Proteomic Techniques for the Discovery of Biomarkers associated with Uveal Melanoma and ...

445 Pages·2014·11.18 MB·English

by  Deirdre O' Flynn

Proteomic Techniques for the Discovery
of Biomarkers associated with Uveal
Melanoma and Cutaneous Melanoma
Disease Progression

Deirdre O‟Flynn

Ph.D 2014
Proteomic Techniques for the Discovery
of Biomarkers associated with Uveal
Melanoma and Cutaneous Melanoma
Disease Progression

A thesis submitted for the degree of Ph.D.
by
Deirdre O‟Flynn, B.A. (Mod.) Microbiology

Research work described in this thesis was performed under the
supervision of
Dr. Paula Meleady and Prof. Martin Clynes

School of Biotechnology
National Institute for Cellular Biotechnology
Dublin City University
May 2014
II
I hereby certify that this material, which I now submit for assessment on the
programme of study leading to the award of Ph.D. is entirely my own work, that I
have exercised reasonable care to ensure that the work is original, and does not to
the best of my knowledge breach any law of copyright, and has not been taken from
the work of others save and to the extent that such work has been cited and
acknowledged within the text of my work.

Signed: ____________________ ID No.: 55007607

Date:

III
This thesis is dedicated to my parents, Michael and Olive.

IV
ACKNOWLEDGEMENTS

I would first like to thank Prof. Martin Clynes and Dr. Paula Meleady for giving me
the chance to do this Ph.D. They have always been supportive, helpful, and
understanding, both in the lab and in life. Thanks so much to both of them for being
such great supervisors.

I would like to thank Mick “The Mass Spec Guru” Henry for all of the hours that he
spent helping and teaching me. And for all of those Saturdays he spent guiding me
through mass spec troubleshooting over the phone while trying to enjoy a round of
golf. I also owe so much gratitude to Annett! She taught me so much and never once
refused to help, all while being a good friend and wonderful scientist.

Thanks also to Paul Dowling for answering my 1,000,001 questions and for
constantly offering good scientific advice. I would also like to thank Clair Gallagher
for help and support with all of the apoptosis assays and Joanne Keenan for her
assistance in the ROS assays.

Thanks to Michael Moriarty and Vincent Lynch for their help, advice and expertise
in any clinical queries I had. I would like to thank Benvon Moran, Susan Kennedy
and Pathma Ramasamy for their help in clinical sample collections, characterisations
and general advice. Thanks also to all of the clinicians and patients involved in the
studies, without which, none of this work would have been possible.

Huge gratitude goes out to Colin Clarke. His (statistically) significant help and
positive attitude made writing the thesis so much easier. Thanks also to Stephen
Madden for his work on the microarray study and to Annemarie for her help with the
immunohistochemistry analysis.

Thanks to Gillian Smith and, previously, Joe Carey for their help in stores and the
prep room. I‟ll miss our little chats Gillian! Thanks also to Carol, Yvonne and
Geraldine in the office for all of their help with everything, particularly at the thesis
printing stage!

Thank you to all of my second floor comrades; Mark, Shane, Paul, Martin, Alan,
Andrew and Gemma. I might not miss the dodgy lunchtime conversations but I will
miss singing along to Whitney, the random frights, the sneaky coffees, and the chats!

Thanks so much to Karen and Trish, also known as Krish. They made the last year
bearable with their constant support, good chats and mostly, the bants which they
provided. Thanks also to Neil who reminded me that a pun can be worse than any
bad day in the lab.

Huge thanks go out to my parents who have always helped me through my education
and who have got me to where I am today. I appreciate every little thing that they‟ve
done for me and their support throughout my Ph.D. has been invaluable. I hope that
I‟ve made you both proud! Thanks also to Ed, “The Other Child”. He has always
helped me without question on any little technical issue, proof reading, and has even
fed me the best burritos and cookies I‟ve ever eaten.

V
Last but not least, thanks to Damian for being the best scientific advisor/proof
reader/steak chef/housekeeper/provider of quality TV shows/boyfriend a gal could
ask for! Through thick and thin, he has supported me more than he will ever know
and never once failed in helping me. Thank you from the bottom of my heart, Dr.
Damo.

VI
ABBREVIATIONS

2-D DIGE - 2-Dimensional Differential Gel Electrophoresis
2-D LC-MS/MS - 2-Dimensional Liquid Chromatography Tandem Mass Spectrometry
2-D PAGE - 2-Dimensional Polyacrylamide Gel Electrophoresis
AJCC - American Joint Committee on Cancer
Arp2/3 - Actin-Related Protein 2/3
AUC - Area Under the Curve
BACE-2 - Beta Secretase 2
BAP1 - BRCA1-associated protein-1
BRAF - V-raf Murine Sarcoma Viral Oncogene Homolog B1
BRB - Blood Retina Barrier
BSA - Bovine Serum Albumin
CAM - Cell-Cell Adhesion Molecules
CBS - Cerebrospinal Fluid
CDK - Cyclin-Dependent Kinase D
CDKN2A - Cyclin-Dependent Kinase Inhibitor 2A
CI - Confidence Interval
CTLA-4 - Cytotoxic T-lymphocyte Associated-Antigen 4
CXCL12 - C-X-C chemokine ligand 12
CXCR4 - C-X-C motif chemokine receptor 4
DAVID - Database for Annotation, Visualization and Integrated Discovery
DCF - 2‟,7‟-dichlorofluorescein
DCFH-DA - 2‟,7‟-dichlorofluorescein-diacetate
DMF - Dimethylformamide
DMO - Diabetic Macular Oedema
DTT - dithiothreitol
ECL - Enhanced Chemiluminescence
ECM - Extracellular Matrix
EDTA - Ethylenediaminetetraacetic Acid
EEF1G - Elongation Factor 1 Gamma
ELISA - Enzyme Linked Immunosorbent Assay
ESI - Electrospray Ionisation
FABP3 - Fatty Acid Binding Protein 3
FCS - Foetal Calf Serum
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FFPE - Formalin-fixed Paraffin Embedded Tissue
FGF2 - Fibroblast Growth Factor 2
GAPDH - Glyceraldehyde-3-Phosphate Dehydrogenase
GEO - Gene Expression Omnibus
GNA11 - Guanine nucleotide-binding protein G(q) subunit alpha-11
GNAQ - G(q) Subunit Alpha
GO - Gene Ontology
gp100 - Glycoprotein 100
GPCR - G-coupled receptor
HDAC - Histone Deacetylase
HDIL-2 - High-Dose Interleukin-2
HPLC - High Performance Liquid Chromatography
IEF - Isoelectric Focusing
IFNɣ - Interferon 1 Gamma
Ig - Immunoglobulin
IGF - Insulin-like growth factor
IGF-1R - Insulin-like growth factor 1 receptor
IHC - Immunohistochemistry
IMAC - Immobilised Metal Affinity Chromatography
IMS - Industrial Methylated Spirits
IPG - Immobiline pH Gradient
IQR - Interquartile Range
KPNB1 - Importin Subunit Beta 1
LC-MS - Liquid Chromatography – Mass Spectrometry
LDH - Lactate Dehydrogenase
LOH - Loss of heterozygosity
m/z - Mass to charge ratio
MALDI - Matrix-Enhanced Laser Desorption/Ionisation
MAPK/ERK - Mitogen-Activated Protein Kinase/Extracellular Signal-Related Kinase
MARS - Multiple Affinity Removal System
MDM2 - Murine Double Minute 2
MEK - Mitogen-Activated Protein Kinase Kinase
MIA - Melanoma inhibitory activity
MIP1α - Macrophage Inhibitory Protein 1 Alpha
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MMP - Matrix Metalloproteases
MRM - Multiple Reaction Monitoring
mRNA - Messenger RNA
MS - Mass Spectrometry
MS/MS - Tandem Mass Spectrometry
mTOR - Target of Rapamycin
MW - Molecular Weight
NPDR - Non-Proliferative Diabetic Retinopathy
NRAS - Neuroblastoma RAS viral (v-ras) oncogene homolog
P13K/AKT - Phosphoinositide 3-kinase/Protein Kinase B
PBS - Phosphate Buffered Saline
PD-1 - Programmed Death-1
PDR - Proliferative Diabetic Retinopathy
PEDF - Pigment Epithelium-Derived Factor
pI - Isoelectric point
PLCβ - β phospholipase C
PMT - Photo Multiplier Tube
PTEN - Phosphatase and Tensin Homolog
PVR - Proliferative Vitreoretinopathy
Rb - retinoblastoma tumour suppressor
RISC - RNA-induced silencing complex
RNAi - RNA Interfernce
RRD - Rhegmatogenous retinal detachment
S/N - Signal to Noise Ratio
S110B - S100 calcium binding protein B
SDS-PAGE - Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
SELDI - Surface Enhanced Laser Desorption/Ionisation
SELENBP1 - Selenium-Binding Protein
SF - Serum Free
SF3B1 - Splicing Factor 3B, subunit 1
SILAC - Stable Isotope Labelling with Amino Acids in Culture
siRNA - Small Interfering RNA
SPA - Sinapinic Acid
TBS - Tris-Buffered Saline
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TIC - Total Ion Chromatogram
TIMP-1 - Tissue inhibitor of metalloproteinase 1
TOF - Time of Flight
TPI1 - Triosephosphate isomerase
UHP - Ultrapure Water
UM - Uveal Melanoma
VEGF - Vascular Endothelial Growth factor
XC - X Correlation

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