991 resultados para non-canonical Wnt
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The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.
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We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.
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PURPOSE: We aimed at further elucidating whether aphasic patients' difficulties in understanding non-canonical sentence structures, such as Passive or Object-Verb-Subject sentences, can be attributed to impaired morphosyntactic cue recognition, and to problems in integrating competing interpretations. METHODS: A sentence-picture matching task with canonical and non-canonical spoken sentences was performed using concurrent eye tracking. Accuracy, reaction time, and eye tracking data (fixations) of 50 healthy subjects and 12 aphasic patients were analysed. RESULTS: Patients showed increased error rates and reaction times, as well as delayed fixation preferences for target pictures in non-canonical sentences. Patients' fixation patterns differed from healthy controls and revealed deficits in recognizing and immediately integrating morphosyntactic cues. CONCLUSION: Our study corroborates the notion that difficulties in understanding syntactically complex sentences are attributable to a processing deficit encompassing delayed and therefore impaired recognition and integration of cues, as well as increased competition between interpretations.
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Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field.
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Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.
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Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^
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During sentence processing there is a preference to treat the first noun phrase found as the subject and agent, unless marked the other way. This preference would lead to a conflict in thematic role assignment when the syntactic structure conforms to a non-canonical object-before-subject pattern. Left perisylvian and fronto-parietal brain networks have been found to be engaged by increased computational demands during sentence comprehension, while event-reated brain potentials have been used to study the on-line manifestation of these demands. However, evidence regarding the spatiotemporal organization of brain networks in this domain is scarce. In the current study we used Magnetoencephalography to track spatio-temporally brain activity while Spanish speakers were reading subject- and object-first cleft sentences. Both kinds of sentences remained ambiguous between a subject-first or an object-first interpretation up to the appearance of the second argument. Results show the time-modulation of a frontal network at the disambiguation point of object-first sentences. Moreover, the time windows where these effects took place have been previously related to thematic role integration (300–500 ms) and to sentence reanalysis and resolution of conflicts during processing (beyond 500 ms post-stimulus). These results point to frontal cognitive control as a putative key mechanism which may operate when a revision of the sentence structure and meaning is necessary
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Una bóveda no canónica es una bóveda que se adapta a una forma distinta de aquella para la que ha sido inicialmente concebida. Bóvedas raras, anormales, no convencionales, habitualmente consideradas excepciones o casos particulares, resultan ser más frecuentes de lo inicialmente esperado. El interés por este tipo de bóvedas surge a raíz de una investigación inicial sobre las bóvedas empleadas para cubrir espacios de planta anular, como en el caso de las girolas de las iglesias. Sin embargo, el problema de la bóveda anular no puede ser abordado directamente, sino como parte de una investigación más general sobre bóvedas que se deforman para adaptarse a una situación anómala. El análisis de las posibilidades que un determinado tipo de bóveda brinda para resolver el abovedamiento de espacios de planta irregular, trascendiendo el problema de la planta anular, es lo que da origen a esta investigación. La cuestión de las bóvedas deformadas forma parte de un contexto mayor, el de la deformación en arquitectura abovedada. Ante una contradicción, la deformación de la bóveda es sólo una de las posibles opciones que esta arquitectura ofrece para resolver un problema de deformación. La tesis se estructura en dos partes: en la primera parte se analizan los conceptos de forma y deformación en el contexto de la arquitectura abovedada con objeto de sentar las bases para una teoría de las bóvedas no canónicas. El objetivo es establecer un punto de partida para la investigación en un campo que todavía no había sido abordado. En la segunda parte se analizan tres tipos de bóveda desde la perspectiva de las bóvedas no canónicas, a partir de un estudio de casos de bóvedas en España entre los siglos XVI y XVIII. El estudio de la deformación en arquitectura abovedada se centra en el problema de la girola, por tratarse de un caso generalizado de deformación, directamente relacionado con el problema de las bóvedas irregulares y cuyo estudio, llamativamente, no había sido llevado a cabo hasta la fecha. Se propone una primera aproximación al problema de la girola, desde un punto de vista puramente morfológico, al margen de consideraciones históricas. En el caso de las bóvedas deformadas, el análisis se centra en tres tipos de bóveda: la bóveda de crucería, la bóveda de arista y la bóveda baída. Estos tres tipos de bóveda, aunque basadas en criterios formales distintos, están íntimamente relacionados entre sí. Por un lado permiten resolver el mismo problema –planta cuadrada delimitada por arcos–, por otro lado es posible establecer una relación formal entre la bóveda de arista y la bóveda baída a través de la bóveda de crucería. El estudio de casos recogido en la segunda parte de la tesis se fundamenta en dos líneas de investigación, la primera sobre soluciones teóricas de bóvedas no convencionales propuestas en los manuscritos y tratados de cantería, y la segunda sobre bóvedas efectivamente construidas, tratado de establecer una comparación entre teoría y práctica, confrontando el grado de relación entre ambas. Sin embargo este doble análisis sólo se ha podido llevar a cabo en contadas ocasiones. Constatamos que las bóvedas no canónicas reflejadas en los tratados son pocas y apenas se han llevado a la práctica, mientras que las soluciones construidas no responden a modelos teóricos propuestos, manifestando un divorcio entre teoría y práctica. El estudio de estas bóvedas permite poner en cuestión la definición tradicional que relaciona los conceptos de ‘bóveda’ y ‘superficie’. Al iniciar el trabajo nos encontramos con un modelo teórico extremadamente rígido que deja fuera un gran número de bóvedas, obligando a agruparlas bajo el término «no canónicas». El trabajo realizado pone en evidencia lo limitado del modelo. El problema no está en la presencia de bóvedas anómalas, que no se adaptan al modelo tradicionalmente propuesto, sino en la extrema rigidez del modelo. ABSTRACT A non canonical vault is a vault adapted to a different form from that for which was originally conceived. These rare, abnormal, unconventional vaults are usually considered as exceptions or special cases. However they prove to be more frequent than it was initially expected. Interest in this type of vaults arises from an initial research on the vaults used to roof annular spaces, such as ambulatories. Nevertheless, the annular vault question cannot be addressed directly, but as a part of a broader research on distorted vaults; a research on vaults deformed to conform an anomalous layout. The analysis of the possibilities that a particular type of vault provides to solve the vaulting of an irregular layout, beyond the problem of the annular plan is the origin of this research. The argument of deformed vaults is part of a greater context, the context of deformation in vaulted architecture. Facing a contradiction, deforming a vault is just one of the options that vaulted architecture offers to solve a problem of deformation. This dissertation is organised in two parts: in the first part we analyse the concepts of form and deformation in the context of vaulted architecture in order to lay the foundations for a non canonical vaults theory. The objective is to establish a starting point for future research in a field that has not been addressed yet. In the second part, we analyse three types of vault from the perspective of non canonical vaults, based on a case study of Spanish vaults between the 16th and 18th Centuries. The analysis of deformation in vaulted architecture focuses on the question of the ambulatory, because it is a generalized example of deformation, directly related to the problem of irregular vaults. Remarkably, the analysis of these spaces had not been conducted to date. We propose a first approach to the question of the ambulatory, from a purely morphological point of view, setting aside historical considerations. The analysis of deformed vaults focuses on three types of vault: the groin vault, the ribbed vault and the sail vault. These three types of vault, although based on different formal criteria, are closely related between them. On the one hand, they allow to solve the same problem –a square perimeter limited by arcs-; on the other hand, it is possible to establish a formal relationship between the groin vault and the sail vault through the ribbed vault. The case study presented in the second part of this dissertation is based on two research lines: theoretical non conventional vaults solutions proposed on stonecutting treatises; and currently built vaults. The aim of this double analysis was to establish a comparison between theory and practice, comparing the degree of relationship between them. Nevertheless, this double analysis has only been carried out on rare occasions. It is noted that non canonical vaults reflected in treaties are few and hardly been employed, while the built solutions do not meet proposed theoretical models, expressing a divorce between theory and practice. The analysis of these vaults allows us to question the traditional definition that connects the concepts of 'vault' and 'surface'. When we began this research, we found an extremely rigid theoretical model that leaved out many vaults, forcing to group them under the term of «non canonical vaults». This research evidences the limitations of the model. The problem is not the presence of abnormal vaults, which cannot adapt to the traditional model, but in the very high stiffness of the model.
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Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/β-catenin signaling pathway, but its mechanism of action remains obscure [Zeng, W., Wharton, K. A., Jr., Mack, J. A., Wang, K., Gadbaw, M., et al. (2000) Nature (London) 403, 789–795]. Here we have cloned a cDNA encoding a mammalian homolog of Drosophila Nkd, mNkd, and demonstrated that mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. We showed that mNkd acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupted convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway.
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Properties of a mutant bacteriophage T2 DNA [N6-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher kcat in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage λ DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BclI and EcoRV endonucleases has been shown on phage λ DNA and with BclI and DpnII endonucleases on yeast chromosomal DNA embedded in agarose.
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A typical G-rich telomeric DNA strand, which runs 5′→3′ toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3′-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3′-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56–62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.
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We investigated whether children’s inhibitory control is associated with their ability to produce irregular verb forms as well as learn from corrective feedback following their use of an over-regularized form. Forty-eight 3.5 to 4.5 year old children were tested on the irregular past tense and provided with adult corrective input via models of correct use or recasts of errors following ungrammatical responses. Inhibitory control was assessed with a three-item battery of tasks that required suppressing a prepotent response in favor of a non-canonical one. Results showed that inhibitory control was predictive of children’s initial production of irregular forms and not associated with their post-feedback production of irregulars. These findings show that children’s executive functioning skills may be a rate-limiting factor on their ability to produce correct forms, but might not interact with their ability to learn from input in this domain. Findings are discussed in terms of current theories of past-tense acquisition and learning from input more broadly.
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Vital Subjects: Race and Biopolitics in Italy is an interdisciplinary study of how racial and colonial discourses shaped the “making” of Italians as modern political subjects in the years between its administrative unification (1861-1870) and the end of the First World War (1919)
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The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.
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Este estudo analisa o processo comunicacional em torno das manifestações populares e da história do santo não-canônico piauiense Motorista Gregório. O objetivo geral consistiu em observar, analisar e relacionar a comunicação popular que faz parte do processo à consolidação da figura do milagreiro. As teorias da cultura, especialmente a Folkcomunicação, assim como as da linguagem, configuraram-se referenciais relevantes nas análises das formas de comunicação dos devotos e dos jornais impressos de Teresina. Para isso, utilizou-se uma combinação de metodologias tais como a de Marques de Melo (2008) para inventariar os ex-votos, além de entrevistas semiestruturadas, coleta de depoimentos e observação participante para reunir dados e classificar as informações obtidas. A análise revelou que a oralidade é a forma de comunicação mais utilizada e que mais tem influencia sobre os devotos, que os jornais impressos reproduzem as histórias do povo e que o santo é criado e formado no cotidiano, no convívio dos familiares, vizinhos e amigos.