cell activation Rho-GTPase activating protein

120 Pages·2017·3 MB·English

by  Brede Ruben Nilsen

Molecular characterization of T-
cell activation Rho-GTPase
activating protein

A protein encoded by multiple sclerosis
susceptibility gene

Fernanda Kropf Correia

Thesis submitted for the degree of Master of Science in
Molecular Bioscience
60 credits

Department of Bioscience Faculty of Mathematics and
Natural Sciences
UNIVERSITY OF OSLO
October / 2017
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Molecular characterization of T-cell
activation Rho-GTPase activating protein

A protein encoded by multiple sclerosis
susceptibility gene

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© Fernanda Kropf Correia
2017
Molecular characterization of T-cell activation Rho-GTPase activating protein
Fernanda Kropf Correia
http://www.duo.uio.no/
Trykk: Reprosentralen, Universitetet i Oslo
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Abstract
Multiple sclerosis is an autoimmune, inflammatory, demyelinating disorder that is believed to
be triggered by an association of genetic and environmental factors, such as smoking and
reduced levels of vitamin D. Genes within the MHC region encoding antigen-presenting
molecules account for the largest component of the genetic risk for MS. Moreover, genome-
wide association studies identified 200 MS risk variants outside the MHC II region. A
selection of these genes has been studied in the current thesis, CLEC16A, IL2RA, CD69, and
TAGAP, with emphasis on TAGAP. These genes have also been demonstrated to be associated
with other autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, Crohn’s
disease, and celiac disease. Furthermore, a number of MS associated genes have vitamin D
binding sites in their genomic region or respond to stimuli via the vitamin D receptor. In this
thesis, we have experimentally determined if CLEC16A, IL2RA, CD69, and TAGAP are
regulated by vitamin D in CD4+ T cells.
In addition, we further investigated the function and subcellular localization of T cell
activation Rho-GTPase activating protein (TAGAP) that is believed to be a member of the
Rho GTPase-activator protein superfamily, as all TAGAP isoforms have a conserved Rho-
GTPase domain. TAGAP is induced upon T cell activation, and due to the Rho-GTPase
domain, it is suggested to have a role in actin formation, cell motility, and subsequent
establishment of cell to cell contacts. Rho GTPases are also involved in TCR-mediated signal
transduction, playing an important role in T-cell development and activation.
The data from the current thesis indicates that vitamin D modulates the expression of IL2RA,
TAGAP, and CD69 in human CD4+ T cells. Moreover, it shows that TAGAP is located in the
cytosol of Jurkat cells, CD4+ T cells, and 293T cells. Preliminary results indicates that
TAGAP might have a role in actin polymerization.

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Acknowledgments
First, I would like to thank my supervisor, Tone Berge, for the guidance and inspiration,
Hanne Harbo, for the leadership example, and a thank you to everyone in the MS-research
group, specially Ina, Anna, Ingvild, Steffan, and Einar, for their help in the lab, their words of
encouragement, and for speaking English.
I would like to thank Ina again for letting me do her the favour of taking care of Kaos, for the
conversations, and for the glasses of wine. A very special thank you to Ingrid, with whom I
shared most of my time in the lab. Thank you for your concern, support, for sharing
frustrations, and for the songs we sang together.
Second, I would like to thank my family in Brazil for understanding my absence, and
encouraging my choices, even if they mean that I have to be far away. I would like to also
thank Jessica, Tabitha, Alice, and Ludmila, for accepting that WhatsApp became our bar
table, and for still sharing and being part of all of our moments.
I would like to thank Kristian, Casper, Verena, Jacob, Nils, and Mariella, for making life
away from home a lot easier. Further, a very special thank you for Cecilie, William, Edward,
and Sebastian for opening their home for me and making me feel part of the family.
Finally, thank you, Charles, for all the love and support. For listening, understanding,
comforting, and, specially, for cooking. I would not have managed without you.

Fernanda Kropf Correia

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List of contents

1 Introduction ........................................................................................................................ 1
1.1 Immune system ............................................................................................................ 1
1.1.1 Innate immunity ................................................................................................... 1
1.1.2 Adaptive immunity ............................................................................................... 2
1.1.2.1 B lymphocytes .................................................................................................... 2
1.1.2.2 T lymphocytes .................................................................................................... 2
1.1.2.2.1 T cell activation ............................................................................................... 4
1.2 The immune system and autoimmune diseases ........................................................... 5
1.3 Multiple sclerosis ......................................................................................................... 6
1.3.1 Multiple sclerosis and the immune system .......................................................... 7
1.3.2 Multiple sclerosis and environmental factors ....................................................... 8
1.3.3 Multiple sclerosis and genetics ............................................................................ 9
1.3.3.1 Vitamin D mechanisms and MS genetics ........................................................ 11
1.4 TAGAP ...................................................................................................................... 12
1.4.1 Genetic association with autoimmune disorders ................................................ 12
1.4.2 TAGAP expression ............................................................................................ 13
1.4.3 TAGAP function ................................................................................................ 13
1.5 Aims........................................................................................................................... 16
2 Methods ............................................................................................................................ 17
2.1 Human cell lines and primary cells ........................................................................... 17
2.1.1 Jurkat cells .......................................................................................................... 17
2.1.1.1 Freezing of Jurkat cells .................................................................................... 17
2.1.1.2 Thawing of Jurkat cells .................................................................................... 18
2.1.1.3 Cultivation of Jurkat cells ................................................................................ 18
2.1.2 293T cells ........................................................................................................... 18
2.1.2.1 Freezing of 293T cells ...................................................................................... 18
2.1.2.2 Thawing of 293T cells ...................................................................................... 19
2.1.2.3 Cultivation of 293T cells .................................................................................. 19
2.1.3 Cells from whole blood ...................................................................................... 20
2.1.3.1 Isolation of peripheral blood mononuclear cells (PBMCs) .............................. 20
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2.1.3.2 Isolation of CD4+ T cells .................................................................................. 21
2.2 Cell Count .................................................................................................................. 23
2.2.1 Counting cells in suspension .............................................................................. 23
2.2.2 Counting adherent cells ...................................................................................... 24
2.3 Cell treatment ............................................................................................................ 24
2.3.1 Activation and vitamin D treatment of CD4+ T cells ......................................... 24
2.3.2 Activation of Jurkat cells .................................................................................... 25
2.4 Cell transfection ......................................................................................................... 26
2.4.1 Transfection of Jurkat cells ................................................................................ 26
2.4.2.1 Preparation of adherent coverslips ................................................................... 28
2.4.2.2 293T cell transfection with Lipofectamine® .................................................... 28
2.5 Staining with fluorochrome ....................................................................................... 30
2.5.1 Actin staining of adherent cells .......................................................................... 30
2.5.2 Immunostaining for flow cytometry .................................................................. 31
2.5.2.1 Staining of cell surface markers for flow cytometry ........................................ 32
2.5.2.2 Intracellular immunostaining for flow cytometry using fluorochrome-
conjugated secondary antibody .................................................................................... 32
2.5.3 Immunostaining for confocal microscopy .......................................................... 34
2.5.3.1 Staining of non-adherent cells .......................................................................... 34
2.5.3.2 Staining of adherent cells ................................................................................. 35
2.6 Nucleic acid experiments ........................................................................................... 36
2.6.1 DNA maxiprep ................................................................................................... 36
2.6.1.1 Agarose gel electrophoresis ............................................................................. 39
2.6.1.2 Restriction enzyme digestion ........................................................................... 40
2.6.2 RNA extraction .................................................................................................. 40
2.6.3 NanoDrop ........................................................................................................... 42
2.6.4 Bioanalyzer ......................................................................................................... 42
2.7 Gene expression analyses .......................................................................................... 45
2.7.1 Complementary DNA (cDNA) synthesis ........................................................... 45
2.7.2 Polymerase chain reaction (PCR) ...................................................................... 46
2.7.3 Quantitative real-time PCR (qPCR) ................................................................... 47
2.8 Protein experiments ................................................................................................... 49
2.8.1 Protein extraction ............................................................................................... 49
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