Professional Education

  • Doctor of Philosophy, Universidade De Coimbra (2009)

Stanford Advisors


All Publications

  • Mutations of GPR126 Are Responsible for Severe Arthrogryposis Multiplex Congenita AMERICAN JOURNAL OF HUMAN GENETICS Ravenscroft, G., Nolent, F., Rajagopalan, S., Meireles, A. M., Paavola, K. J., Gaillard, D., Alanio, E., Buckland, M., Arbuckle, S., Krivanek, M., Maluenda, J., Pannell, S., Gooding, R., Ong, R. W., Allcock, R. J., Carvalho, E. D., Carvalho, M. D., Kok, F., Talbot, W. S., Melki, J., Laing, N. G. 2015; 96 (6): 955-961


    Arthrogryposis multiplex congenita is defined by the presence of contractures across two or more major joints and results from reduced or absent fetal movement. Here, we present three consanguineous families affected by lethal arthrogryposis multiplex congenita. By whole-exome or targeted exome sequencing, it was shown that the probands each harbored a different homozygous mutation (one missense, one nonsense, and one frameshift mutation) in GPR126. GPR126 encodes G-protein-coupled receptor 126, which has been shown to be essential for myelination of axons in the peripheral nervous system in fish and mice. A previous study reported that Gpr126(-/-) mice have a lethal arthrogryposis phenotype. We have shown that the peripheral nerves in affected individuals from one family lack myelin basic protein, suggesting that this disease in affected individuals is due to defective myelination of the peripheral axons during fetal development. Previous work has suggested that autoproteolytic cleavage is important for activating GPR126 signaling, and our biochemical assays indicated that the missense substitution (p.Val769Glu [c.2306T>A]) impairs autoproteolytic cleavage of GPR126. Our data indicate that GPR126 is critical for myelination of peripheral nerves in humans. This study adds to the literature implicating defective axoglial function as a key cause of severe arthrogryposis multiplex congenita and suggests that GPR126 mutations should be investigated in individuals affected by this disorder.

    View details for DOI 10.1016/j.ajhg.2015.04.014

    View details for Web of Science ID 000355931200009

    View details for PubMedID 26004201

  • Differential Requirement for irf8 in Formation of Embryonic and Adult Macrophages in Zebrafish PLOS ONE Shiau, C. E., Kaufman, Z., Meireles, A. M., Talbot, W. S. 2015; 10 (1)


    Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. In particular, genetic tools to analyze the role of Irf8 in zebrafish macrophage development at larval and adult stages are lacking. We generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils and excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.

    View details for DOI 10.1371/journal.pone.0117513

    View details for Web of Science ID 000349122100063

    View details for PubMedID 25615614

  • The phosphate exporter xpr1b is required for differentiation of tissue-resident macrophages. Cell reports Meireles, A. M., Shiau, C. E., Guenther, C. A., Sidik, H., Kingsley, D. M., Talbot, W. S. 2014; 8 (6): 1659-1667


    Phosphate concentration is tightly regulated at the cellular and organismal levels. The first metazoan phosphate exporter, XPR1, was recently identified, but its in vivo function remains unknown. In a genetic screen, we identified a mutation in a zebrafish ortholog of human XPR1, xpr1b. xpr1b mutants lack microglia, the specialized macrophages that reside in the brain, and also displayed an osteopetrotic phenotype characteristic of defects in osteoclast function. Transgenic expression studies indicated that xpr1b acts autonomously in developing macrophages. xpr1b mutants display no gross developmental defects that may arise from phosphate imbalance. We constructed a targeted mutation of xpr1a, a duplicate of xpr1b in the zebrafish genome, to determine whether Xpr1a and Xpr1b have redundant functions. Single mutants for xpr1a were viable, and double mutants for xpr1b;xpr1a were similar to xpr1b single mutants. Our genetic analysis reveals a specific role for the phosphate exporter Xpr1 in the differentiation of tissue macrophages.

    View details for DOI 10.1016/j.celrep.2014.08.018

    View details for PubMedID 25220463

  • Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes PLOS GENETICS Colombie, N., Gluszek, A. A., Meireles, A. M., Ohkura, H. 2013; 9 (6)


    In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.

    View details for DOI 10.1371/journal.pgen.1003562

    View details for Web of Science ID 000321222600038

    View details for PubMedID 23785300

  • Kebab: Kinetochore and EB1 Associated Basic Protein That Dynamically Changes Its Localisation during Drosophila Mitosis PLOS ONE Meireles, A. M., Dzhindzhev, N. S., Ohkura, H. 2011; 6 (9)


    Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.

    View details for DOI 10.1371/journal.pone.0024174

    View details for Web of Science ID 000294686100020

    View details for PubMedID 21912673

  • Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations BMC CANCER Preto, A., Goncalves, J., Rebocho, A. P., Figueiredo, J., Meireles, A. M., Rocha, A. S., Vasconcelos, H. M., Seca, H., Seruca, R., Soares, P., Sobrinho-Simoes, M. 2009; 9


    Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF(V600E) mutation. In BRAF(V600E) mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27(Kip1). Specific inhibition of BRAF by RNAi in cells with BRAF(V600E) mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF(V600E) mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.Our results in thyroid cancer cells, namely those harbouring BRAF(V600E) mutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF(V600E) mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.

    View details for DOI 10.1186/1471-2407-9-387

    View details for Web of Science ID 000271857000001

    View details for PubMedID 19878585

  • Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis JOURNAL OF CELL BIOLOGY Meireles, M., Fisher, K. H., Colombie, N., Wakefield, J. G., Ohkura, H. 2009; 184 (6): 777-784


    The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex.

    View details for Web of Science ID 000264774800002

    View details for PubMedID 19289792

  • Conventional and molecular cytogenetics of human non-medullary thyroid carcinoma: characterization of eight cell line models and review of the literature on clinical samples BMC CANCER Ribeiro, F. R., Meireles, A. M., Rocha, A. S., Teixeira, M. R. 2008; 8


    Cell lines are often poorly characterized from a genetic point of view, reducing their usefulness as tumor models. Our purpose was to assess the genetic background of eight commonly used human thyroid carcinoma models and to compare the findings with those reported for primary tumors of the gland.We used chromosome banding analysis and comparative genomic hybridization to profile eight non-medullary thyroid carcinoma cell lines of papillary (TPC-1, FB2, K1 and B-CPAP), follicular (XTC-1) or anaplastic origin (8505C, C643 and HTH74). To assess the representativeness of the findings, we additionally performed a thorough review of cytogenetic (n = 125) and DNA copy number information (n = 270) available in the literature on clinical samples of thyroid carcinoma.The detailed characterization of chromosomal markers specific for each cell line revealed two cases of mistaken identities: FB2 was shown to derive from TPC-1 cells, whereas K1 cells have their origin in cell line GLAG-66. All cellular models displayed genomic aberrations of varying complexity, and recurrent gains at 5p, 5q, 8q, and 20q (6/7 cell lines) and losses at 8p, 13q, 18q, and Xp (4/7 cell lines) were seen. Importantly, the genomic profiles were compatible with those of the respective primary tumors, as seen in the meta-analysis of the existing literature data.We provide the genomic background of seven independent thyroid carcinoma models representative of the clinical tumors of the corresponding histotypes, and highlight regions of recurrent aberrations that may guide future studies aimed at identifying target genes. Our findings further support the importance of routinely performing cytogenetic studies on cell lines, to detect cross-contamination mishaps such as those identified here.

    View details for DOI 10.1186/1471-2407-8-371

    View details for Web of Science ID 000264053500001

    View details for PubMedID 19087340

  • A microtubule interactome: Complexes with roles in cell cycle and mitosis PLOS BIOLOGY Hughes, J. R., Meireles, A. M., Fisher, K. H., Garcia, A., Antrobus, P. R., Wainman, A., Zitzmann, N., Deane, C., Ohkura, H., Wakefield, J. G. 2008; 6 (4): 785-795


    The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

    View details for DOI 10.1371/journal.pbio.0060098

    View details for Web of Science ID 000255368600015

    View details for PubMedID 18433294

  • Molecular and genotypic characterization of human thyroid follicular cell carcinoma-derived cell lines THYROID Meireles, A. M., Preto, A., Rocha, A. S., Rebocho, A. P., Maximo, V., Pereira-Castro, I., Moreira, S., Feijao, T., Botelho, T., Marques, R., Trovisco, V., Cirnes, L., Alves, C., Velho, S., Soares, P., Sobrinho-Simoes, M. 2007; 17 (8): 707-715


    Our aim was to characterize the molecular and genotypic profile of eight thyroid carcinoma-derived cell lines-TPC1, FB2, B-CPAP, K1, XTC-1, C643, 8505C, and Hth74-in order to use them as in vitro models of thyroid carcinogenesis.We evaluated the expression of five thyroid-specific genes (Tg, TSHr, TPO, PAX8, and TTF-1) to establish the cell lineage and to assess the differentiation status of each of the cell lines. We screened for mutations in the most relevant oncogenes/tumor suppressor genes affected in thyroid carcinogenesis: RAS, BRAF, CTNNB1, and TP53 along with RET/PTC rearrangements. Considering the putative relevance in general carcinogenesis, we have also studied other molecules such as EGFR, PI3K, RAF-1, and THRB. To determine the genetic identity of the cell lines, we performed genotypic analysis.The panel of cell lines we have studied displayed activation of several oncogenes (BRAF, RAS, RET/PTC) and inactivation of tumor suppressor genes (TP53) known to be important for thyroid carcinogenesis. Two of the cell lines-TPC1 and FB2-shared the same genotypic profile, probably representing clones of an ancestor cell line (TPC1).Due to their different molecular alterations, these cell lines represent a valuable tool to study the molecular mechanisms underlying thyroid carcinogenesis. We suggest that genotypic analyses should be included as a routine procedure to guarantee the uniqueness of each cell line used in research.

    View details for DOI 10.1089/thy.2007.0097

    View details for Web of Science ID 000249145300003

    View details for PubMedID 17725429

  • Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients' age but not with tumour aggressiveness VIRCHOWS ARCHIV Trovisco, V., SOARES, P., Preto, A., de Castro, I. V., Lima, J., Castro, P., Maximo, V., Botelho, T., Moreira, S., Meireles, A., Magalhas, J., Abrosimov, A., Cameselle-Teijeiro, J., Sobrinho-Simoes, M. 2005; 446 (6): 589-595


    A high prevalence of the BRAF(V600E) somatic mutation was recently reported in several series of papillary thyroid carcinomas (PTC). This mutation appears to be particularly prevalent in PTC with a predominantly papillary architecture. Another BRAF mutation (K601E) was detected in a follicular adenoma and in some cases of the follicular variant of PTC. The few studies on record provided controversial data on the relationship between the occurrence of BRAF mutations and clinicopathologic parameters such as gender, age and tumour staging. In an attempt to clarify such controversies we decided to enlarge our previous series to 315 tumours or tumour-like lesions diagnosed in 280 patients, including a thorough analysis of several clinicopathologic features. The BRAF(V600E) mutation was exclusively detected in PTC with a papillary or mixed follicular/papillary architecture both of the conventional type (46%) and of other histotypes, such as microcarcinoma (43%), Warthin-like PTC (75%) and oncocytic variant of PTC (55%). The BRAF(K601E) mutation was detected in four of the 54 cases of the follicular variant of PTC (7%). The mean age of patients with conventional PTC harbouring BRAF(V600E) (46.7 years) was significantly higher (P<0.0001) than that of patients with conventional PTC without BRAF(V600E) (29.5 years). The BRAF (BRAF(V600E)) mutated PTC did not exhibit signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) and were in fact less often multicentric than PTC without the mutation.

    View details for DOI 10.1007/s00428-005-1236-0

    View details for Web of Science ID 000229907600002

    View details for PubMedID 15902486