Faculdade de Medicina e Ciências Biomédicas
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- High prevalence of double Plasmodium falciparum dhfr mutations at codons 108 and 59 in the Sistan-Baluchistan province, IranPublication . Zakeri, S; Gil, José Pedro; Bereckzy, S; Djadid, ND; Bjorkman, A
- Silicon strip detectors for two-dimensional soft X-ray imaging at normal incidencePublication . Mendes, PR; Abreu, MC; Baldazzi, G; Bollini, D; Rodriguez, AEC; Dabrowski, W; Garcia, AD; Gambaccini, M; Giubellino, P; Gombia, M; Grybos, P; Idzik, M; Marzari-Chiesa, A; Montano, LM; Prino, F; Ramello, L; Rodrigues, Susana; Sitta, M; Sousa, P; Swientek, K; Taibi, A; Tuffanelli, A; Wheadon, R; Wiacek, PA simple prototype system for static two-dimensional soft X-ray imaging using silicon microstrip detectors irradiated at normal incidence is presented. Radiation sensors consist of single-sided silicon detectors made from 300 mum thick wafers, read by RX64 ASICs. Data acquisition and control is performed by a Windows PC workstation running dedicated LabVIEW routines, connected to the sensors through a PCI-DIO-96 interface. Two-dimensional images are obtained by scanning a lead collimator with a thin slit perpendicular to the strip axis, along the whole detector size; the several strip profiles (slices) taken at each position are then put together to form a planar image. Preliminary results are presented, illustrating the high-resolution imaging capabilities of the system with soft X-rays. (C) 2003 Elsevier B.V. All rights reserved.
- Endogenous Cerberus activity is required for anterior head specification in XenopusPublication . Silva, Ana Cristina; Filipe, Mário; Kuerner, Klaus-Michael; Steinbeisser, Herbert; Belo, José A.We analyzed the endogenous requirement for Cerberus in Xenopus head development. 'Knockdown' of Cerberus function by antisense morpholino oligonucleotides did not impair head formation in the embryo. In contrast, targeted increase of BMP, Nodal and Wnt signaling in the anterior dorsal-endoderm (ADE) resulted in synergistic loss of anterior head structures, without affecting more posterior axial ones. Remarkably, those head phenotypes were aggravated by simultaneous depletion of Cerberus. These experiments demonstrated for the first time that endogenous Cerberus protein can inhibit BMP, Nodal and Wnt factors in vivo. Conjugates of dorsal ectoderm (DE) and ADE explants in which Cerberus function was 'knocked down' revealed the requirement of Cerberus in the ADE for the proper induction of anterior neural markers and repression of more posterior ones. This data supports the view that Cerberus function is required in the leading edge of the ADE for correct induction and patterning of the neuroectoderm.
- The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axisPublication . Marques, Sara; Borges, Ana; Silva, Ana Cristina; Freitas, Sandra; Cordenonsi, M.; Belo, José A.Correct establishment of the left/right (L/R) body asymmetry in the mouse embryo requires asymmetric activation of the evolutionarily conserved Nodal signaling cascade in the left lateral plate mesoderm (L-LPM). Furthermore, the presence of Nodal in the node is essential for its own expression in the L-LPM. Here, we have characterized the function of cerl-2, a novel Nodal antagonist, which displays a unique asymmetric expression on the right side of the mouse node. cerl-2 knockout mice display multiple laterality defects including randomization of the L/R axis. These defects can be partially rescued by removing one nodal allele. Our results demonstrate that Cerl-2 plays a key role in restricting the Nodal signaling pathway toward the left side of the mouse embryo by preventing its activity in the right side.
- Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizerPublication . Echevarria, D; Martinez, S; Marques, S; Lucas-Teixeira, V; Belo, JAThe pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium. (C) 2004 Elsevier Inc. All rights reserved.
- Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endodermPublication . Kimura-Yoshida, C.; Nakano, H.; Okamura, D.; Nakao, K.; Yonemura, S.; A. Belo, José; Aizawa, S.; Matsui, Y.; Matsuo, I.The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt- misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.
- N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase expression during early mouse embryonic developmentPublication . Salgueiro, Ana Marisa; Filipe, Mario; Belo, José A.N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is an enzyme which is known to help build up the GlcA beta 1-3GalNAc(4,6-bisSO(4)) unit of chondroitin sulfate E (CSE). This enzymatic activity has been reported in squid cartilage and in human serum, but has never been reported as an enzyme required during early mouse development. On the other hand, CSE has been shown to bind with strong affinity to Midkine (MK). The latter is a heparin-binding growth factor which has been found to play important regulatory roles in differentiation and morphogenesis during mouse embryonic development. We have analyzed the expression pattern of the GalNAc4S-6ST gene during early mouse embryonic development by whole mount in situ hybridization. The results show that GalNAc4S-6ST is differentially expressed in the anterior visceral ectoderm at stage E5.5 and later becomes restricted to the embryonic endoderm, especially in the prospective midgut region. During the turning process, expression of GalNAc4S-6ST gene is detected in the forebrain, branchial arches, across the gut tube (hindgut, midgut and foregut diverticulum), in the vitelline veins and artery and in the splanchnopleure layer. These results open the possibility of a role for GalNAc4S-6ST during early mouse development.
- Developmental expression of Shisa-2 in Xenopus laevisPublication . Silva, Ana-Cristina; Filipe, Mario; Vitorino, Marta; Steinbeisser, Herbert; Belo, José A.Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.
- Comparative expression of mouse and chicken shisa homologues during early developmentPublication . Filipe, Mario; Gonçalves Dias da Silva, Lisa; Bento, Margaret; Silva, Ana Cristina; Belo, José A.During vertebrate embryogenesis, fibroblast growth factor (FGF) and Wnt signaling have been implicated in diverse cellular processes, including cell growth, differentiation, and tissue patterning. The recently identified Xenopus Shisa protein promotes head formation by inhibiting Wnt and FGF signaling through its interaction with the immature forms of Frizzled and FGF receptors in the endoplasmic reticulum, which prevents their posttranslational maturation. Here, we describe the mouse and chicken homologues of Xenopus Shisa. The mouse and chicken Shisa proteins share, respectively, 33.6% and 33.8% identity with the Xenopus homolog. In situ hybridization analysis shows that mouse shisa is expressed throughout embryonic development, predominantly in the anterior visceral endoderm, headfolds, somites, forebrain, optic vesicle, and limb buds. Cross-species comparison shows that the expression pattern of cshisa closely mirrors that of mshisa. Our observations indicate that the Shisa family genes are typically expressed in tissues known to require the modulation of Wnt and FGF signaling. Developmental Dynamics 235:2567-2573, 2006. (c) 2006 Wiley-Liss, Inc.
- Influence of consecutive-day blood sampling on polymerase chain reaction-adjusted parasitological cure rates in an antimalarial-drug trial conducted in TanzaniaPublication . Martensson, Andreas; Ngasala, Billy; Ursing, Johan; Veiga, M. Isabel; Wiklund, Lisa; Membi, Christopher; Montgomery, Scott M.; Premji, Zul; Farnert, Anna; Bjorkman, AndersWe assessed the influence that consecutive-day blood sampling, compared with single-day blood sampling, had on polymerase chain reaction (PCR)-adjusted parasitological cure after stepwise genotyping of merozoite surface proteins 2 (msp2) and 1 (msp1) in 106 children in Tanzania who had uncomplicated falciparum malaria treated with either sulfadoxine-pyrimethamine or artemether-lumefantrine; 78 of these children developed recurrent parasitemia during the 42-day follow-up period. Initial msp2 genotyping identified 27 and 33 recrudescences by use of single-and consecutive-day sampling, respectively; in subsequent msp1 genotyping, 17 and 21 of these episodes, respectively, were still classified as recrudescences; these results indicate a similar sensitivity of the standard single-day PCR protocol-that is, 82% (27/33) and 81% (17/21), in both genotyping steps. Interpretation of PCR-adjusted results will significantly depend on methodology.