Publications by authors named "Telma Lopes"

13 Publications

  • Page 1 of 1

Notch/Her12 signalling modulates, motile/immotile cilia ratio downstream of in zebrafish left-right organizer.

Elife 2017 09 6;6. Epub 2017 Sep 6.

CEDOC, Chronic Diseases Research Centre, NOVA Medical School - Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal.

Foxj1a is necessary and sufficient to specify motile cilia. Using transcriptional studies and slow-scan two-photon live imaging capable of identifying the number of motile and immotile cilia, we now established that the final number of motile cilia depends on Notch signalling (NS). We found that despite all left-right organizer (LRO) cells express and the ciliary axonemes of these cells have dynein arms, some cilia remain immotile. We identified that this decision is taken early in development in the Kupffer's Vesicle (KV) precursors the readout being transcription. We demonstrate that overexpression of either or Notch intracellular domain (NICD) increases the number of immotile cilia at the expense of motile cilia, and leads to an accumulation of immotile cilia at the anterior half of the KV. This disrupts the normal fluid flow intensity and pattern, with consequent impact on expression pattern and left-right (L-R) axis establishment.
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http://dx.doi.org/10.7554/eLife.25165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608511PMC
September 2017

Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development.

Cell Rep 2017 05;19(7):1467-1478

Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland. Electronic address:

Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1) B cell development (pre-B cell, naive B cell, plasma cell), (2) antigen exposure (three structurally different proteins), and (3) four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size) extracted from antibody repertoire sequencing data (400 million reads). Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells) and antigen-driven (e.g., 40% for clonal diversity in plasma cells) predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.
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http://dx.doi.org/10.1016/j.celrep.2017.04.054DOI Listing
May 2017

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant Physiol 2016 08 29;171(4):2371-8. Epub 2016 Jun 29.

CIBIO/InBIO-Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, 4485-661 Vairão, Portugal (S.B., T.M.-C., J.G.G., M.S.); Instituto de Investigação e Inovação em Saúde, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal (I.C., A.L.G., P.D.);Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal (I.C., M.S.);Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal (R.G., T.L., C.A., C.B., N.P.M.);REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal (P.A., P.V.); andREQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal (I.M.V., J.A.R.)

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.
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http://dx.doi.org/10.1104/pp.16.01028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972299PMC
August 2016

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

Anal Chem 2016 Jan 6;88(2):1222-9. Epub 2016 Jan 6.

ETH Zurich , Department of Biosystems Science and Engineering, Bio Engineering Laboratory, Mattenstrasse 26, CH-4058 Basel, Switzerland.

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.
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http://dx.doi.org/10.1021/acs.analchem.5b03513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610554PMC
January 2016

Rmax: A systematic approach to evaluate instrument sort performance using center stream catch.

Methods 2015 Jul 4;82:64-73. Epub 2015 Mar 4.

Flow Cytometry Unit, Spanish National Cancer Research Center (CNIO), Madrid, Spain.

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument's sort count. Yield and Recovery are often neglected, mostly due to difficulties in their measurement. Purity of the sort product is often cited alone but is not sufficient to evaluate sorting performance. All of these three performance metrics require re-sampling of the sorted fraction. But, unlike Purity, calculating Yield and/or Recovery calls for the absolute counting of particles in the sorted fraction, which may not be feasible, particularly when dealing with rare populations and precious samples. In addition, the counting process itself involves large errors. Here we describe a new metric for evaluating instrument sort Recovery, defined as the number of particles sorted relative to the number of original particles to be sorted. This calculation requires only measuring the ratios of target and non-target populations in the original pre-sort sample and in the waste stream or center stream catch (CSC), avoiding re-sampling the sorted fraction and absolute counting. We called this new metric Rmax, since it corresponds to the maximum expected Recovery for a particular set of instrument parameters. Rmax is ideal to evaluate and troubleshoot the optimum drop-charge delay of the sorter, or any instrument related failures that will affect sort performance. It can be used as a daily quality control check but can be particularly useful to assess instrument performance before single-cell sorting experiments. Because we do not perturb the sort fraction we can calculate Rmax during the sort process, being especially valuable to check instrument performance during rare population sorts.
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http://dx.doi.org/10.1016/j.ymeth.2015.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503806PMC
July 2015

Independent recruitment of Igh alleles in V(D)J recombination.

Nat Commun 2014 Dec 17;5:5623. Epub 2014 Dec 17.

Epigenetics and Soma Laboratory, Instituto Gulbenkian de Ciência, Rua da Quinta Grande, n° 6, 2780-156 Oeiras, Portugal.

How the vast majority of B cells express only one of the two alleles at their immunoglobulin loci remains a biological puzzle. Here, in mice reconstituted with a single haematopoietic stem cell, we demonstrate that each of the two immunoglobulin heavy chain (Igh) alleles has a similar probability to be the first to undergo V(H) to DJ(H) rearrangement. We also observe this similar probability in clones from multipotent and common lymphoid precursors. The extreme biases in the expression of the alleles that we find in more differentiated subsets are mostly due to constraints imposed by early rearrangements. Our data demonstrate that each of the two Igh alleles in a B cell behaves independently of the other, up to the moment when a successful rearrangement in one allele triggers a feedback mechanism that prevents further recombination.
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http://dx.doi.org/10.1038/ncomms6623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351640PMC
December 2014

Expression profile of microRNAs regulating proliferation and differentiation in mouse adult cardiac stem cells.

PLoS One 2013 17;8(5):e63041. Epub 2013 May 17.

Instituto Gulbenkian de Ciência, Oeiras, Portugal.

The identification of cardiac cells with stem cell properties changed the paradigm of the heart as a post mitotic organ. These cells proliferate and differentiate into cardiomyocytes, endothelial and vascular smooth muscle cells, providing for cardiac cell homeostasis and regeneration. microRNAs are master switches controlling proliferation and differentiation, in particular regulating stem cell biology and cardiac development. Modulation of microRNAs -regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnologic and therapeutic applications. To obtain insights into the regulatory networks active in cardiac stem cells, we characterized the expression profile of 95 microRNAs with reported functions in stem cell and tissue differentiation in mouse cardiac stem cells, and compared it to that of mouse embryonic heart and mesenchymal stem cells. The most highly expressed microRNAs identified in cardiac stem cells are known to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. We report a subset of differentially expressed microRNAs that are proposed to act as regulators of differentiation and proliferation of adult cardiac stem cells, providing novel insights into active gene expression networks regulating their biological properties.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063041PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656880PMC
December 2013

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei.

Plant Methods 2012 Oct 17;8(1):44. Epub 2012 Oct 17.

Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156, Oeiras, Portugal.

Unlabelled:

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis.

Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure.

Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.
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http://dx.doi.org/10.1186/1746-4811-8-44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502443PMC
October 2012

Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA.

Cell 2012 Sep 20;151(1):194-205. Epub 2012 Sep 20.

Howard Hughes Medical Institute-Gordon and Betty Moore Foundation, Watson School of Biological Sciences, Cold Spring Harbor Laboratory, NY 11724, USA.

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.
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http://dx.doi.org/10.1016/j.cell.2012.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697483PMC
September 2012

A rapid FACS-based strategy to isolate human gene knockin and knockout clones.

PLoS One 2012 29;7(2):e32646. Epub 2012 Feb 29.

Instituto Gulbenkian de Ciência, Oeiras, Portugal.

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032646PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290580PMC
July 2012

Modulation of fungal sensitivity to staurosporine by targeting proteins identified by transcriptional profiling.

Fungal Genet Biol 2011 Dec 5;48(12):1130-8. Epub 2011 Oct 5.

IBMC - Instituto de Biologia Molecular e Celular, Portugal.

An analysis of the time-dependent genetic response to the death-inducer staurosporine was performed in Neurospora crassa by transcriptional profiling. Staurosporine induced two major genes encoding an ABC transporter and a protein with similarity to regulatory subunits of potassium channels. The transcriptional response is dependent on the activity of a novel transcription factor. Deletion mutants in differentially expressed genes displayed altered sensitivity to staurosporine, underscoring significant proteins involved in the response to the drug. A null-mutant of the ABC transporter (abc3) is extremely sensitive to staurosporine, accumulates more staurosporine than the wild type strain and is defective in energy-dependent export of the drug, indicating that the ABC3 protein is the first described staurosporine transporter. It was located in the plasma membrane by immunofluorescence microscopy. The combination of inhibitors of ABC transporters or of potassium channels with staurosporine leads to an enhanced activity against N. crassa and pathogenic fungi paving the way to the development of more potent and specific antifungals. Our results highlight the general use of transcriptional profiling for the identification of novel proteins involved in cell death and their potential use as drug targets.
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http://dx.doi.org/10.1016/j.fgb.2011.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230747PMC
December 2011

Mo(II) complexes: a new family of cytotoxic agents?

J Inorg Biochem 2010 Nov 17;104(11):1171-7. Epub 2010 Jul 17.

Departamento de Química e Bioquímica, CQB, Faculdade de Ciências, Universidade de Lisboa, Campo Grande 1749-016 Lisboa, Portugal.

Several molybdenum complexes, [Mo(η(3)-C(3)H(5))X(CO)(2)(N-N)] (N-N = 1,10-phenanthroline, phen: X = CF(3)SO(3)T1, X = Br B1, X = Cl C1; N-N = 2,2'-bipyridyl, X = CF(3)SO(3)T2, X = Br B2) and [W(η(3)-C(3)H(5))Br(CO)(2)(phen)] (W1) have been synthesized and characterized. Their antitumor properties have been tested in vitro against human cancer cell lines cervical carcinoma (HeLa) and breast carcinoma (MCF-7) using a metabolic activity test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT), leading to IC(50) values ranging from 3 to 45 μM, approximately. Most complexes exhibited significant antitumoral activity. Complexes B1 and T2 were chosen for subsequent studies aiming to understand their mechanism of action. Cellular uptake of molybdenum and octanol/water partition assays revealed that both B1 and T2 exhibit a selective uptake by cells and intermediate partition coefficients. The binding constants of B1 and T2 with ct DNA, as determined by absorption titration, are 2.08 (± 0.98) × 10(5) and 3.68 (± 2.01)x 10(5)M(-1), respectively. These results suggest that they interact with DNA changing its conformation and possibly inducing cell death, and may therefore provide a valuable tool in cancer chemotherapy.
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http://dx.doi.org/10.1016/j.jinorgbio.2010.07.006DOI Listing
November 2010

[Pulmonary epithelioid hemangioendothelioma - rarity, diagnosis and treatment difficulties].

Rev Port Pneumol 2009 Nov-Dec;15(6):1167-74

Centro Hospitalar de Lisboa Norte (CHLN) - Hospital de Pulido Valente, Alameda das Linhas de Torres, Lisboa.

The authors report a case of a primary pulmonary epithelioid haemangioendothelioma (EHE) in a 51 year-old man, a mechanic, who complained of a dry cough followed by constitutional symptoms and dyspnoea. Patient underwent a series of diagnostic exams including surgical biopsy and pulmonary tuberculosis was diagnosed. He was prescribed tuberculosis drugs for three weeks. Following clinical and imagiology deterioration, the case was reviewed by pathologists who concluded the pulmonary biopsy revealed an intermediate/high grade pulmonary EHE/angiosarcoma. The patient underwent three cycles of chemotherapy with carboplatin, etoposide and bevacizumab with no complications. He died seven months after onset of symptoms and seven weeks after definitive diagnosis. The authors wish to highlight the rarity of this pulmonary neoplasm and the importance of clinical suspicion, and the diagnosis and treatment difficulties in addition to the potential benefits of antiangiogenic drugs.
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http://dx.doi.org/10.1016/s0873-2159(15)30198-7DOI Listing
January 2010
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