Correlation between β-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma

Aberrant Wnt-signaling caused by mutants of β-catenin, a key regulator of the canonical Wnt-signaling pathway, is frequently detected in cancer. Only recently, it was suggested that in hepatocellular carcinoma (HCC) the expression of the target gene glutamine synthetase (GS) is a highly reliable marker for the identification of β-catenin mutations. In order to prove this hypothesis, 52 samples from human hepatocellular carcinomas were analysed for the activation of β-catenin and the expression of GS. In total, 45 samples stained positive for cytoplasmic/nuclear β-catenin. A strong correlation between expression of GS and activated β-catenin (100% of nuclear and 84% of cytosolic) was found. However, among 35 GS positive tumors that were analysed for β-catenin mutations no mutations were detected in 25 GS-positive carcinomas although 24 out of the 25 carcinomas exhibited at least abnormal expression of β-catenin. Since the mutational analysis identified 9 different point mutations of the β-catenin gene including the rare mutation H36P and the yet unknown mutation P44A it was asked whether these mutations may differently effect β-catenin target genes. Therefore, expression plasmids for different mutations were constructed and cotransfected with the TOP-flash luciferase reporter and a reporter carrying the GS-5'-enhancer. The experiments confirmed that there are differences between different β-catenin target sequences and different β-catenin mutations. In addition, the failure that the endogenous expression of GS in GS-negative cells was not induced by the transient transfection experiment indicated that the effect of β-catenin on the GS-5'-enhancer is only one aspect of gene activation induced by β-catenin

Relatório de Estágio

O presente documento surge como um culminar do percurso formativo da Prática Educativa Supervisionada, em contexto de Educação Pré-Escolar e Ensino do 1.º Ciclo do Ensino Básico, onde serão explicitados referentes teóricos e legais, mobilizando conhecimentos científicos, pedagógicos e didáticos, por meio a articular a teoria e a prática desenvolvida. Salienta-se a importância concebida a todo este processo, que emergiu da metodologia de investigação-ação, uma vez que possibilitou o desenvolvimento de práticas pedagógicas refletidas a partir da observação e reflexão contínua dos contextos educativos, através das grelhas de observação e diários de formação, e que impulsionou a análise das ações desenvolvidas de forma crítica e analítica. Neste sentido, e tendo em conta os desafios da atualidade que o docente tem vindo a ultrapassar para transformar os paradigmas educacionais, urge a necessidade de formar profissionais com uma nova visão abrangente no que se refere a uma perspetiva socioconstrutivista. O perfil duplo de docente abrange o desenvolvimento de profissionais que devem formar cidadão ativos na sociedade, fornecendo-lhes durante o percurso escolar, ferramentas e competências essenciais para a vida. Destaca-se, ainda, um trabalho colaborativo desenvolvido ao longo deste processo e que permitiu o crescimento a nível pessoal e profissional.This research paper results from a formative path in Supervised Educational Practice, carried out within the scope of the Master in Pre-School Education and Teaching of the 1st Cycle of the Basic Education. In this study, theoretical and legal references are explained, assembling scientific, pedagogical, and didactic knowledge to connect theory and practice. The importance of this entire process, which emerged from the action research methodology, is highlighted as it enabled the development of pedagogical practices. The study focus on the observation and continuous analysis of the educational context, through the use of observation grids and training diaries, which enhanced the analysis of the actions developed critically and analytically. In this sense and considering the current challenges that the teacher faces to transform educational paradigms, there is an urgent need to train professionals with a new comprehensive vision concerning a socio-constructivist perspective. The dual teacher profile encompasses the development of professionals who must train active citizens in society, providing them with essential tools and skills for life during their school career. It is also noteworthy the collaborative work developed throughout this process, which allowed personal and professional growth

Deficiency of Vasodilator-Stimulated Phosphoprotein (VASP) Increases Blood-Brain-Barrier Damage and Edema Formation after Ischemic Stroke in Mice

Background: Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification. Methodology/Principal Findings: Focal cerebral ischemia was induced in Vasp2/2 mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan’s Blue tracer was applied to visualize the extent of blood-brainbarrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p,0.05) and edema volumes (1.7 mm360.5 mm3 versus 0.8 mm360.4 mm3; p,0.0001) were significantly enhanced in Vasp2/2 mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm3617.3 mm3 versus 39.3 mm3610.7 mm3, respectively; p,0.01) and a non significant trend (p.0.05) towards worse neurological outcomes. Conclusion: Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage

Deficiency of von Willebrand factor protects mice from ischemic stroke

We recently demonstrated that blockade of the platelet adhesion receptor glycoprotein (GP) Ibalpha protects mice from ischemic stroke. Although von Willebrand factor (VWF) is the major ligand for GPIbalpha, GPIbalpha can engage other counterreceptors on endothelial cells, platelets, and leukocytes (eg, Mac-1 or P-selectin) potentially involved in stroke outcome. To further analyze whether VWF is of particular relevance for stroke development, VWF(-/-) mice underwent 60 minutes of middle cerebral artery occlusion. After 24 hours, VWF(-/-) mice had significantly smaller infarctions (P< .05) and less severe neurologic deficits (P< .01) compared with controls. This effect was sustained after 1 week, and intracranial bleeding was absent in VWF(-/-) mice as revealed by serial magnetic resonance imaging. Hydrodynamic injection of a VWF-encoding plasmid restored the susceptibility for stroke in VWF(-/-) mice. This study indicates that VWF is critically involved in cerebral ischemia. Hence, targeted inhibition of the GPIbalpha-VWF pathway might become a promising therapeutic option.status: publishe

The %age of GS-expressing tumors with nuclear (N), cytosolic (C) and normal membranous (M) β-catenin expression is indicated

Numbers on top indicate the total amount of HCCs in each group.<p><b>Copyright information:</b></p><p>Taken from "Correlation between β-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma"</p><p>http://www.molecular-cancer.com/content/7/1/21</p><p>Molecular Cancer 2008;7():21-21.</p><p>Published online 18 Feb 2008</p><p>PMCID:PMC2287186.</p><p></p

Inhibition of bradykinin receptor B1 protects mice from focal brain injury by reducing blood–brain barrier leakage and inflammation

Kinins are proinflammatory and vasoactive peptides that are released during tissue damage and may contribute to neuronal degeneration, inflammation, and edema formation after brain injury by acting on discrete bradykinin receptors, B1R and B2R. We studied the expression of B1R and B2R and the effect of their inhibition on lesion size, blood–brain barrier (BBB) disruption, and inflammatory processes after a focal cryolesion of the right parietal cortex in mice. B1R and B2R gene transcripts were significantly induced in the lesioned hemispheres of wild-type mice (P<0.05). The volume of the cortical lesions and neuronal damage at 24 h after injury in B1R−/− mice were significantly smaller than in wild-type controls (2.5±2.6 versus 11.5±3.9 mm3, P<0.001). Treatment with the B1R antagonist R-715 1 h after lesion induction likewise reduced lesion volume in wild-type mice (2.6±1.4 versus 12.2±6.1 mm3, P<0.001). This was accompanied by a remarkable reduction of BBB disruption and tissue inflammation. In contrast, genetic deletion or pharmacological inhibition of B2R had no significant impact on lesion formation or the development of brain edema. We conclude that B1R inhibition may offer a novel therapeutic strategy after acute brain injuries