und führen während des Eindringens von Salmonella zur F-Aktin Umlagerung. Durch Untersuchungen des Einflusses einiger T3SS-1 Effektorproteine auf die Zerstörung der MV konnte nachgewiesen werden, dass allein die Translokation von SopE die MV-Auslöschung verursachte und ausreichend für die Wiederherstellung der Invasion war. Echtzeitlebend-zellmikroskopie zeigte, dass MV ausgelöscht werden während Membranausstülpungen (Ruffles) gebildet werden. Diese Ruffle-Bildung vereinfachte ein paralleles Eindringen nicht-invadierender Stämme von Salmonella. Es konnte beobachtet werden, dass die Ausschaltung von Villin und Myosin 1a durch shRNA in C2BBe1 Zellen die Invasionsrate von Salmonella ermäßigte. Darüber hinaus wurde Ezrin zu den intrazellulären Bakterien aber nicht zur apikalen Seite rekrutiert. Außerdem verhinderte die durch das SopE verursachte Umlagerung des F-Aktins, welche die MV-Auflösung zur Folge hatte, die Makropinozytose der infizierten Zellen. Es lässt sich daraus schließen, dass die Zerstörung der MV für eine effiziente Invasion von Salmonella nötig ist. Die F-Aktin Umlagerung begünstigt zudem das Eindringen von nicht-invadierenden Bakterien. Des Weiteren benötigt Salmonella MV-Proteine zur F-Aktin Polymerisierung und Invasion in polarisierten Epithelzellen, was die Makropinozytose der Zellen beeinträchtigt. Möglicherweise tragen diese Phänotypen zur Infektion in vivo bei und verursachen das klinische Bild des Durchfalls.