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

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

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

Individualised therapy of angiotensin converting enzyme (ACE) inhibitors in stable coronary artery disease: overview of the primary results of the PERindopril GENEtic association (PERGENE) study

In patients with stable coronary artery disease (CAD) without overt heart failure, ACE inhibitors are among the most commonly used drugs as these agents have been proven effective in reducing the risk of cardiovascular events. Considerable individual variations in the blood pressure response to ACE inhibitors are observed and as such heterogeneity in clinical treatment effect would be likely as well. Assessing the consistency of treatment benefit is essential for the rational and cost-effective prescription of ACE inhibitors. Information on heterogeneities in treatment effect between subgroups of patients could be used to develop an evidence-based guidance for the installation of ACE-inhibitor therapy. Obviously, therapy should only be applied in those patients who most likely will benefit. Attempts to develop such treatment guidance by using clinical characteristics have been unsuccessful. No heterogeneity in risk reduction by ACE inhibitors has been observed in relation to relevant clinical characteristics. A new approach to such ‘guided-therapy’ could be to integrate more patient-specific characteristics such as the patients’ genetic information. If proven feasible, pharmacogenetic profiling could optimise patients’ benefit of treatment and reduce unnecessary treatment of patients. Cardiovascular pharmacogenetic research of ACE inhibitors in coronary artery disease patients is in a formative stage and studies are limited. The PERGENE study is a large pharmacogenetic substudy of the EUROPA trial, aimed to assess the achievability of pharmacogenetic profiling. We provide an overview of the main results of the PERGENE study in terms of the genetic determinants of treatment benefit and blood pressure response. The main results of the PERGENE study show a pharmacogenetic profile related to the treatment benefit of perindopril identifying responders and non-responders to treatment

Kinin-B2 Receptor Mediated Neuroprotection after NMDA Excitotoxicity Is Reversed in the Presence of Kinin-B1 Receptor Agonists

Background: Kinins, with bradykinin and des-Arg 9-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg 9-bradykinin as well as Lys-des-Arg 9-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-Daspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings: Bradykinin at 10 nM and 1 mM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg 9-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spik

Cyr61/CCN1 Is Regulated by Wnt/β-Catenin Signaling and Plays an Important Role in the Progression of Hepatocellular Carcinoma

Abnormal activation of the canonical Wnt signaling pathway has been implicated in carcinogenesis. Transcription of Wnt target genes is regulated by nuclear β-catenin, whose over-expression is observed in Hepatocellular Carcinoma (HCC) tissue. Cyr61, a member of the CCN complex family of multifunctional proteins, is also found over-expressed in many types of tumor and plays dramatically different roles in tumorigenesis. In this study, we investigated the relationship between Cyr61 and β-catenin in HCC. We found that while Cyr61 protein was not expressed at a detectable level in the liver tissue of healthy individuals, its expression level was elevated in the HCC and HCC adjacent tissues and was markedly increased in cancer-adjacent hepatic cirrhosis tissue. Over-expression of Cyr61 was positively correlated with increased levels of β-catenin in human HCC samples. Activation of β-catenin signaling elevated the mRNA level of Cyr61 in HepG2 cells, while inhibition of β-catenin signaling reduced both mRNA and protein levels of Cyr61. We identified two TCF4-binding elements in the promoter region of human Cyr61 gene and demonstrated that β-catenin/TCF4 complex specifically bound to the Cyr61 promoter in vivo and directly regulated its promoter activity. Furthermore, we found that over-expression of Cyr61 in HepG2 cells promoted the progression of HCC xenografts in SCID mice. These findings indicate that Cyr61 is a direct target of β-catenin signaling in HCC and may play an important role in the progression of HCC

Regular frequency computations

Die vorliegende Arbeit beschäftigt sich mit Häufigkeitsberechnungen, einem rekursionstheoretischen Begriff, der in den 60er Jahren von Rose eingeführt wurde. Eine Funktion f ist berechenbar mit Frequenz m/n, wenn es einen Algorithmus gibt, der für je n verschiedene Eingaben n Ausgabewerte berechnet, von denen mindestens m mit den zugehörigen Funktionswerten von f übereinstimmen. Eine erste natürliche Fragestellung war: gibt es nicht-berechenbare Funktionen, die mit einer Frequenz nahe 1 berechnet werden können? Trakhtenbrot beantwortete diese Frage 1963 negativ, indem er zeigte, dass eine Funktion mit Frequenz echt größer als 1/2 bereits berechenbar im herkömmlichen Sinne ist. Andererseits gibt es überabzählbar viele Funktionen, die sich mit Frequenz 1/2 berechnen lassen. Also ist dieses Ergebnis optimal. Die Forschung auf diesem Gebiet intensivierte sich daraufhin: Wissenschaftler wie Dëgtev, Kinber und Trakhtenbrot selbst (in den 70er und 80er Jahren) und Beigel, Gasarch, Hinrichs, Kummer, Stephan, Tantau und Wechsung (ab den 90er Jahren) beschäftigten sich mit verschiedenen Aspekten von Häufigkeitsberechnungen und verwandten Berechenbarkeitsbegriffen. In der vorliegenden Arbeit beschäftigen wir uns vorwiegend mit regulären Häufigkeitsberechnungen, also solchen, die von endlichen Automaten vorgenommen werden können. Kinber untersuchte diesen Aspekt als erster im Jahre 1976 und behauptete, dass sich Trakhtenbrots Resultat auf endliche Automaten überträgt. Sein folgerte dies aus einem allgemeineren Resultats über separierbare Sprachen, das sich allerdings als falsch herausstellte (ein Gegenbeispiel wurde 2002 von Tantau angegeben). (Zwei disjunkte Sprachen A und B heißen separierbar, wenn ein Algorithmus alle Wörter aus A akzeptiert und alle Wörter aus B ablehnt). Die Frage, ob das Analogon von Trakhtenbrots Resultat für endliche Automaten gilt, stellte sich also von neuem. Diese Dissertation enthält folgende Ergebnisse. In Kapitel 2 geben wir zwei Beweise für Trakhtenbrots Resultat an. Zunächst präsentieren wir seinen Originalbeweis, um dann einen neuen kombinatorischen Beweis zu geben, der einen großen Vorteil besitzt: er erlaubt die Übertragung des Ergebnisses auf endliche Automaten. Zwei kleinere Resultate beschließen dieses Kapitel: der Nachweis, dass es überabzählbar viele nicht häufigkeitsberechenbare Sprachen gibt, und eine Darstellung des Zusammenhangs von Häufigkeitsberechnungen und sog. mulit-selektiven Mengen. In Kapitel 3 arbeiten wir den Fehler in Kinbers Beweis über separierbare Sprachen heraus und geben ein konkretes Gegenbeispiel an. Dann untersuchen wir die Separierbarkeit von sog. Pfad- und Anti-Pfadsprachen, die wie folgt definiert sind: sei alpha ein unendliches Wort über dem Alphabet { 0, 1 }; dann ist A(alpha) die Menge der endlichen Präfixe von alpha, und B(alpha) die Menge der Wörter von A(alpha), bei denen das letzte Bit negiert wurde. Wir zeigen, dass A(alpha) und B(alpha) genau dann separiert werden können, wenn die 1-Positionen von alpha berechnet werden können. Andererseits gibt es überabzählbar viele alpha, für die A(alpha) und B(alpha) mit Frequenz 1/2 berechnet werden können. Wenn (ab drei Eingaben) nur ein Fehler zugelassen ist, dann sind A(alpha) und B(alpha) bereits rekursiv. Dieses Ergebnis überträgt sich auch auf endliche Automaten. Zum Abschluss dieses Kapitels zeigen wir, dass sich Kinbers Vermutung (dass Trakhtenbrots Resultat auch für Sprachen gilt, die durch endliche Automaten separiert werden können) nicht retten lässt: für jede Konstante q q und ein alpha derart, dass A(alpha) und B(alpha) durch endliche Automaten mit Frequenz m/n separiert werden können, jedoch nicht rekursiv separierbar sind. In Kapitel 4 untersuchen wir verschiedene Aspekte regulärer Häufigkeitsberechnungen. Wir zeigen zunächst, dass sich Trakhtenbrots Resultat auf endliche Automaten überträgt, indem wir den neuen Beweis aus Kapitel 2 nochmals genauer betrachten. Anschließend zeigen wir, dass aperiodische Automaten, die Häufigkeitsberechnungen durchführen, nur aperiodische reguläre Sprachen berechnen können (aperiodische Sprachen entsprechen sternfreien Sprachen). Dann beweisen wir ein sog. Iterationskriterium, das vergleichbar mit dem bekannten Pumping-Lemma für reguläre Sprachen ist und uns für viele konkrete Beispielsprachen den Nachweis erlaubt, dass diese nicht häufigkeitsberechenbar sind. Im letzten Teil untersuchen wir dann Abschlusseigenschaften der Klasse der regulär häufigkeitsberechenbaren Sprachen: wir zeigen, dass sie eine boolesche Algebra bilden, jedoch nicht unter Spiegelung abgeschlossen sind. Darüberhinaus ist die Vereinigung zweier Sprachen, die mit Frequenz 1/n erkennbar sind, in der Regel nicht 1/n-erkennbar. Wir beweisen eine untere Schranke, die sehr nah an der besten bekannten oberen Schranke liegt.This thesis deals with frequency computations, a recursion theoretic notion that was introduced by Rose in 1960. A function f is said to be computable with frequency m/n, if there is an algorithm that, for every n distinct input values, computes n output values, and at least m of these values coincide with the corresponding values of the function f. A first question arose naturally: are there non-computable functions that are computable with a frequency close to 1? Trakhtenbrot answered this questions negatively in 1963 by showing that a function computable with frequency strictly greater than 1/2 is already computable in the usual sense. On the other hand, there are uncountably many functions computable with frequency 1/2, thus this result is optimal. Research in this area intensified after Trakhtenbrot's result, with persons like Dëgtev, Kinber and Trakhtenbrot himself during the 70s and early 80s, and Beigel, Gasarch, Hinrichs, Kummer, Stephan, Tantau, Wechsung and others from the 90s onwards, working on different aspects of frequency computations and related notions. In this work we mostly deal with regular frequency computations, which are frequency computations performed by finite automata. Kinber was the first to investigate this direction in 1976, and claimed that Trakhtenbrot's result carries over to finite automata. He proved this as a corollary of a more general result concerning the separation of languages via finite automata, which turned out to be wrong (a counterexample was given by Tantau in 2002). (Two disjoint languages A and B are seperable if an algorithm accepts all words from A and rejects all words from B.) Thus, the question whether the analogon of Trakhtenbrot's result holds for finite automata posed itself once more. In this thesis, we prove the following results. In Chapter 2 we give two proofs of Trakhtenbrot's theorem. First, we present a self-contained version of his original proof, and second, we give a new combinatorial proof which has one major advantage over Trakhtenbrot's: it allows the transfer of this result to finite automata. Two minor results end this chapter: the existence of uncountably many non-frequency computable languages, and a connection between frequency computations and so-called multi-selective languages. In Chapter 3 we work out the error in Kinber's proof about seperable languages, and give a concrete counterexample. Afterwards, we investigate the separation of so-called path and anti-path languages, which are defined as follows: let alpha be an infinite word over the alphabet { 0, 1 }; then A(alpha) is the set of finite prefixes of alpha, and B(alpha) is the set of words from A(alpha) with the last bit toggled. We show that A(alpha) and B(alpha) are seperable if and only if the positions of alpha containing a 1 are computable. On the other hand, there are uncountably many alpha for which A(alpha) and B(alpha) are seperable with frequency 1/2. If it is possible to seperate A(alpha) and B(alpha) with one error (given at least three inputs), then these languages are already recursive. This result carries over to finite automata as well. We end this chapter by showing that Kinber's claim, a Trakhtenbrot-like result for the seperation of languages by finite automata, fails badly: for any constant q q and an alpha such that A(alpha) and B(alpha) can be seperated by finite automata with frequency m/n, but are not recursively seperable. In Chapter 4 we investigate different aspects of regular frequency computations. First we show that Trakhtenbrot's result for general frequency computations carries over to finite automata, by a close inspection of the new proof given in Chapter 2. We then show that aperiodic automata can--in the frequency computation sense - only compute aperiodic regular languages (the class of aperiodic languages is equivalent to the class of star-free languages). Next we prove a so-called iteration criterion, which is comparable to the well-known pumping lemma for regular languages, and allows us to show for many concrete example languages that they are not frequency computable. Then, in the last part, we investigate closure properties of the class of languages frequency computable by finite automata: we show that they form a Boolean algebra, but are not closed under the reversal operation. Furthermore, the union of two languages which are computable with frequency 1/n is, in general, not computable with frequency 1/n. We give a lower bound which is very close to the best-known upper bound