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  • Bruno Vellutini 23:33 on 2014/04/29 Permalink
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    Ran assembly with test data from MaSuRCA. It worked smoothly.

     
  • Bruno Vellutini 20:17 on 2014/04/29 Permalink
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    Washed and fed the larvae of Lineus longissimus. In the old jar the pilidium larvae have a well-formed juvenile with eyes, it will soon settle. In the second jar the juveniles are visible, but in a much earlier stage.

    20140429_210448

    I will let these metamorphose and fix this pilidium with well-developed juveniles after nice from the second jar.

     
  • Bruno Vellutini 20:10 on 2014/04/29 Permalink
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    Mounted and took some pictures of Membranipora in situs Chema did. Pattern looks sharp and with no background in the early stages. evx is restricted to one ectodermal cell during early gastrulation and 2 large lateral vegetal cells get activated later and it ends in the midgut and hindgut in early larva.

    20140429_192744

     
  • Bruno Vellutini 17:01 on 2014/04/29 Permalink
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    Cloning Ttra frizzled, Nano hox1, dlx, wnts 

    Need to clone the remaining frizzleds from Terebratalia and wnts from Novocrania. Set overnight regular PCR with:

    Nano hox1 Nano wnt4 Nano wnt7 Nano dlx Ttra fz5/8 Ttra fz7

    30/04/14

    20140430_175703

    Unfortunately hox1 and the wnts did not work. I should change something in the PCR. I extracted dlx and frizzleds and set a ligation for 4h at RT.

    Transformed and plated the 3 genes and incubated at 37 °C.

    01/05/14

    Pic colonies for colony PCR. Run gel and set bacteria to grow.

    20140501_150448

    02/05/14

    Extracted minipreps and set sequencing PCR.

     
  • Bruno Vellutini 17:10 on 2014/04/28 Permalink
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    Fixed 20d Lineus. Larval epidermis is clearly visible in smaller embryos. More than half of the embryos had already hatched from the larval epidermis, specially fat embryos. I have relaxed the worms before fixation doing 30 min of MgCl2 in the cold room. Fixed in 4% FA diluted in magnesium chloride.

     
  • Bruno Vellutini 19:53 on 2014/04/15 Permalink
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    I took a look at Lineus longissimus pilidium larvae and observed the following.

    The larvae from the culture with stirring at 19 °C look the best. They are larger with food and more responsive. This can be due to the higher temperature. But I think stirring keeps the algae floating and optimizes the feeding. On the two large jars in the incubator there was a smoodge of Rhodomonas on the bottom while the bottom of the fritzen room was relatively clean.

    Larvae on the bowl looked ok at the naked eye (swimming and so), but at the scope they are small and with contracted lobes. Larvae on the beaker are better, but not as good as the large jars. The large jars in the incubator have happy larva, but they are smaller and seem to have gotten less food.

    I tried pipetting the bottom before sieving the beaker, but it was not effective. And maybe the larvae will try to eat the dirt! So for now it seems better to keep the bottom untouched.

    I used the filter of 50µm from FHL and the small or larger rubber tube (much faster) to remove half of the volume of the water. Replaced with UV filtered water at RT from our facility,

     
  • Bruno Vellutini 14:15 on 2014/04/15 Permalink
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    Looking again at the wild caught cocoons from Lineus viridis I realized that, in fact, the eggs were not advanced blastula, but zygotes! Since then they haven’ t started cleaving! The egg mass that was laid on the day that we put then in the cold room had only one embryo at early cleavage, the rest were still 1 cell.

    So, we don’t know the actual fertilization rate of wild cocoons! I put them back at 14 °C to see what happens. Hopefully they start cleaving.

     
  • Bruno Vellutini 11:37 on 2014/04/15 Permalink
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    Submitted the application to the EMBO Light sheet microscopy course.

    Here are my motivation letter and CV.

    Curriculum Vitae: Bruno Cossermelli Vellutini

    Bruno C. Vellutini
    Thormøhlens Gate 55
    5008 Bergen
    Norway

    Mobile: +47 4508-9464
    Email: bruno.vellutini@sars.uib.no

    Summary of online presence
    ==========================

    Research interests
    ==================

    • Evolutionary developmental biology of marine invertebrates
    • Body patterning of larvae and evolution of life cycles
    • Gene regulatory networks and developmental diversity
    • Biodiversity and science outreach

    Education
    =========

    • 2011-Today: PhD Student @ Sars International Centre for Marine Molecular

    Biology and University of Bergen. Bergen, Norway.

    • 2006-2008: MSc Student @ Department of Zoology of the Institute of

    Biosciences of the University of São Paulo and Marine Biology Center of
    University of São Paulo. São Paulo, Brazil.

    • 2001-2005: BSc Student @ Institute of Biosciences of the University of São

    Paulo, Brazil.

    PhD thesis
    ==========

    • Title: Comparative development of spiralian larvae.
    • Supervisor: Andreas Hejnol
    • Summary: I am investigating the embryonic origin and fate of larval tissues

    in bryozoans, brachiopods, and nemerteans using microscopy and gene
    expression patterns to better understand the developmental diversity of
    Spiralia.

    MSc thesis
    ==========

    • Title: Development and reproductive cycle of the sea biscuit _Clypeaster

    subdepressus_ (Echinodermata: Echinoidea) from São Sebastião, SP

    • Supervisor: Alvaro E. Migotto
    • Summary: I documented the embryonic, larval, and juvenile development of a

    sea biscuit with emphasis on the morphology of post-metamorphic juveniles.
    Histological analysis was conducted to evaluate the gonadal maturation of
    adults during an year.

    Work experience
    ===============

    Positions
    ———

    • Jan-Feb 2014: Visiting Scholar @ Dunn Lab, Department of Ecology and Evolutionary

    Biology, Brown University. Providence, RI, USA. [Gene expression profiling of
    priapulid development and phylogenomics of acoels.]

    • 2011-Today: PhD Student @ Sars Centre and University of Bergen. Bergen,

    Norway. [Comparative developmental biology of larval body patterns in
    different spiralian species. In situ hybridization, antibody staining,
    confocal imaging, transcriptome]

    • 2010-2011: Editor, programmer, and front-end developer @ Marine Biology

    Center of University of São Paulo. São Sebastião, SP, Brazil. [Cifonauta is a
    metadata-driven image database for marine biology photos and videos.
    Development is based on Python using Django web framework and PostgreSQL
    database.] http://cifonauta.cebimar.usp.br/

    • 2006-2008: MSc Student @ Department of Zoology of the Institute of

    Biosciences of the University of São Paulo and Marine Biology Center of
    University of São Paulo. São Paulo and São Sebastião, SP, Brazil [Research on
    the development of echinoderms. Artificial fertilization and maintenance of
    embryos and larvae of echinoderms, still and video documentation with optical
    microscopy, histological techniques for tissue analysis.]

    • 2007-2008: Co-organizer and web developer @ Marine Biology Center of

    University of São Paulo. São Sebastião, SP, Brazil. [Itinerant photographic
    exhibit of marine organisms.] http://www.usp.br/cbm/oceano/

    • 2001-2004: Intern Researcher @ Evolutive Histophysiology Laboratory,

    Institute of Biomedical Sciences of University of São Paulo. São Paulo, SP,
    Brazil. [Innate immune response of Tropical and Antarctic animals.]

    Courses
    ——-

    • 2013: Marine Models in Evolution and Development. EMBO Practical Course.

    Fiskebäckskil, Sweden. [4 credits]

    • 2012: 8th MIC Confocal Microscopy Course. Department of Biomedicine,

    University of Bergen. Bergen, Norway [24h]

    • 2010: Concepts and Model Organisms in Regenerative Biology. Universidad de

    Chile and Pontificia Universidad Catolica de Chile. Santiago, Chile.
    Satellite course of Latin American Society for Developmental Biology 2010
    meeting. [76h]

    • 2008: Mini-course theoretical-practical on image processing and embryo

    manipulation. Institute of Biomedical Sciences (ICB) and Heart Institute
    (INCOR), University of São Paulo. São Paulo, SP, Brazil. During the II
    Meeting of Developmental Biology Students. [8h]

    • 2006: Comparative Invertebrate Embryology (BIOL536). Friday Harbor

    Laboratories, University of Washington. Friday Harbor, WA, USA. Summer
    Session A. [9 credits]

    • 2006: Larval Biology (BIOL533). Friday Harbor Laboratories, University of

    Washington. Friday Harbor, WA, USA. Summer Session B. [9 credits]

    Conferences, posters, and presentations
    —————————————

    • 2014: Annual Meeting of the Society for Integrative and Comparative Biology

    (SICB). Austin, Texas. Beyond boundaries: expression of “segment polarity”
    genes during larval lobe development in brachiopods. [oral presentation]

    • 2012: Bergen Marine Invertebrate Symposium. Bergen, Norway.
    • 2012: Scalidophora meeting. Bergen, Norway.
    • 2012: 4th Euro Evo Devo meeting. Lisbon, Portugal. Germ cell development in

    non-spiralian lophotrochozoans: insights from a bryozoan and a brachiopod.
    [poster presentation]

    • 2010: V International Meeting of the Latin American Society for Developmental

    Biology. Santa Cruz, Chile. Juvenile development of the sea biscuit
    _Clypeaster subdepressus_ (Echinodermata: Clypeasteroida). [poster
    presentation]

    • 2010: Workshop on Marine Diversity, FAPESP. São Paulo, SP, Brazil.
    • 2008: II Meeting of Developmental Biology Students. São Paulo, SP, Brazil.
    • 2008: 8th Larval Biology Symposium. Lisbon, Portugal. Larval and juvenile

    development of the sea biscuit _Clypeaster subdepressus_ (Echinodermata:
    Clypeasteroida). [oral presentation]

    • 2008: XXVII Brazilian Congress of Zoology. Curitiba, PR, Brazil. Embryonic

    and larval development of the sea biscuit _Clypeaster subdepressus_
    (Echinodermata: Clypeasteroida). Metamorphosis and post-metamorphic
    development of the sea biscuit _Clypeaster subdepressus_ (Echinodermata:
    Clypeasteroida). [2x poster presentation]

    • 2007: III International Symposium of Developmental Biology. Uberaba, MG,

    Brazil. Life cycle of the sea biscuit _Clypeaster subdepressus_
    (Echinodermata: Clypeasteroida). [poster presentation]

    • 2007: I Meeting of Developmental Biology of University of São Paulo. Ribeirão

    Preto, SP, Brazil. Development of the sea biscuit _Clypeaster subdepressus_.
    [oral presentation]

    • 2005: 15th International Society of Developmental Biologists Congress.

    Sydney, Australia.

    • 2005: 2nd International Meeting of the Latin American Society for

    Developmental Biology. Guarujá, SP, Brazil.

    Teacher assistant
    —————–

    • 2007: Diversity and evolution of marine invertebrates (9200001). Marine

    Biology Center of University of São Paulo. São Sebastião, SP, Brazil. [2nd
    Semester, 6 credits]

    • 2005: Invertebrates I (BIZ0210). Institute of Biosciences of University of

    São Paulo. São Paulo, SP, Brasil. [2nd Semester, 8 credits]

    Honors, awards, and scholarships
    ——————————–

    • 2013: SICB Symposium Funding Support. Company of Biologists. Society for

    Integrative and Comparative Biology Annual Meeting in Austin TX, USA 2014.
    Symposium: The cell’s view of animal body plan evolution.

    • 2011-Today: PhD Research Fellow. Faculty of Mathematics and Natural Sciences.

    University of Bergen. Comparative development of spiralian larvae.

    • 2011: Winner, 3rd Place (Category 4: Photomicrographs). I Prêmio

    Fotografia-Ciência & Arte CNPq. Photomicrograph “Sea biscuit juvenile”
    http://www.cnpq.br/saladeimprensa/noticias/2011/0927b.htm

    • 2009: Winner, 5th Place. Nikon Small World Photomicrography Competition.

    Photomicrograph “Oral surface of a young sea star”
    http://nikonsmallworld.com/detail/year/2009/5

    • 2009: Honorable mention. Nikon Small World Photomicrography Competition.

    Photomicrograph “Pluteus larva of a sea biscuit”
    http://nikonsmallworld.com/detail/year/2009/32

    • 2008: Honorable mention. Olympus BioScapes Digital Imaging Competition.

    Photomicrograph “Larval skeleton of _Clypeaster subdepressus_
    http://www.olympusbioscapes.com/staticgallery/2008/hm58.html

    • 2007: Honorable mention, 2nd place. III Simpósio Internacional de Biologia do

    Desenvolvimento. Uberaba, MG, Brazil. Poster “Life cycle of the sea biscuit
    _Clypeaster subdepressus_”.

    • 2006-2008: MSc Scholarship. Fundação de Amparo à Pesquisa do Estado de São

    Paulo (FAPESP). 2006/01898-7. Development and reproductive cycle of the sea
    biscuit _Clypeaster subdepressus_ (Echinodermata: Echinoidea) from São
    Sebastião, SP.

    • 2005: ISDB Travel Sponsorship Package. Company of Biologists. 15th

    International Society of Developmental Biologists Congress in Sydney,
    Australia.

    • 2002-2004: Scientific Initiation Scholarship. Conselho Nacional de

    Desenvolvimento Científico e Tecnológico (PIBIC-CNPq). Characterization of
    the phagocytic activity of coelomocytes of the sea urchin _Lytechinus
    variegatus_ (Lamarck – 1816) in vivo and in vitro.

    Science Outreach
    —————-

    • 2010-Today: Cifonauta. Co-author, programmer, and front-end developer. CNPq.

    A metadata-driven image database for marine biology.
    http://cifonauta.cebimar.usp.br/

    • 2007-Today: Ocean: hidden life. Co-organizer and web developer. Fundo de

    Cultura e Extensão da Universidade de São Paulo. Itinerant photo exhibit of
    marine organisms. http://www.usp.br/cbm/oceano/

    Languages
    =========

    • Portuguese: Native language
    • English: Fluent [read/speak/write]

    Skills
    ======

    • Microscopy: light microscopy, confocal and SEM.
    • Imaging: photomicrography, video and timelapse, 3D reconstruction (ImageJ).
    • Molecular: gene cloning, in situ hybridization, antibody staining, transcriptome assembly.
    • Programming: python and bash, ImageJ macros, R (basics)}

    Journal articles
    ================

    Vellutini, B. C. & Migotto, A. E. Embryonic, larval, and juvenile development
    of the sea biscuit Clypeaster subdepressus (Echinodermata: Clypeasteroida).
    PLoS One 5, e9654 (2010). http://dx.doi.org/10.1371/journal.pone.0009654

    Fukuzawa, A. H. et al. The role of hemocytes in the immunity of the spider
    Acanthoscurria gomesiana. Dev. Comp. Immunol. 32, 716–725 (2008).
    http://dx.doi.org/10.1016/j.dci.2007.11.002

    Silva, J. R. M. C. da et al. Unspecific immune response of antarctic ectotermic
    animals under polar temperatures. Oecologia Brasiliensis 11, 110–121 (2007).
    http://www.ppgecologia.biologia.ufrj.br/oecologia/index.php/oecologiabrasiliensis/article/viewArticle/124

    Cunha da Silva, J. R. M. et al. Microscopical study of experimental wound
    healing in Notothenia coriiceps (Cabeçuda) at 0 degrees C. Cell Tissue Res.
    321, 401–410 (2005). http://dx.doi.org/10.1007/s00441-005-1139-z

    Da Silva, J. R. M. C. et al. Kinetics of induced wound repair at 0°C in the
    Antarctic fish (Cabeçuda) Notothenia coriiceps. Polar Biol. 27, 458–465 (2004).
    http://dx.doi.org/10.1007/s00300-004-0611-7

    Magazine articles & others
    ==========================

    Migotto, A. E. & Vellutini, B. C. Cifonauta, um banco de imagens para a
    Biologia Marinha. Boletim Informativo da Associação Brasileira de Biologia
    Marinha 4, 7–12 (2011).

    Vellutini, B. C. Bolacha no mar? Que história é essa? Ciência Hoje das Crianças
    204, 8–11 (2009).

    Lindner, A., Migotto, A. E., Vellutini, B. C. & Silva-Neto, I. D. Vida
    escondida. O Telescópio 2–3 (2008).

    Vellutini, B. C. & Migotto, A. E. A Sea Biscuit’s Life. (2008).
    doi:10.4016/8126.01

    Background & skills
    ===================

    I am a PhD student at the Sars Centre for Marine Molecular Biology associated
    to the Molecular and Computational Biology Research School of University of
    Bergen, Norway. My ultimate research goal is to understand how changes in
    development are related to the evolution of animal forms. I am currently
    focused on the evolution of development and larval body patterns of non-model
    marine invertebrates, such as bryozoans, brachiopods, and priapulids.

    My academic background is based on invertebrate zoology and comparative
    embryology. Throughout my master’s project I gained some experience with light
    microscopy documentation techniques of live embryos using photography, video,
    and time-lapse imaging. I also experimented with 3D reconstruction
    reconstruction of larvae which triggered my interest for image processing and
    programming.

    One of my goals as a doctorate student is to broaden my approaches for evo-devo
    by learning different molecular techniques and more advanced imaging methods. I
    am currently using light and confocal microscopy to compare the morphology and
    gene expression patterns between different brachiopod larvae and I am tracing
    the cell lineage of a bryozoan using recordings from a 4D light microscope.

    This course fits my immediate and future research interests in several aspects.
    Since embryogenesis is an interactive process, comprehending the dynamics of
    development is paramount to fully understand its underlying mechanisms. Thus, I
    am looking forward to learn how light sheet microscopy can provide a platform
    for scrutinizing developmental processes. Also, the possibility of integrating
    live imaging and gene expression patterns is truly attractive to me.

    Likewise, I am particularly interested in getting familiar with state-of-art
    methods for cell lineage reconstruction, including computational methods and
    open source microscopy setups, and in discussing how they can be applied to
    non-model organisms.

    I believe the course will provide solid grounds for light sheet microscopy that
    are aligned with my career goals. It will be a great opportunity to learn,
    collaborate, and get stimuli for new research ideas which will positively
    impact my future work.

    Proposed experiment
    ===================

    Spiral cleavage is the remarkably conserved iconic pattern of embryonic cell
    divisions and developmental fate map characteristic of a major branch of
    invertebrates, the Spiralia. In a typical spiralian embryo, the anteroposterior
    axis is established in the zygote while the dorsoventral axis is specified
    either by early asymmetric cleavage (unequally-cleaving species) or by cellular
    interactions at the 32-cell stage of equally-cleaving species. In the latter, a
    vegetal cell (macromere) intrudes the blastocoel and contacts the apical cells
    (micromeres). After the interaction, the macromere becomes an embryonic
    organizer and induces the dorsoventral polarity of the embryo from that moment
    onwards.

    Even though such key event in spiralian development was described almost forty
    years ago there is no real-time imaging depicting these cellular interactions.

    More interestingly, the dorsoventral axis determination in spiralians seems to
    be dependent on the mitogen-activated protein kinase (MAPK) signaling cascade.
    The MAPK pathway is active in the organizer cell in equally-cleaving molluscs
    and embryos treated with an inhibitor of the pathway become radialized (i.e.,
    fail to establish the dorsoventral axis).

    What is really happening at the onset of dorsoventral axis specification in
    terms of developmental dynamics? Can we further understand the role of MAPK
    pathway when comparing cellular interactions under normal and experimental
    conditions?

    Approaching these questions requires live imaging with a high spatial and
    temporal resolution. Thus, light sheet microscopy is a promising technique to
    accomplish these recordings with live spiralian embryos and reveal the
    mechanisms of dorsoventral axis specification of spiral cleavage.

    The experiment has two objectives:

    1. Describe the interactions between macromeres and micromeres during
    dorsoventral specification of a spiralian embryo.
    2. Compare normal development (above) to the developmental dynamics of embryos
    treated with an inhibitor of the MAPK pathway.

    An equally-cleaving annelid or mollusc where the macromere interaction is
    expected to occur would be the ideal animal model. However, I am excited to try
    these experiments with the bryozoan species that I am working with. Although
    there are logistic challenges that need to be solved like the transport and
    maintenance of the animals in sea water. A third alternative is using an
    unequally-cleaving spiralian (e.g., _Platynereis_) to verify the effects of
    MAPK inhibition which would focus the experiment on the second objective. The
    development in these animals is relatively fast and the recordings would only
    require early stages, so the comparison between different treatments could be
    done within the course period.

     
  • Bruno Vellutini 20:11 on 2014/04/14 Permalink
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    Wrote the motivation letter for the light sheet microscopy course.

     
  • Bruno Vellutini 12:12 on 2014/04/14 Permalink
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    Moved adult Lineus from the cold room back to the incubator at 14 °C to see if they start laying eggs.

     
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