Neonatal hyperbilirubinemia: in vivo characterization of ... - Simone Vodret - FINAL...

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    Neonatal hyperbilirubinemia: in vivo characterization of the mechanisms

    of bilirubin neurotoxicity and pharmacological treatments

    Simone Vodret

    A Thesis submitted in fulfilment of the requirements of the Open

    University (UK) for the degree of Doctor of Philosophy

    The Open University (UK)

    International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

    Director of the studies: Dr. Andrs Fernando Muro External supervisor: Dr. Libor Vitek

    29th November 2016

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    ACKNOWLEDGEMENTS

    I would like to thank Dr. Andrs Fernando Muro, the director of the study, for the

    opportunity to work in his lab. The shared knowledge and the constructive

    discussions over the last four years make me grow not only from the scientific point

    of view, but also as a person. I would also like to thank Dr. Giulia Bortolussi, for her

    patience in teaching and the assistance in performing experiments. Also without

    her, this work would not have been possible. I would like to thank the ICGEB for the

    financial support.

    My gratitude goes to Dr. Libor Vitek, my external supervisor, for the support and the

    discussions during our meeting and the preparation of the manuscript. I would like

    to thank Prof. Henkjan Verkade, for the successful collaboration. My

    acknowledgements also go to Jana Japrov and Andrea Schreuder, for the

    determination of tissue UCB and free bilirubin, respectively.

    I would also like to thank all the past and present colleagues of the Mouse Molecular

    Genetics lab, Fabiola Porro, Luka Bockor, Alessia De Caneva, Alessandra Iaconcig,

    Vipin Singh Rawat, Riccardo Seppia Sola, for the help and the laughs at the end

    of each Internal Internal Seminar session. My gratitude goes to the staff of the

    ICGEB animal facility, Willy De Mattia and Stefano Artico (also the captain of the

    ICGEB football team), for their help.

    I would like to thank Luca, for all the time and the books we shared, Zain, who

    provided me half of the lunches of the last year, Chiara, for her carelessness we all

    need in our life, and Andrea, who shared with me any given Sunday supporting

    Roma.

    I would like to thank my sisters, Cecilia and Caterina, for being together despite the

    distance, and Lucia and Carlo, for their support in the darkest moments. Thanks to

    my friends, Dino, Frinky, Miglio, Bob, Giorgio and Gino, you guys are my family.

    My gratitude goes also to Giulia, I am lucky to share my life with her.

    Finally, I would like to thank Arturo, without you there would not have been me.

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    TABLE OF CONTENTS

    ACKNOWLEDGEMENTS ................................................................................ 2

    TABLE OF CONTENTS ................................................................................... 3

    LIST OF FIGURES .......................................................................................... 7

    LIST OF TABLES ............................................................................................. 9

    ABBREVIATIONS .......................................................................................... 10

    ABSTRACT .................................................................................................... 12

    INTRODUCTION ........................................................................................... 14

    1.1 Bilirubin metabolism .................................................................................... 15

    1.2 UGT1 gene locus ........................................................................................ 17

    1.3 Bilirubin toxicity and disease ....................................................................... 18

    1.3.1 Hereditary causes of unconjugated hyperbilirubinemia......................... 18

    1.3.2 Non-genetic causes of unconjugated hyperbilirubinemia ...................... 19

    1.3.3 Neonatal jaundice ................................................................................. 20

    1.3.4 Management of hyperbilirubinemia ....................................................... 21

    1.3.5 Bf .......................................................................................................... 22

    1.3.6 Bilirubin-induced neurological dysfunction (BIND) ................................ 23

    1.3.7 Kernicterus ............................................................................................ 24

    1.4 Animal models to study hyperbilirubinemia ................................................. 24

    1.4.1 Non-genetic animal models of hyperbilirubinemia ................................ 25

    1.4.2 Gunn rat ................................................................................................ 25

    1.4.3 Ugt1-/- mouse model (gene disruption by a neomycin cassette) ........... 26

    1.4.4 Ugt1-/- mouse model (1 base deletion) .................................................. 27

    1.5 Standard therapeutic treatments for hyperbilirubinemia .............................. 30

    1.5.1 Phenobarbital treatment ........................................................................ 30

    1.5.2 Phototherapy ........................................................................................ 30

    1.5.3 Immunoglobulins ................................................................................... 31

    1.5.4 Exchange transfusion ........................................................................... 32

    1.6 Experimental treatments .............................................................................. 32

    1.6.1 Orlistat and ursodeoxycholic acid ......................................................... 33

    1.6.2 Human serum albumin .......................................................................... 33

    1.6.3 Minocycline ........................................................................................... 35

    1.6.4 Other treatments ................................................................................... 36

    1.7 Mechanisms of bilirubin neurotoxicity .......................................................... 37

    1.7.1 Neurodegeneration ............................................................................... 37

    1.7.2 Oxidative stress .................................................................................... 38

    1.7.3 ER stress .............................................................................................. 41

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    1.7.4 Neuroinflammation ................................................................................ 43

    1.7.5 Autophagy ............................................................................................. 45

    AIM OF THE THESIS .................................................................................... 47

    MATERIALS AND METHODS ....................................................................... 48

    2.1 Chemicals and standard solutions: .............................................................. 49

    2.2 Animals........................................................................................................ 50

    2.3 Genomic DNA extraction from mouse tail biopsies ..................................... 51

    2.4 Polymerase chain reaction protocol to detect and discriminate different genotypes .......................................................................................................... 51

    2.5 Animals treatments ...................................................................................... 52

    2.5.1 Phototherapy treatment ........................................................................ 53

    2.5.2 Minocycline treatment ........................................................................... 53

    2.5.3 Human serum albumin treatment .......................................................... 53

    2.5.4 Carbon tetrachloride treatment ............................................................. 53

    2.6 Biochemical analyses of plasma samples ................................................... 54

    2.6.1 Plasma total bilirubin measurement ...................................................... 54

    2.6.2 Free bilirubin measurement .................................................................. 54

    2.6.3 Plasma albumin measurement ............................................................. 55

    2.6.4 Aminotransferases ................................................................................ 55

    2.7 Tissue bilirubin measurement ...................................................................... 56

    2.8 Preparation of total RNA from the mouse cerebellum ................................. 57

    2.8.1 Quantification and quality control of RNA ............................................. 57

    2.8.2 Reverse transcription (RT) .................................................................... 57

    2.9 Quantitative real-time RT-PCR .................................................................... 58

    2.10 Preparation of total protein extracts ........................................................... 61

    2.11 SDS-PAGE and Western blot .................................................................... 61

    2.12 Histological analysis .................................................................................. 62

    2.12.1 Nissl staining ....................................................................................... 63

    2.12