Activation of the Fibroblast Growth Factor Receptor 3 in Bladder Cancer

The identification of frequent FGFR3 mutations in superficial bladder cancer suggests that mutation of the FGFR3 gene is a key genetic event in the development of noninvasive bladder tumors. Furthermore, FGFR3 mutations were associated with a good prognosis, suggesting that the activation of FGFR3 has a beneficial effect during urothelial tumor formation. In this thesis, two aspects of FGFR3 mutations in bladder cancer have been investigated. First, the potential use of FGFR3 mutations in bladder cancer diagnosis, prognosis, and in surveillance of patients with bladder cancer has been explored. The second, more important aim was to get insight in the functional role of mutant FGFR3 in bladder carcinogenesis. For clinical studies, a simple and fast method for FGFR3 mutation detection was developed that is fast, easy, more sensitive and less laborious than previous techniques. With this technique, several studies described in this thesis were done on the role of FGFR3 in bladder carcinogenesis and patient outcome. We analyzed FGFR3 mutations in urothelial hyperplasia, a precursor of low-grade papillary carcinoma, and found that FGFR3 mutations are already present in hyperplastic lesions. Furthermore, we reported a low number of chromosomal aberrations in bladder tumors with an FGFR3 mutation, which suggests that FGFR3 mutated tumors are genetically stable, in contrast to most tumors that do not carry this mutation, and we showed that this genetic stability of FGFR3 mutant tumors was paralleled by a normal differentiation of urothelial cells, since a normal staining pattern for cytokeratin 20, a marker for terminal differentiation of urothelium, was correlated with bladder cancers carrying the FGFR3 mutation. We also described the relation of molecular markers to bladder cancer patient outcome. The subset of FGFR3 mutated tumors with a normal CK20 staining pattern rarely progressed, providing a combination of two molecular markers that is able to define the group of prognostically favorable low-grade noninvasive papillary tumors. Upper urinary tract tumors (i.e. in the ureter and renal pelvis) may be genetically different from bladder tumors. The FGFR3 mutation frequency was, however, equally high (~50%) in all urothelial tract tumors, and FGFR3 mutation status was an independent predictor for progression and disease-specific survival in tumors from the ureter. Finally, we report the results of an experimental study analyzing the expression of a mutant FGFR3 receptor transfected in a human bladder cancer cell line. The most striking effect was that cells expressing the mutant FGFR3 receptor display both loss of integrin expression and increased apoptosis when cultured in Matrigel. This would suggest that interaction of bladder cancer cells expressing mutant FGFR3 with Matrigel (i.e. basement membrane substances) does not permit their survival. The latter would also explain the clinical finding that FGFR3 mutant bladder cancers have a comparatively low tendency to become invasive. In conclusion, this thesis describes both clinical and functional aspects of the activation of FGFR3 in bladder cancer. The results confirm the association of mutant FGFR3 with well-differentiated, noninvasive bladder tumors with few genetic alterations and a good prognosis

Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice

DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease

Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1 D173A mice

DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease

The principle of situated practice in literacy learning: students’ perspectives

O artigo resulta de uma investigação realizada no âmbito de uma iniciativa governamental destinada a melhorar os níveis de literacia nas séries iniciais do ensino fundamental em Portugal. A investigadora estudou as representações dos alunos sobre essa experiência por meio da realização de entrevistas em grupo. Este artigo analisa os dados referentes às representações dos alunos sobre uma das dimensões pedagógicas centrais da aprendizagem da literacia, nomeadamente a constituída pela prática situada. A análise qualitativa revela representações muito positivas sobre a prática que situou a aprendizagem, tendo os alunos expressado opiniões e sentimentos extremamente favoráveis sobre a prática de aprendizagem de literacia que experimentaram. A análise dos dados desvelou ainda que o contexto que situou a aprendizagem foi ativo, lúdico, colaborativo e mediado pelas TIC. Esses resultados fundamentam, do ponto de vista único dos próprios aprendentes, uma redefinição do entendimento atual do princípio da prática situada da literacia nas séries iniciais do ensino fundamental, no sentido do reconhecimento da centralidade da ludicidade nessa aprendizagem.This article derives from research developed in the context of the implementation of a governmental initiative aimed to enhance literacy learning in primary education in Portugal. The researcher studied students’ representations about their learning experience through group interviews. This article focuses on data concerning students’ representations about one of the central pedagogical dimensions of literacy learning, namely situated practice. Qualitative analysis revealed students’ very positive representations about the practice which situated their learning, as they expressed extremely favourable opinions and feelings. Data analysis further unveiled that the context of learning was active, playful, collaborative, and mediated by ICT. Such results provide foundations for a theoretical redefinition of current conceptions of situated practice by evidencing the centrality of playfulness as learning practice in the education of the first grades of primary education. This is an original contribution made from the perspectives of learners themselves(undefined)info:eu-repo/semantics/publishedVersio

The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination

Antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are similarly initiated in B cells with the generation of U:G mismatches by activation-induced cytidine deaminase but differ in their subsequent mutagenic consequences. Although SHM relies on the generation of nondeleterious point mutations, CSR depends on the production of DNA double-strand breaks (DSBs) and their adequate recombination through nonhomologous end joining (NHEJ). MLH1, an ATPase member of the mismatch repair (MMR) machinery, is emerging as a likely regulator of whether a U:G mismatch progresses toward mutation or DSB formation. We conducted experiments on cancer modeled ATPase-deficient MLH1G67R knockin mice to determine the function that the ATPase domain of MLH1 mediates in SHM and CSR. Mlh1(GR/GR) mice displayed a significant decrease in CSR, mainly attributed to a reduction in the generation of DSBs and diminished accumulation of 53BP1 at the immunoglobulin switch regions. However, SHM was normal in these mice, which distinguishes MLH1 from upstream members of the MMR pathway and suggests a very specific role of its ATPase-dependent functions during CSR. In addition, we show that the residual switching events still taking place in Mlh1(GR/GR) mice display unique features, suggesting a role for the ATPase activity of MLH1 beyond the activation of the endonuclease functions of its MMR partner PMS2. A preference for switch junctions with longer microhomologies in Mlh1(GR/GR) mice suggests that through its ATPase activity, MLH1 also has an impact in DNA end processing, favoring canonical NHEJ downstream of the DSB. Collectively, our study shows that the ATPase domain of MLH1 is important to transmit the CSR signaling cascade both upstream and downstream of the generation of DSBs

Fibroblast Growth Factor Receptor 3 Mutations in Bladder Tumors Correlate with Low Frequency of Chromosome Alterations

The aim of this study was to analyze the distribution of FGFR3 mutations in bladder tumors of different grade and stage and determine the relation of mutations to chromosomal alterations detected by comparative genomic hybridization (CGH). One hundred bladder cancer samples served as templates for manual microdissection. DNA was isolated from dissected samples containing at least 80% tumor cells. Mutations in FGFR3 were analyzed by SNaPshot analysis. CGH was carried out according to standard protocols. FGFR3 mutations were detected in 45 of 92 samples (48.9%). Concerning T-category, the following mutation frequencies occurred: pTa, 69%; pT1, 38%; and pT2-3, 0%. The mutation frequency was significantly associated with tumor grade: G1, 72%; G2, 56%; and G3, 4%. In pTaG1 tumors, mutations were found in 74%. A significantly lower number of genetic alterations per tumor detected by CGH was associated with FGFR3 mutations (2 vs 8). This association was also seen in pTaG1 tumors: 2.5 (with mutation) vs 7.5 (without mutation). FGFR3 mutations characterize noninvasive low-risk tumors of low malignancy. The low malignant potential of these tumors is underlined by a low number of genetic alterations per tumor. Therefore, FGFR3 represents a valuable prognostic marker of tumors with low malignant potential and can be used as surrogate marker for the detection of genetically stable bladder tumors

FGFR3 and PIK3CA mutations are involved in the molecular pathogenesis of solar lentigo

Solar lentigines (SL) are frequent benign skin lesions appearing on sun-exposed areas especially in elderly people and therefore represent a hallmark of (photo)aged skin. It has been proposed that SL may subsequently evolve into adenoid seborrhoeic keratosis (SK). However, little is known about the genetic basis of SL. In human SK, FGFR3 and PIK3CA mutations have recently been identified. To analyse SL for potential FGFR3 and PIK3CA mutations. We screened 30 SL for FGFR3 mutations using a SNaPshot((R)) multiplex assay. For PIK3CA mutations we used direct sequencing of exon 9 and a SNaPshot((R)) assay for the H1047R hotspot mutation (exon 20). Because psoralen plus ultraviolet A (PUVA) lentigines show the V600E BRAF hotspot mutation, we additionally investigated this mutation in SL by allele-specific polymerase chain reaction. FGFR3 mutations were detected in five of 30 (17%) SL and PIK3CA mutations in two of 28 (7%) SL. None of 28 SL available for BRAF analysis revealed the V600E mutation. Our results suggest that FGFR3 and PIK3CA mutations are involved in the pathogenesis of SL. The occurrence of these mutations in both SL and SK suggests a common genetic basis. Our findings furthermore substantiate previous speculations that UV exposure may be a causative factor for FGFR3 and PIK3CA mutations in human skin