Publications by authors named "Cláudia Bispo"

8 Publications

  • Page 1 of 1

Characterization of Coelomic Fluid Cell Types in the Starfish Using a Flow Cytometry/Imaging Combined Approach.

Front Immunol 2021 18;12:641664. Epub 2021 Mar 18.

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.
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http://dx.doi.org/10.3389/fimmu.2021.641664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013778PMC
March 2021

Isolation of Arabidopsis Pollen, Sperm Cells, and Vegetative Nuclei by Fluorescence-Activated Cell Sorting (FACS).

Methods Mol Biol 2017 ;1669:193-210

Instituto Gulbenkian de Ciência, Rua da Quinta Grande Nº6, 2780-156, Oeiras, Portugal.

Efficient methods to isolate highly purified Arabidopsis thaliana pollen and the subcellular components of the male gametophyte (the vegetative nucleus and two sperm cells) have enabled genome-scale studies revealing a highly dynamic reprogramming of the transcriptome and epigenome during pollen development. Here, we describe the isolation of uninucleate microspores, mature pollen, as well as sperm cells and vegetative nuclei by Fluorescence-Activated Cell Sorting.
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http://dx.doi.org/10.1007/978-1-4939-7286-9_16DOI Listing
May 2018

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

Genome-Wide Analysis of PAPS1-Dependent Polyadenylation Identifies Novel Roles for Functionally Specialized Poly(A) Polymerases in Arabidopsis thaliana.

PLoS Genet 2015 Aug 25;11(8):e1005474. Epub 2015 Aug 25.

Universität Potsdam, Institut für Biochemie und Biologie, Potsdam-Golm, Germany.

The poly(A) tail at 3' ends of eukaryotic mRNAs promotes their nuclear export, stability and translational efficiency, and changes in its length can strongly impact gene expression. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerases, PAPS1, PAPS2 and PAPS4. As shown by their different mutant phenotypes, these three isoforms are functionally specialized, with PAPS1 modifying organ growth and suppressing a constitutive immune response. However, the molecular basis of this specialization is largely unknown. Here, we have estimated poly(A)-tail lengths on a transcriptome-wide scale in wild-type and paps1 mutants. This identified categories of genes as particularly strongly affected in paps1 mutants, including genes encoding ribosomal proteins, cell-division factors and major carbohydrate-metabolic proteins. We experimentally verified two novel functions of PAPS1 in ribosome biogenesis and redox homoeostasis that were predicted based on the analysis of poly(A)-tail length changes in paps1 mutants. When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A)-tail length and transcript abundance in paps1 mutants in our analysis. This suggests that their coexpression reflects at least partly the preferential polyadenylation of these transcripts by PAPS1 versus the other two poly(A)-polymerase isoforms. Thus, transcriptome-wide analysis of poly(A)-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1. Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression.
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http://dx.doi.org/10.1371/journal.pgen.1005474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549238PMC
August 2015

Light depolarization measurements in malaria: A new job for an old friend.

Cytometry A 2015 May 23;87(5):437-45. Epub 2015 Mar 23.

Molecular Microbiology and Infection Unit, Instituto De Medicina Molecular, Faculdade De Medicina, Lisbon, Portugal.

The use of flow cytometry in malaria research has increased over the last decade. Most approaches use nucleic acid stains to detect parasite DNA and RNA and require complex multi-color, multi-parameter analysis to reliably detect infected red blood cells (iRBCs). We recently described a novel and simpler approach to parasite detection based on flow cytometric measurement of scattered light depolarization caused by hemozoin (Hz), a pigment formed by parasite digestion of hemoglobin in iRBCs. Depolarization measurement by flow cytometry was described in 1987; however, patent issues restricted its use to a single manufacturer's hematology analyzers until 2009. Although we recently demonstrated that depolarization measurement of Hz, easily implemented on a bench top flow cytometer (Cyflow), provided useful information for malaria work, doubts regarding its application and utility remain in both the flow cytometry and malaria communities, at least in part because instrument manufacturers do not offer the option of measuring depolarized scatter. Under such circumstances, providing other researchers with guidance as to how to do this seemed to offer the most expeditious way to resolve the issue. We accordingly examined how several commercially available flow cytometers (CyFlow SL, MoFLo, Attune and Accuri C6) could be modified to detect depolarization due to the presence of free Hz on solution, or of Hz in leukocytes or erythrocytes from rodent or human blood. All were readily adapted, with substantially equivalent results obtained with lasers emitting over a wide wavelength range. Other instruments now available may also be modifiable for Hz measurement. Cytometric detection of Hz using depolarization is useful to study different aspects of malaria. Adding additional parameters, such as DNA content and base composition and RNA content, can demonstrably provide improved accuracy and sensitivity of parasite detection and characterization, allowing malaria researchers and eventually clinicians to benefit from cytometric technology.
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http://dx.doi.org/10.1002/cyto.a.22659DOI Listing
May 2015

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells.

J Biotechnol 2013 Dec 24;168(4):436-9. Epub 2013 Oct 24.

IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica (ITQB), Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.
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http://dx.doi.org/10.1016/j.jbiotec.2013.10.020DOI Listing
December 2013