http://hdl.handle.net/10400.7/508 2020-03-07T13:47:41Z 2020-03-07T13:47:41Z Calarco, Joseph P. Borges, Filipe Donoghue, Mark T.A. Van Ex, Frédéric Jullien, Pauline E. Lopes, Telma Gardner, Rui Berger, Frédéric Feijó, José A. Becker, Jörg D. Martienssen, Robert A. http://hdl.handle.net/10400.7/708 2016-11-09T03:00:23Z 2012-09-28T00:00:00Z

Título: Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA Autor: Calarco, Joseph P.; Borges, Filipe; Donoghue, Mark T.A.; Van Ex, Frédéric; Jullien, Pauline E.; Lopes, Telma; Gardner, Rui; Berger, Frédéric; Feijó, José A.; Becker, Jörg D.; Martienssen, Robert A. Resumo: 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.

2012-09-28T00:00:00Z Borges, Filipe Gardner, Rui Lopes, Telma Calarco, Joseph P Boavida, Leonor C Slotkin, R Martienssen, Robert A Becker, Jörg D http://hdl.handle.net/10400.7/688 2017-12-06T19:14:10Z 2012-10-17T00:00:00Z

Título: FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei Autor: Borges, Filipe; Gardner, Rui; Lopes, Telma; Calarco, Joseph P; Boavida, Leonor C; Slotkin, R; Martienssen, Robert A; Becker, Jörg D Resumo: 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.

2012-10-17T00:00:00Z Gomes-Santos, Carina S S Itoe, Maurice A Afonso, Cristina Henriques, Ricardo Gardner, Rui Sepúlveda, Nuno Simões, Pedro D Raquel, Helena Almeida, António Paulo Moita, Luis F Frischknecht, Friedrich Mota, Maria M http://hdl.handle.net/10400.7/649 2016-06-15T02:00:18Z 2012-01-06T00:00:00Z

Título: Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes Autor: Gomes-Santos, Carina S S; Itoe, Maurice A; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D; Raquel, Helena; Almeida, António Paulo; Moita, Luis F; Frischknecht, Friedrich; Mota, Maria M Resumo: Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

2012-01-06T00:00:00Z Stankovic, Ana Jansen, Lars E.T. http://hdl.handle.net/10400.7/610 2016-05-19T02:00:14Z 2012-12-31T00:00:00Z

Título: Reductionism at the vertebrate kinetochore Autor: Stankovic, Ana; Jansen, Lars E.T. Resumo: The kinetochore forms the site of attachment for mitotic spindle microtubules driving chromosome segregation. The interdependent protein interactions in this large structure have made it difficult to dissect the function of its components. In this issue, Hori et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201210106) present a novel and powerful methodology to address the sufficiency of individual proteins for the creation of a functional de novo centromere.

2012-12-31T00:00:00Z Mata, João F. Lopes, Telma Gardner, Rui Jansen, Lars E. T. http://hdl.handle.net/10400.7/609 2016-05-19T02:00:17Z 2012-02-29T00:00:00Z

Título: A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones Autor: Mata, João F.; Lopes, Telma; Gardner, Rui; Jansen, Lars E. T. Resumo: 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.

2012-02-29T00:00:00Z