Made available in DSpace on 2016-08-29T15:37:23Z (GMT). No. of bitstreams: 1 tese_5526_MARIA APARECIDA DA SILVA.pdf: 3362386 bytes, checksum: 9e40b51a695b6e8a58969240cbd6e47c (MD5) Previous issue date: 2012-02-27 / A hematúria enzoótica bovina (HEB) é causada pela ingestão crônica de Pteridium aquilinum e caracteriza-se pela presença de sangue na urina e desenvolvimento de lesões na bexiga, sendo responsável por grandes perdas econômicas. A intoxicação por esta planta também pode ocorrer em humanos. Objetivou-se avaliar lesões de bexigas de animais com HEB na região sul do Espírito Santo. Para isto, foram avaliadas 350 bexigas de bovinos em matadouro frigorífico e, destas, selecionadas 46 que apresentavam lesões macroscópicas e/ou hematúria. Amostras de cada bexiga foram fixadas em formol a 10% submetidas ao processamento histológico de rotina e classificadas histomorfologicamente. A imunoistoquímica foi realizada com anti-vimentina, anti-citoqueratina, anti-CD31, anti-VEGF e anti-uroplaquina apenas nas 26 bexigas que revelaram neoplasia. Lesões não neoplásicas foram observadas em 100% das amostras e neoplásicas em 56,52%. A presença de neoplasias foi significativa (p<0,05) na porção caudal da bexiga. As neoplasias encontradas foram carcinoma urotelial; carcinoma in situ, adenocarcinoma, hemangioma, mixoma e hemangiossarcoma. Houve maior frequência de displasia, metaplasia de células claras, inflamação e espessamento vascular em bexigas com neoplasia. A expressão de citoqueratina foi significativa (p<0,05) nas neoplasias epiteliais e vimentina nas mesenquimais. A marcação da uroplaquina III diferiu nos diversos tipos neoplásicos e revelou-se típica e atípica enquanto que a do CD31 foi significativa (p<0,05) nas neoplasias mesenquimais vasculares. Houve diferença significativa (p<0,05) na quantidade de vasos extratumorais marcados pelo VEGF entre os mixomas e adenocarcinomas e nos vasos intratumorais marcados por CD31 e VEGF nos diferentes tipos tumorais. Houve correlação positiva entre os vasos extra e intratumorais nos hemangiomas, hemangiossarcomas e mixomas marcados pelo CD31; nos hemangiomas, mixomas e adenocarcinomas marcados pelo VEGF; entre a expressão de vimentina e CD31 e entre citoqueratina e uroplaquina. Conclui-se que bexigas de bovinos com HEB apresentam lesões não neoplásicas e neoplásicas, isoladas ou associadas. Os biomarcadores auxiliam na diferenciação da histogênese das neoplasias epiteliais e mesenquimais vasculares. Uroplaquina demonstrou-se efetiva para a avaliação da integridade urotelial e os marcadores vasculares (CD31 e VEGF) para a integridade endotelial e para o prognóstico.

Abstract Fetal alcohol syndrome (FAS) is a pattern of anomalies in affected children due to maternal alcohol administration at vulnerable stages of fetal development. Intrauterine growth restriction and facial malformation are the presenting phenotypes of FAS. In this investigation, novel pathomechanisms of intrauterine growth restriction and facial malformation were the primary aims. We found by a FAS mouse model that AceCS1 gene expression and polyamines are the immediate targets of fetal alcohol exposure. The AceCS1 product is a precursor for lipid synthesis and protein acetylation and possibly, for polycation acetylation. We cloned the Mus musculus nuclear-cytosolic AceCS1 gene, and showed that its expression is developmentally regulated with a dynamic localization in the cytosolic and nuclear compartment. The enzyme plays an essential role in de novo synthesis of acetyl Coenzyme A. Fetal alcohol administration targets nutrient supplying networks, which are localized at critical barriers. The main findings were reduced surface of the labyrinthine zone, destruction of gap junctions in the hemotrichorial placenta, reduced syncytiotrophoblastic cell layers and loosening of interaction between cell layers and embryo endothelial cells, reduced Reichert’s membrane thickness with discontinued Reichert’s trophoblast and loss of interaction by Reichert’s-parietal cells, reduction of capillary network and reduced vascularization in the brain area, and perturbed neural crest migration and formation of neural tube defect. Alteration of angiogenesis -regulating proteins such as VEGF, PlGF, PECAM was detected in FAS, with no significant changes in placental angiogenesis of the labyrinthine zone, but up-regulation of VEGF/PlGF caused permeability changes in the placenta and yolk sac. On the other hand, the PECAM pool in embryos’ brain was reduced, which in turn led to decreased angiogenesis and vascularization. / Tiivistelmä Sikiön alkoholisyndrooma (engl. Fetal alcohol syndrome, FAS) on joukko muutoksia, joita esiintyy äidin raskaudenaikaisen alkoholin käytön seurauksena, kun käyttö osuu sikiökehityksen kannalta kriittiseen vaiheeseen. Kohdunsisäisen kasvun rajoittuminen ja kasvojen epämuodostumat ovat FAS:n tyypillisimpiä ilmentymiä. Tässä tutkimuksessa pyrittiin löytämään uusia patomekanismeja kohdunsisäisen kasvun rajoittumiselle ja kasvojen epämuodostumille. Hiiren FAS-mallin avulla selvisi, että sikiön altistuminen alkoholille vaikuttaa suoraan AceCS1-geenin ilmentymiseen ja polyamiinien pitoisuuteen. AceCS1-geenin tuote on esiaste lipidien synteesissä ja proteiinien asetylaatiossa sekä mahdollisesti myös polykationien asetylaatiossa. Työssä myös kloonattiin hiiren (Mus musculus) AceCS1-geeni, jonka tuotetta esiintyy sekä tumassa että solulimassa. Lisäksi osoitettiin, että geenin ekspressio oli kehityksen aikana säädelty tuottamaan entsyymiä dynaamisesti eri paikkoihin solussa. Entsyymillä on lisäksi merkittävä osuus asetyyli-koentsyymi-A:n de novo–synteesissä. Sikiön altistuminen alkoholille kohdistuu sellaisten ravintoaineiden saatavuuteen, jotka sijaitsevat kriittisesti tärkeissä kudosrajapinnoissa. Päälöydöksinä olivat vähentynyt labyrinttikudoksen pinta-ala, gap-liitosten tuhoutuminen istukan veriesteessä (hemotrichorial?), ohentunut trofoblastisolujen kerros ja Reichertin kalvon paksuus, harventunut hiusverisuonten verkosto sekä verisuonitus aivojen alueella sekä hermopienan solujen siirtymishäiriö ja hermostoputken sulkeutumishäiriö. Verisuonten muodostumista (angiogeneesiä) säätelevien proteiinien (kuten VEGF, PlGF, PECAM) muutoksia todettiin FAS:ssa, mutta merkittäviä muutoksia ei havaittu istukan verisuonten muodostumisessa. VEGF/PlGF-suhteen suureneminen muutti istukan ja ruskuaispussin verisuonten läpäisevyyttä. Toisaalta sikiöiden aivojen PECAM-määrä pieneni, mikä johti verisuonten ja verisuoniverkoston muodostumisen vähenemiseen.

Made available in DSpace on 2016-12-23T13:53:40Z (GMT). No. of bitstreams: 1 Maria Aparecida da Silva.pdf: 3366725 bytes, checksum: 59d418ef608b4e75da9a6ba6a1fdb233 (MD5) Previous issue date: 2012-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bovine enzootic hematuria (BEH) is caused by chronic ingestion of Pteridium aquilinum and is characterized by the presence of blood in the urine and development of lesions in the urinary bladder and is responsible for economic losses. Poisoning by this plant can also occur in humans. The objective was to evaluate the lesions in bladders of animals with BEH in the south region of the Espírito Santo. For this, were evaluated 350 bladders of bovines in a slaughterhouse and, of these, selected 46 that had macroscopic lesions and/or hematuria. Samples of each bladder were fixed in formalin 10% submitted to histological processing and classified by histomorphology. The immunohistochemistry was performed with anti-vimentin, anti-cytokeratin, anti-CD31, anti-VEGF and anti-uroplakin only in the 26 bladders that presented neoplastic lesions. Non-neoplastic lesions were observed in 100% of samples and the neoplastic in 56.52%. The presence of tumors was significant (p<0.05) in the caudal portion of the bladder. Detected neoplastic types were urothelial carcinoma, in situ carcinoma, adenocarcinoma, hemangioma, myxoma and hemangiosarcoma. There was a higher frequency of dysplasia, clear cell metaplasia, inflammation and vascular thickening in bladders with neoplasm. The expression of cytokeratin was significant (p<0.05) in epithelial neoplasms and vimentin in mesenchymal neoplasms. Uroplakin III differed in varied types of neoplastic lesions and showed to be typical and atypical while that of CD31 was significantly (p <0.05) in vascular mesenchymal neoplasms. A significant difference (p <0.05) in the number of vessels extratumorais stained by VEGF between myxomas and adenocarcinomas, and in intratumoral vessels stained by CD31 and VEGF in the different tumor types. Positive correlation existed between the number of intra- and extratumoral vessels in hemangiomas, hemangiosarcomas, and myxomas stained by CD31; between hemangiomas, myxomas, and adenocarcinomas stained with VEGF; between the expression of vimentin and CD31 and between cytokeratin and uroplakin. It is concluded that bladders from bovines with BEH have non-neoplastic and neoplastic lesions, isolated or associated. Biomarkers aid in the differentiation of the histogenesis of epithelial and vascular mesenchymal neoplasms. Uroplakin demonstrated to be effective for the assessment of integrity urothelial, and vascular markers (CD31 and VEGF) for endothelial integrity and for prognosis / A hematúria enzoótica bovina (HEB) é causada pela ingestão crônica de Pteridium aquilinum e caracteriza-se pela presença de sangue na urina e desenvolvimento de lesões na bexiga, sendo responsável por grandes perdas econômicas. A intoxicação por esta planta também pode ocorrer em humanos. Objetivou-se avaliar lesões de bexigas de animais com HEB na região sul do Espírito Santo. Para isto, foram avaliadas 350 bexigas de bovinos em matadouro frigorífico e, destas, selecionadas 46 que apresentavam lesões macroscópicas e/ou hematúria. Amostras de cada bexiga foram fixadas em formol a 10% submetidas ao processamento histológico de rotina e classificadas histomorfologicamente. A imunoistoquímica foi realizada com anti-vimentina, anti-citoqueratina, anti-CD31, anti-VEGF e anti-uroplaquina apenas nas 26 bexigas que revelaram neoplasia. Lesões não neoplásicas foram observadas em 100% das amostras e neoplásicas em 56,52%. A presença de neoplasias foi significativa (p<0,05) na porção caudal da bexiga. As neoplasias encontradas foram carcinoma urotelial; carcinoma in situ, adenocarcinoma, hemangioma, mixoma e hemangiossarcoma. Houve maior frequência de displasia, metaplasia de células claras, inflamação e espessamento vascular em bexigas com neoplasia. A expressão de citoqueratina foi significativa (p<0,05) nas neoplasias epiteliais e vimentina nas mesenquimais. A marcação da uroplaquina III diferiu nos diversos tipos neoplásicos e revelou-se típica e atípica enquanto que a do CD31 foi significativa (p<0,05) nas neoplasias mesenquimais vasculares. Houve diferença significativa (p<0,05) na quantidade de vasos extratumorais marcados pelo VEGF entre os mixomas e adenocarcinomas e nos vasos intratumorais marcados por CD31 e VEGF nos diferentes tipos tumorais. Houve correlação positiva entre os vasos extra e intratumorais nos hemangiomas, hemangiossarcomas e mixomas marcados pelo CD31; nos hemangiomas, mixomas e adenocarcinomas marcados pelo VEGF; entre a expressão de vimentina e CD31 e entre citoqueratina e uroplaquina. Conclui-se que bexigas de bovinos com HEB apresentam lesões não neoplásicas e neoplásicas, isoladas ou associadas. Os biomarcadores auxiliam na diferenciação da histogênese das neoplasias epiteliais e mesenquimais vasculares. Uroplaquina demonstrou-se efetiva para a avaliação da integridade urotelial e os marcadores vasculares (CD31 e VEGF) para a integridade endotelial e para o prognóstico

Altered fluid flow, which is found in branches and curvatures of arteries, results in abnormal forces on the endothelial cells (EC). These forces have been shown to alter EC gene expression and phenotype and to activate several cellular structures including G-proteins, ion channels, adhesion molecules, and caveolae. Recently, PECAM-1 has been implicated as the primary sensor of hemodynamic forces in EC. Shear stress rapidly induces tyrosine phosphorylation of PECAM-1 and the recruitment of SHP-2. These events appear to contribute to shear-activation of ERK1/2. Additionally, PECAM-1 has been shown to form a mechanosensory signaling complex with VE-cadherin, VEGFR2, and &#946;catenin which plays a role in adhesion molecule expression and regulation of NF-&#954;B. Past work has shown that caveolae membrane domains also serve as mechanotransduction sites that regulate many of these same second messengers. Based on these novel observations, we hypothesize that the PECAM-1 mediated mechanotransduction requires caveolar membrane domains to effectively propagate mechano-signals. In this study, we intended to specifically test this hypothesis by 1) evaluating the role of caveolae in shear stress-induced PECAM-1 tyrosine phosphorylation, recruitment of SHP-2, and formation of a signaling complex with VE-cadherin, VEGFR2, and &#946;catenin and 2) determining the functional significance of PECAM-1 compartmentalization within caveolae with regard to changes in endothelial cell phenotype induced by atherogenic patterns of flow. Here, we have identified a pool of PECAM-1 which localizes within lipid rafts and caveolar membranes. This pool of PECAM-1 was shown to be activated by tyrosine phosphorylation and recruitment of mechanosignaling complex members in response to shear stress. We were also able to demonstrate complex formation in an in vivo model of disturbed blood flow. The significance of PECAM-1 compartmentalization to these membrane microdomains was demonstrated in endothelial cells treated with raft/caveolae disrupting compounds where shear stress-induced PECAM-1 tyrosine phosphorylation was markedly attenuated. Finally, we attempted to generate an adenovirus expressing a mutant form of PECAM-1 which was unable to target to lipid rafts in order to determine the importance of PECAM-1 localization in lipid rafts and caveolae on its downstream signaling in response to shear stress. Results from these studies provide new knowledge as to how endothelial cells respond to changing hemodynamic parameters, which could provide greater insight into how flow influences vascular homeostasis. / Physiology

L'intégrité de l'endothelium vasculaire est maintenue par des structures d'adhérence jonctionelle, notamment les jonctions adhérentes, dont la VE-cadhérine est le constituant majeur. Cette cadhérine est exclusivement exprimée dans les cellules endothéliales et joue un rôle clé au cours de la formation du plexus vasculaire. Les cellules endothéliales expriment également une autre cadhérine : la N-cadhérine. Bien que possédant une forte homologie structurale avec la VE-cadhérine, celle-ci possède une localisation subcellulaire originale pour une cadhérine classique puisqu'elle est localisée de manière diffuse au niveau de la membrane. Dans le développement vasculaire, elle pourrait ainsi permettre le recrutement des cellules murales par l'endothelium conduisant à la stabilisation du vaisseau. Mon travail de thèse a été d'analyser les propriétés différentielles de ces deux cadhérines dans la cellule endothéliale ainsi que le mécanisme d'exclusion jonctionelle de la N-cadhérine. Nous avons montré que la VE- et la N-cadhérine interagissent similairement à l'- et à la -caténine mais à l'inverse, p120 caténine lie préférentiellement la VE-cadhérine. Cette propriété confère à la VE-cadhérine la capacité d'exclure la N-cadhérine de la jonction interendothéliale. D'autre part, nous avons montré qu'une petite proportion de la VE-cadhérine est enchâssée dans des microdomaines riches en cholestérol : les radeaux lipidiques, à laquelle p120 est très fortement associée. Cette fraction pourrait représenter un pool de VE-cadhérine assurant la stabilité de la jonction et le maintien de la force cohésive entre cellules endothéliales. Nous avons également montré dans un modèle de différenciation cellulaire dérivé des cellules ES murines (embryonic stem cell), que l'expression de la VE-cadhérine était nécessaire à l'expression d'autres gènes endothéliaux : PECAM, Flk-1 et Tie-1. La VE-cadhérine exercerait un contrôle transcriptionel de l'expression de ces gènes mais le mécanisme exact de régulation n'a pas pu être décrypté. L'ensemble de ce travail permet donc de montrer un autre visage de la VE-cadhérine dans la biologie de l'endothelium qui serait de réguler des protéines-clé de l'endothelium.

No description available.

The increasing demand on healthcare from pregnancies complicated by gestational diabetes (GDM) and obesity is caused in large part by fetal macrosomia (FM). Alterations to the vasculature of the placenta leading to changes to nutrient flux may be more frequent when GDM and obesity occur concomitantly. However, the impact of obesity as an independent comorbidity is poorly understood. The current study sought to characterise structural and functional changes in placenta from pregnancies complicated by GDM and/or obesity and examine the involvement of miRs in this phenomenon, as the phenotype of vascular smooth muscle (VSM) has been documented to be influenced by microRNA (miR) expression. Patients were stratified according to the presence or absence of GDM and/or obesity, which resulted in four groups. Morphometric analysis of CD31 immuno-stained placentas showed that pregnancies complicated by GDM or obesity both had a higher mean sum ratio of the area of the lumen compared to the endothelium. No relationship was found with FM. The ratio increased with maternal body mass index (BMI) in all pregnancies. Immunohistochemistry with a panel of VSM markers suggested an altered phenotype of VSM in pregnancies complicated by GDM and/or obesity. RT-QPCR and immunoblotting showed a higher expression of smooth muscle myosin (SM-MHC), h-caldesmon (HC) and alpha smooth muscle actin (ASMA) in pregnancies complicated by obesity, consistent with a greater contractile capacity. This was most marked when obesity occurred without GDM.Studies were conducted on two miRs, miR-145, which is associated with VSM in many vascular tissues, and the snoRNA-derived species miR-664a-3p, which microarray studies had shown to be higher in placentas from pregnancies complicated by GDM. Dicer and dyskerin, components of the snoRNA-derived miR biogenesis pathway, were increased and reduced respectively in GDM placenta. However, studies in cultured placental villous explants suggested that neither miR species was regulated by glucose, insulin or IGF-I. Placental mesenchymal cells are the developmental precursors of VSM. In primary culture, these cells expressed both miRs. To determine the function of miR-664a-3p, a nucleofection protocol was developed in a fetal mesenchymal cell line, WI38, and applied to first-trimester placental mesenchymal cells. Preliminary proteomic analysis after nucleofection-mediated knockdown of miR-664a-3p suggested a series of novel candidate target proteins for this uncharacterised miR species. Blood vessel structure and VSM phenotype are both altered in pregnancies complicated by GDM and/or obesity. The significance of apparently higher level of contractile proteins with wider vessel lumens in obesity requires further investigation. Translational regulation by miRs including miR-145 and miR-664a-3p is implicated in these alterations. In future, targeted therapies that alter miR levels in the placenta may be useful in control of fetal overgrowth such as FM.

Mantle cell lymphoma (MCL) is a subtype of B-non-Hodgkin's lymphoma, characterized by often relapses. Despite an Ibrutinib (a Bruton's kinase inhibitor) implementation into salvage therapy, these patients often relapse with biologically highly aggressive disease and very poor prognosis. An increased activation of alternative metabolic pathways was described as one of ibrutinib-resistance mechanisms. Some of these pathways have also significant proangiogenic activity (e.g. PI3K-AKT-mTOR). In presented study, we established and standardized a real-time ultrasound and photoacoustic imaging of neovascularization and tissue oxygenation of subcutaneous MCL tumors in mice. Ultrasound and photoacoustic imaging is a fast, non-invasive method for angiogenesis evaluation in subcutaneous tumors with huge preclinical potential. Using MCL mice models, we also demonstrated the importance of CD31/PECAM-1 expression for engraftment, growth and spread of MCL cells in vivo. The level of CD31 expression in primary MCL cell (obtained directly from MCL patients) positively correlates with extent of extranodal involvement. CD31 facilitates survival and regulates extranodal spread of mantle cell lymphoma. We found that increased VEGFA expression causes not only increased microvessel density due to higher sprouting...

No description available.

Multipotent mesenchymal stem cells (MSCs), found in many adult tissues, may be useful for regenerative medicine applications. Their identification and purification have been difficult due to their low frequency and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove contaminating hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOp) population that was also enriched for other mesenchymal precursors, including MSCs (Itoh and Aubin, 2009). To further enhance enrichment, we positively selected BMSCs and HipOps for CD73, a putative MSC marker, which resulted in no significant additional enrichment for osteoprogenitors when the population was tested in vitro. However, we also found that HipOps were enriched in vascular endothelial cells, and that removing these cells by further negative selection with CD31/PECAM resulted in a CD31(-) HipOp population with higher osteogenic capacity than HipOps in vitro and in vivo.