19 research outputs found

    Alla ricerca delle basi molecolari del Diabete di Tipo 1: sequenziamento di ultima generazione in gemelli monozigoti concordanti e discordanti per la malattia

    Get PDF
    During my three years of PhD, I worked on a project which aims to clarify the molecular basis of Type 1 Diabetes (T1D), an autoimmune chronic disease showing a strikingly and, when compared to other Mediterranean regions, anomalously high incidence in the isolated founder population from Sardinia. The project, supervised by Prof. Francesco Cucca, involves the transcriptome, metilome and TCR repertoire analysis in CD4+ and CD8+ cells fromSardinian monozygotic twins (MZ), both concordant and discordant for T1D.The aim, during my PhD course, was to clarify how changes in transcriptional activity may contribute to the disease. CD4+ and CD8+ cells were collected from 3 concordant and 2 discordant Sardinian twin pairs, andtotal RNAwas isolated. The polyadenilated fraction was then sequenced and reads were mapped to the human genome, normalized, and scaled for library size and gene length. Gene expression levels were then quantified. Samples were sequenced with a mean coverage of ~60 millions of clusters, reads composition showed the expected behavior for PolyA(+) libraries. Nine genes were found differentially expressed between discordant MZ only in CD8+ cells. Results needs to be validated in a larger sample and further studied to confirm and understand the putative role of the 9 genes in T1D. The transcriptome, the metilome and TCR repertoire analysis in studies with MZ pairs concordant and discordant could contribute to the understanding of the pathogenesis of T1D

    The Forkhead Transcription Factor Foxl2 Is Sumoylated in Both Human and Mouse: Sumoylation Affects Its Stability, Localization, and Activity

    Get PDF
    The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues

    Esa Caves: training astronauts for space exploration

    Get PDF
    The first spaceflight was several decades ago, and yet extraterrestrial exploration is only at the beginning and has mainly been carried out by robotic probes and rovers sent to extraterrestrial planets and deep space. In the future human extraterrestrial exploration will take place and to get ready for long periods of permanence in space, astronauts are trained during long duration missions on the International Space Station (ISS). To prepare for such endeavours, team training activities are performed in extreme environments on Earth, as isolated deserts, base camps on Antarctica, or stations built on the bottom of the sea, trying to simulate the conditions and operations of space. Space agencies are also particularly interested in the search of signs of life forms in past or present extreme natural environments, such as salt lakes in remote deserts, very deep ocean habitats, submarine volcanic areas, sulphuric acid caves, and lava tubes. One natural environment that very realistically mimics an extraterrestrial exploration habitat is the cave. Caves are dark, remote places, with constant temperature, many logistic problems and stressors (isolation, communication and supply difficulties, physical barriers), and their exploration requires discipline, teamwork, technical skills and a great deal of behavioural adaptation. For this reason, since 2008 the European Space Agency has carried out training activities in the subterranean environment and the CAVES project is one of those training courses, probably the most realistic one. CAVES stands for Cooperative Adventure for Valuing and Exercising human behaviour and performance Skills, and is meant as a multidisciplinary multicultural team exploration mission in a cave. It has been developed by ESA in the past few years (2008-2011) and is open for training of astronauts of the ISS Partner Space Agencies (USA, Russia, Japan, Canada, and Europe). Astronauts are first trained for 5 days to explore, document and survey a karst system, then take on a cave exploration mission for 6 days underground. A team of expert cave instructors, a Human Behaviour and Performance facilitator, scientists and video reporters, ensure that all tasks are performed in complete safety and guides all these astronauts\u27 activities. During the underground mission the astronauts\u27 technical competences are challenged (exploring, surveying, taking pictures), their human behaviour and decision-making skills are debriefed, and they are required to carry out an operational programme which entails performing scientific tasks and testing equipment, similarly to what they are required to do on the ISS. The science program includes environmental and air circulation monitoring, mineralogy, microbiology, chemical composition of waters, and search for life forms adapted to the cavern environment. The CAVES 2012 Course will be explained and the first interesting scientific results will be presented

    Abstracts from the Food Allergy and Anaphylaxis Meeting 2016

    Get PDF

    ESA CAVES: TRAINING ASTRONAUTS FOR SPACE EXPLORATION

    No full text
    The first spaceflight was several decades ago, and yet extraterrestrial exploration is only at the beginning and has mainly been carried out by robotic probes and rovers sent to extraterrestrial planets and deep space. In the future human extraterrestrial exploration will take place and to get ready for long periods of permanence in space, astronauts are trained during long duration missions on the International Space Station (ISS). To prepare for such endeavours, team training activities are performed in extreme environments on Earth, as isolated deserts, base camps on Antarctica, or stations built on the bottom of the sea, trying to simulate the conditions and operations of space. Space agencies are also particularly interested in the search of signs of life forms in past or present extreme natural environments, such as salt lakes in remote deserts, very deep ocean habitats, submarine volcanic areas, sulphuric acid caves, and lava tubes. One natural environment that very realistically mimics an extraterrestrial exploration habitat is the cave. Caves are dark, remote places, with constant temperature, many logistic problems and stressors (isolation, communication and supply difficulties, physical barriers), and their exploration requires discipline, teamwork, technical skills and a great deal of behavioural adaptation. For this reason, since 2008 the European Space Agency has carried out training activities in the subterranean environment and the CAVES project is one of those training courses, probably the most realistic one. CAVES stands for Cooperative Adventure for Valuing and Exercising human behaviour and performance Skills, and is meant as a multidisciplinary multicultural team exploration mission in a cave. It has been developed by ESA in the past few years (2008–2011) and is open for training of astronauts of the ISS Partner Space Agencies (USA, Russia, Japan, Canada, and Europe). Astronauts are first trained for 5 days to explore, document and survey a karst system, then take on a cave exploration mission for 6 days underground. A team of expert cave instructors, a Human Behaviour and Performance facilitator, scientists and video reporters, ensure that all tasks are performed in complete safety and guides all these astronauts’ activities. During the underground mission the astronauts’ technical competences are challenged (exploring, surveying, taking pictures), their human behaviour and decision-making skills are debriefed, and they are required to carry out an operational programme which entails performing scientific tasks and testing equipment, similarly to what they are required to do on the ISS. The science program includes environmental and air circulation monitoring, mineralogy, microbiology, chemical composition of waters, and search for life forms adapted to the cavern environment. The CAVES 2012 Course will be explained and the first interesting scientific results will be presented

    Esa Caves: training astronauts for SPACE exploration

    No full text
    The first spaceflight was several decades ago, and yet extraterrestrial exploration is only at the beginning and has mainly been carried out by robotic probes and rovers sent to extraterrestrial planets and deep space. In the future human extraterrestrial exploration will take place and to get ready for long periods of permanence in space, astronauts are trained during long duration missions on the International Space Station (ISS). To prepare for such endeavours, team training activities are performed in extreme environments on Earth, as isolated deserts, base camps on Antarctica, or stations built on the bottom of the sea, trying to simulate the conditions and operations of space. Space agencies are also particularly interested in the search of signs of life forms in past or present extreme natural environments, such as salt lakes in remote deserts, very deep ocean habitats, submarine volcanic areas, sulphuric acid caves, and lava tubes. One natural environment that very realistically mimics an extraterrestrial exploration habitat is the cave. Caves are dark, remote places, with constant temperature, many logistic problems and stressors (isolation, communication and supply difficulties, physical barriers), and their exploration requires discipline, teamwork, technical skills and a great deal of behavioural adaptation. For this reason, since 2008 the European Space Agency has carried out training activities in the subterranean environment and the CAVES project is one of those training courses, probably the most realistic one. CAVES stands for Cooperative Adventure for Valuing and Exercising human behaviour and performance Skills, and is meant as a multidisciplinary multicultural team exploration mission in a cave. It has been developed by ESA in the past few years (2008–2011) and is open for training of astronauts of the ISS Partner Space Agencies (USA, Russia, Japan, Canada, and Europe). Astronauts are first trained for 5 days to explore, document and survey a karst system, then take on a cave exploration mission for 6 days underground. A team of expert cave instructors, a Human Behaviour and Performance facilitator, scientists and video reporters, ensure that all tasks are performed in complete safety and guides all these astronauts’ activities. During the underground mission the astronauts’ technical competences are challenged (exploring, surveying, taking pictures), their human behaviour and decision-making skills are debriefed, and they are required to carry out an operational programme which entails performing scientific tasks and testing equipment, similarly to what they are required to do on the ISS. The science program includes environmental and air circulation monitoring, mineralogy, microbiology, chemical composition of waters, and search for life forms adapted to the cavern environment. The CAVES 2012 Course will be explained and the first interesting scientific results will be presented

    FOXL2 interacts with sumoylation machinery, is sumoylated and co-localize with SUMO-1 and PIAS1.

    No full text
    <p><i>A., B.</i> FOXL2 interacts with PIAS1 and UBC9: COS-7 cells were co-transfected with pCRUZ-HA-FOXL2 and pCRUZ-myc-PIAS1 (A), or with pCRUZ-myc-FOXL2 and pCRUZ-HA-UBC9 (B) or with HA and myc empty vectors (−). Lysates were immunoprecipitated with anti-myc agarose conjugated antibody and analysed by western blotting with anti-FOXL2, anti-PIAS1 and anti-UBC9 antibodies. Mock IP consisted in an immunoprecipitation with only protein-A beads, without antibody. Expression of all proteins was also analysed in total lysates (input). <i>C.</i> FOXL2 is sumoylated in transfected COS-7 cells: COS-7 cells were transfected with pCRUZ-myc-FOXL2 (0.5 ”g) alone or with pCRUZ-HA-SUMO-1 (4.5 ”g), or with HA and myc empty vectors (−). Lysates were immunoprecipitated with anti-myc agarose conjugated antibody, and analysed by western blotting using anti-FOXL2 antibody (upper panel) or anti-SUMO-1 antibody (lower panel). Both antibodies recognized band(s) of about 105-160-kDa indicated by brackets (<b>[</b>), not present in the transfection of FOXL2 alone. <i>D.,E.</i> FOXL2 is sumoylated <i>in vivo</i> in physiological conditions: (D) Immunoprecipitation was done using anti-FOXL2 antibody on α-T31 cell lysate and the eluate analyzed by western blotting using anti-FOXL2 antibody (left panel) or anti-SUMO-1 antibody (right panel). Both antibodies recognized band(s) of about 105–160-kDa indicated by brackets (<b>[</b>). The asterisk indicates the FOXL2 signal from the previous hybridization. (E) 4-week old mouse ovaries were lysed with or without NEM, 60 ”g of protein was loaded on SDS PAGE, electrophoresed, and then immunoblotted. Western blotting with an anti-Foxl2 antibody showed a 45 kDa band corresponding to native Foxl2 and a slower migrating band of about 105–160 kDa, also recognised by anti-Sumo-1 antibody (not shown). <i>F.</i> FOXL2 and SUMO-1 co-localize in the nucleus: pCRUZ-myc-FOXL2 and pCRUZ-HA-SUMO-1 were co-transfected into COS7 cells, and immunofluorescence was performed using anti-myc and anti-HA antibody. In red (Alexa 633) is shown FOXL2, in green (Alexa 488) SUMO-1. The yellow colour indicates co-localization, and is particularly seen in spots resembling PML bodies. <i>G.</i> Wild type FOXL2 co-localizes with PIAS1 in the nucleus: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 and pCRUZ-HA-PIAS1. The intracellular distribution of FOXL2 (red) and PIAS1 (green) was detected by indirect immunofluorescence with mouse anti-myc and rabbit anti-HA primary antibodies and Alexa Fluor 633 anti-mouse and Alexa Fluor 488 anti-rabbit secondary antibodies. <i>H.</i> PIAS1 enhances FOXL2 sumoylation: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 and pCRUZ-HA-PIAS1. And lysed with or without NEM. Lysates were immunoblotted with anti-FOXL2 antibody and densitometric analysis was performed on sumoylated band using Image J software. Densitometric analysis is reported compared to that in lane 1.</p

    FOXL2 stability is increased by SUMO-1 and is not dependent on proteasome-mediated degradation.

    No full text
    <p><i>A.</i> Sumoylation regulates FOXL2 stability: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 (0.5 ”g) and increasing amounts of pCRUZ-HA-UBC9 or pCRUZ-HA-PIAS1(3.5–4.5 ”g) or pCRUZ-HA-SUMO-1 (4.5 ”g). Lysates treated with and without NEM were analysed by immunoblotting with anti-FOXL2 antibody. The bracket shows slower migrating bands of about 105–160 kDa. Densitometric analysis was performed on sumoylated band using Image J software and is reported compared to that in lane 1. <i>B.</i> FOXL2 stabilization is mediated by SUMO-1 in a dose-dependent manner: COS7 were transfected with 500 ng of pCRUZ-myc-FOXL2 and increasing amounts of pCRUZ-HA-SUMO-1 (0, 1, 2, 3, 4 ”g). Immunoblotting against myc shows that FOXL2 increases with the augmentation of SUMO-1. In the lower panel anti-actin is used as loading control. <i>C.</i> FOXL2 stability is not dependent on ubiquitination and proteasome-mediated degradation: COS7 cells were transfected with pCRUZ-HA-FOXL2 alone and with pCRUZ-HA-SUMO-1 in quadruplicate. After 12 h of transfection, cells were lysed (t0) or treated with cycloheximide (inhibitor of protein biosynthesis), cycloheximide and MG132 (proteasome-inhibitor) or neither. After 36 h cells were lysed and 50 ug of protein were blotted and hybridization performed with anti-myc antibody.</p
    corecore