Cover Page
The handle http://hdl.handle.net/1887/45594 holds various files of this Leiden University dissertation
Author: Holst, Stephanie
Title: Glycomic signatures of colorectal cancer
Issue Date: 2017-01-24
A ppendix
L ist O f A bbreviAtiOns
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(2-)AA, 2-aminobenzoic acid (2-)AB, 2-aminobenzamide Ac, acetyl group
ACN, acetonitrile AFP, α-fetoprotein AUC, area under the curve CA, carbohydrate antigen
CCLE, Cancer Cell Line Encyclopedia CDX, caudal-related homeobox protein CE, capillary electrophoresis
CEA, Carcinoembryonic antigen CHCA, α-2,4-hydroxycinammic acid COX, cyclooxygenase
CRC, colorectal cancer CSC, cancer stem cells CV, cross-validation DC, dendritic cells
DC-SIGN, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin DHB, 2,5-dihydroxybenzoic acid
DMEM, Dulbecco's Modified Eagle DMSO, dimethyl sulfoxide
DR, death receptors DTT, Ddithiothreitol ECM, extracellular matrix
EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
EGF(R), epidermal growth factor (receptor) ELISA, enzyme-linked immunosorbent assay EMT, epithelial-to-mesenchymal transition ESI, electrospray ionization
EtOH, ethanol FBS, fetal bovine serum FCS, fetal calf serum
FFPE, formalin-fixed paraffin embedded FGF(R), fibroblast growth factor (receptor) Fig., figure
(i)FOBT, (immunological) faecal occult blood testing
FT-ICR, fourier transform ion cyclotron resonance Fuc, F, dHex, fucose
FUT, fucosyltransferases GAG, glycosylaminoglycans Gal, galactose
GalCer, galactosylceramides Galectin, galactose binding proteins GalNAc, N-acetylgalactosamine GALT, GalT, galactosyltransferase GALNT, N-acetylgalactosaminyltransferase GMDS, GDP-mannose-4,6-dehydratase Glc, glucose
GlcCer, glucosylceramides GlcNAc, N-acetylglucosamine
GNT, GnT, N-acetylglucosaminyltransferase GPI, glycosylphosphatidylinositol
GSL-glycan, glycosphingolipid derived glycan GT(s), glycosyltransferase(s)
GuHCl, guanidine hydrochloride H&E, hematoxylin and eosin
HBP, hexosamine biosynthetic pathway Hex, H, hexose
HexNAc, N, N-acetylhexosamine HIF, hypoxia inducible factor HILIC, hydrophilic interaction liquid
chromatography
HNF, hepatocyte nuclear factor HOBt, 1-hydroxybenzotriazole
hTERT-HPNE, immortalized human pancreatic duct epithelial-like cell line with human telomerase reverse transcriptase (hTERT)
IL, interleukin
IMDM, Iscove’s Modiied Dulbecco’s Medium ITO, indium-tin-oxide
LacNAc, N-acetyllactosamine LeX/A/Y/B, Lewis X/A/Y/B antigen LC, liquid chromatography
LacdiNAc, LDN, N,N-diacetyllactosediamine (GalNAcβ1,4GlcNAc)
LMS, leiomyosarcoma Man, mannose
MALDI-TOF, matrix-assisted laser desorption/
ionization time-of-flight
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MBP, mannan (mannose)-binding protein MCA, multilevel component analysis MET, mesenchymal-to-epithelial transition MFI, mean fluorescence intensity
MGAT3, mannosyl (beta-1,4-)-glycoprotein beta- 1,4-N-acetylglucosaminyltransferase MGAT4, mannosyl-(alpha-1,3-)-glycoprotein-β-
1,4-N-acetylglucosaminyltransferase
MGAT5, mannosyl (alpha-1,6-)-glycoprotein beta- 1,6-N-acetyl-glucosaminyltransferase MGL, macrophage galactose binding lectin MHC, major histocompatibility complex MMP, matrix metalloproteinase MRM, multiple reaction monitoring MS, mass spectrometry
MSCA, multivariate simultaneous component analysis
MS/MS, tandem mass spectrometry MSI, mass spectrometry imaging MSI, microsatellite instability
mQ, milli-Q, ultrapure water of "Type 1"
N, Asn, asparagine
NaHCO3, sodium bicarbonate Neu, neuraminidase
NeuAc, S, N-acetylneuraminic acid NeuGc, N-glycolylneuraminic acid NK cells, natural killer cells PCA, principal component analysis PaTu-S, Pa-Tu-8988S
PaTu-T, Pa-Tu-8988T
PDAC, pancreatic duct adenocarcinoma PG, prostaglandin
PGC, porous graphitized carbon PNGase F, Peptide N-Glycosidase F
PLS-DA, partial least squares-discriminant analysis PSA, prostate specific antigen
PVDF, polyvinylidene difluoride
rEGCase, recombinant endoglycoceramidase RIA, radioimmunoassay
RMS, root mean square ROC, receiver operating curve RP, reversed phase
RPMI, Roswell Park Memorial Institute RT, room temperature
s, sialyl- S, Ser, serine SA, Sia, sialic acid SD, standard deviation
SIGLEC, sialic acid-binding immunoglobulin-type lectins
S/N, signal-to-noise SPE, solid phase extraction ST, sialyltransferase Su, sulfate group
superDHB, 2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid (1:9 w/w) T, Thr, threonine
T antigen, Thomsen-Friedenreich antigen Tn antigen, Thomsen-nouvelle antigen TAM, tumor associated macrophages TFA, trifluoroacetic acid
TGF(R), transforming growth factor (receptor) TIC, total ion current
TIMP, tissue inhibitor of metalloproteinase TNF, tumor necrosis factor
TNM, tumor-lymph node-metastasis classification system
TRAIL(R), TNF-related apoptosis-inducing ligand (receptor)
UDP, uridine diphosphate UV-scaling, unit variance scaling
VEGF(R), vascular endothelial growth factor (receptor)
VIL1, vilin VIM, vimentin
VIP, variable importance in the projection ZEB, zinc finger E-Box binding homeobox
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Glycans form an essential part of the outer layer of a cell and are thus involved in cellular recognition and other biological processes such as cancer development and progression. Changes in the glycan phenotype have been recognized as a hallmark of cancer and have been the subject of many clinical (biomarker discovery) studies. Alterations in the glycome associated with colorectal cancer (CRC) have been reviewed in Chapter 1 of this thesis and the most common observations for N-, O-, and glycosphingolipid (GSL)-glycans can be summarized as: For N-glycans i) increased β1,6-branching and (poly-)N-acetyllactosamine extensions, ii) increase in pauci-mannosidic and high-mannose type N-glycans, iii) decrease of bisecting N-acetylglucosamine. For O-glycans: i) increase in core 1 structures, ii) increase in truncated structures such as (sialyl) T-antigen, (sialyl) Tn-antigen, iii) higher density of O-glycans. For GSL-glycans: i) decrease in disialylated structures, ii) decreased abundance of globo- type structures with exception of Gb3. Alterations observed in all three classes were increased sialylation and fucosylation as well as elevated levels of (sialyl) Lewis type antigens and type-2 chain glycans.
The analysis of GSL-glycans from CRC tumor and corresponding control tissues from the same patients described in Chapter 2 complemented a previous study on N-glycans from the same tissue samples. Whereas increased fucosylation and (sialyl) Lewis type structures seemed to be a typical indication of change common to different classes of glycans and to diverse cancers, sulfation for example, was found to be increased in N-glycans from colon tumor tissues, while it was reduced in GSL- glycan profiles from the same tissues. This both emphasizes the complexity of glycosylation in cancer and demonstrates its specificity — with important implications for clinical applications.
For the described study, GSL-glycans were extracted from tissue homogenates, thereby losing spatial information. In contrast, mass spectrometry imaging (MSI) is a growing technology which allows the spatial characterization of molecules within a tissue while keeping its structure intact. This permits to associate certain molecules with specific tissue features and facilitates to resolve tumor heterogeneity. In this context, Chapter 6 describes the development of an in-situ linkage-specific sialic acid derivatization by double amidation. This novel workflow has largely improved the MSI N-glycan analysis by stabilizing labile sialic acids which are prone to in-source/post-source decay and often cause biases in the glycan profiles. The linkage discrimination of sialic acids further enables a more reliable identification of cancer-associated sialyl Lewis antigens, thereby providing more insight into the glycobiology of the tissues. Analyzing CRC and leiomyosarcoma formalin-fixed paraffin-embedded tissues with this workflow highlighted the association between different N-glycans structures and tumor, stroma, necrotic, and/or collagen-rich tissue areas, with N-glycans carrying differently linked sialic acid showing distinctive distributions.
For functional studies, cell lines are essential model systems. However, we realized that the glycomic characterization of cell lines has been largely neglected, although their use as a glycobiological model system is routine. Moreover, most protocols for cellular glycomics use several million cells, which are not always available. Therefore, in Chapter 3, we developed a new, high-sensitive sample preparation method, which consists of the release of N-glycans from proteins, extracted from smaller number of cells (0.25-1x10E6) and immobilized on a PVDF-membrane, followed by their linkage-specific sialic acid derivatization, purification, and MALDI-TOF-MS analysis. Applying this method to a number of CRC cell lines revealed characteristic N-glycan profiles for each of the tested cell lines, making generalization and interpretation of in vitro experiments challenging. Comparison with the glycosylation from tissues, however, revealed a large overlap between the expressed complex type N-glycans – though in different ratios – demonstrating the potential of these CRC cell lines as models. One characteristic of the cellular N-glycome was the high abundance of high-mannose type glycans, which was partly due to intracellular precursors, but also due to their presence on the cell surface. In addition, we found an association of multi-fucosylated N-glycans and corresponding fucosyltransferases with CDX1 mRNA expression, a
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homeobox protein associated with differentiation and gut homeostasis. This association between high fucosylation and CDX1 mRNA was confirmed in a different set of CRC cell lines, described in Chapter 4. Interestingly, hepatocyte nuclear factors 4A and 1A — previously identified as regulators of antenna fucosylation — were found to be positively associated with high CDX1 mRNA expression, revealing new insights into potential regulatory mechanisms. We further expanded our research in Chapter 5 and characterized the N-glycan profiles of four pancreatic cancer cells in comparison to a normal pancreatic duct cell line. This has revealed pronounced differences between the normal and pancreatic cancer cells as well as between the cancer cells themselves, which were characterized by different metastatic behavior, and thus suggests that distinct mechanisms are involved.
In the presented cell line studies not only the potential of (CRC) cell lines as model systems was evaluated, but also the quality and robustness discussed. Differences in the cellular N-glycome resulting from culture conditions and origin need to be further investigated, and standardization as well as better characterization are fundamental to improve the use of cell lines as model systems.
Major progress in glyco(proteo)mics has been made during the last two decades, facilitating the analysis and expanding our understanding of the structure and function of glycans. Nevertheless, constant efforts are being made to identify glycan signatures in exploration of their potential as biomarkers for targeted applications in diagnostics, vaccination, treatments, and others. Likewise, improving the methods for quantitation and sensitivity is an ongoing process. The results of this thesis contribute to this progress by having developed new methods for the sensitive N-glycan analysis of cell line samples as well as intact tissue sections by MSI. The application of these methods has revealed new insights into (CRC) tumor glycobiology, but these need to be validated before they can ultimately be applied in the clinics. Furthermore, expanding the studies to other glycan classes, such as O-glycans, glycolipids, and/or glycosylaminoglycans, as well as combining different omics-technologies will aid in understanding both cancer glycobiology and the regulation of glycosylation, as it has been demonstrated that transcription factors as well as metabolic changes influence glycosylation. Chapter 7 discusses the role of glycans in the clinical setting, the potential of our findings, and future perspectives in the field of clinical glycomics.
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Glycanen vormen een belangrijk deel van de buitenkant van een cel en zijn betrokken bij cellulaire interacties en een verscheidenheid aan biologische processen. Veranderingen in het geheel aan glycanen (het glycoom) zijn erkend als indicator voor kanker en zijn daardoor onderwerp van veel klinische (biomarker identificatie) studies. In Hoofdstuk 1 van dit proefschrift worden met colorectaal carcinoom (CRC) geassocieerde modificaties van het glycoom besproken. De belangrijkste waarnemingen voor N-, O- en glycosfingolipide (GSL)-glycanen kunnen worden samengevat als: Voor N-glycanen i) een toename van β1,6-vertakkingen en poly-N-acetyllactosamine verlengingen, ii) een toename van pauci- mannosidic en high-mannose type N-glycanen, en iii) een afname van een extra N-acetylglucosamine (ofwel: een bisecting N-acetylglucosamine). Voor de O-glycanen geldt: i) een toename van Core 1 structuren, ii) een toename van verkorte structuren, zoals het (sialyl) T-antigen en het (sialyl) Tn-antigen, en iii) een toename van de hoeveelheid O-glycanen. Voor de GSL-glycanen geldt: i) een afname van di- gesialyleerde structuren, en ii) een afname van globo-type structuren met uitzondering van Gb3. In alle drie de klassen werden een verhoogde sialylering en fucosylering en een toename van (gesialyleerde) Lewis type antigenen en type-2 glycanen waargenomen.
De analyse van GSL-glycanen uit CRC tumorweefsel en uit naastgelegen controleweefsel van dezelfde patiënten, zoals beschreven in Hoofdstuk 2, vormt een aanvulling op een eerder onderzoek naar N-glycanen in dezelfde weefselmonsters. Terwijl verhoogde fucosylering en een toename van (gesialyleerde) Lewis type structuren kenmerkend lijken te zijn voor zowel verschillende soorten glycanen als voor verschillende tumoren, werd daarentegen in weefsel van CRC bij N-glycanen een verhoogde sulfatering en bij GSL-glycaan profielen een verlaagde sulfatering vastgesteld. Dit onderstreept zowel de complexe rol van glycosylering in kanker, als de specificiteit hiervan, wat van belang is voor de uiteindelijke toepassing in de kliniek.
Voor de beschreven studie werden de GSL-glycanen geëxtraheerd uit weefsel-homogenaten waardoor de ruimtelijke informatie verloren gaat. Massa spectrometrie imaging (MSI) is een vrij nieuwe onderzoeksmethode om de moleculaire samenstelling van weefsel te analyseren met behoud van de structuur. Met behulp van deze techniek kunnen bepaalde moleculen in gedefinieerde weefselgebieden geassocieerd worden met tumorheterogeniteit. In Hoofdstuk 6 wordt de ontwikkeling van een in-situ derivatisering van siaalzuren door middel van dubbele amidering ten behoeve van een verbindings- specifieke siaalzuur bepaling beschreven. Recente verbeteringen van de methode hebben geleid tot een verbeterde MSI N-glycan analyse door stabilisatie van labiele siaalzuren; deze zijn gevoelig voor verval tijdens de massaspectrometrische bepaling, hetgeen kan resulteren in een ongewenste verandering van glycaan profielen. Met de nieuwe methode kunnen de verschillende verbindingen van de siaalzuren worden aangetoond, wat tot een robuuste identificatie van carcinoom-geassocieerde sialyl Lewis antigenen leidt en het inzicht in de glycobiologie van de weefsels vergroot. Met de beschreven methode konden bij de analyse van formaline-gefixeerd paraffine-ingebed CRC en leiomyosarcoma weefsels voor verschillende N-glycanen associaties met tumor-, stroma-, necrotische- en collageenrijke weefselgebieden worden aangetoond; N-glycanen met andere siaalzuurverbindingen vertoonden eveneens karakteristieke distributies.
Cellijnen zijn essentiële modelsystemen, die vaak worden gebruikt bij glycobiologisch onderzoek. Wij hebben echter geconstateerd dat de glycoomtypering van cellijnen grotendeels achterwege is gebleven. Verder beschrijven de meeste methoden voor cellulaire glycomics het gebruik van enkele miljoenen cellen terwijl dit materiaal niet altijd beschikbaar is. Daarom is in Hoofdstuk 3 een methode ontwikkeld voor het vrijmaken van N-glycanen op een PVDF-membraan gevolgd door verbindings-specifieke siaalzuur derivatisering, zuivering en MALDI-TOF-MS analyse voor een kleiner aantal cellen (0.25-1x10E6). Toen deze methode werd toegepast op een reeks CRC cellijnen werd een grote verscheidenheid aan typische N-glycaan profielen geïdentificeerd, waardoor de interpretatie
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en generalisatie van in-vitro experimenten een uitdaging wordt. Anderzijds laat een vergelijking van deze resultaten met de resultaten verkregen van weefsels grote overeenkomsten zien tussen de expressie van complexe N-glycanen (hoewel met verschil in ratio’s), hetgeen de potentie van deze CRC cellijnen als modelsystemen laat zien. Kenmerkend voor het cellulaire N-glycoom was de verhoogde hoeveelheid van high-mannose glycanen, deels van intracellulaire oorsprong en deels verklaard door hun aanwezigheid op het celoppervlak. Daarnaast beschrijven wij een associatie tussen multi- gefucosyleerde N-glycanen, en daarmee verwante fucosyltransferases, met CDX1 mRNA expressie, een homeobox eiwit dat geassocieerd is met differentiatie en darm homeostase. Deze bevindingen werden bevestigd met een nieuwe reeks CRC cellijnen, zoals beschreven in Hoofdstuk 4. Hepatocyte nuclear factor 4A en 1A zijn eerder geïdentificeerd als regulatoren van antenne fucosylering en geassocieerd met een hoge CDX1 mRNA expressie, wat nieuw inzicht in een potentieel regelmechanisme verschaft.
In Hoofdstuk 5 werd ons onderzoek verder uitgebreid naar het karakteriseren van het N-glycaan profiel van vier pancreascarcinoom cellijnen in vergelijking met normale pancreas cellijnen. Hierbij werden uitgesproken verschillen geconstateerd tussen cellijnen met een verschillend metastase gedrag: een indicatie dat meerdere mechanismen hierbij een rol spelen.
In de besproken cellijnstudies is niet alleen de potentiële rol van (CRC) cellijnen als modelsystemen geëvalueerd, ook zijn de kwaliteit en robuustheid ervan belicht. Verschillen in het cellulaire N-glycoom veroorzaakt door kweekomstandigheden en/of herkomst van de cellen dienen verder te worden onderzocht. Voor het verbeteren van het gebruik van cellijnen als modelsystemen zijn standaardisatie en een verbeterde karakterisering hiervan vereist.
Ondanks dat er grote vooruitgang is geboekt in onze kennis over glycosylering en de ontwikkeling van technologieën voor glycaananalyses, blijft het onderzoeksveld sterk in beweging.
Er wordt voortdurend gewerkt aan het identificeren en analyseren van glycaanveranderingen en de toepasbaarheid hiervan in relatie tot vaccinaties en andere therapeutische en/of diagnostische doeleinden. Het verbeteren van de kwantificering en gevoeligheid van de technieken blijft echter een continu proces. De resultaten in dit proefschrift verschaffen vernieuwende methodes voor gevoelige N-glycaananalyse van cellijnen en analyse van intact weefsel met behulp van MSI.
Toepassing van deze methoden heeft nieuw inzicht in de glycobiologie van (CRC) tumoren opgeleverd, maar deze dienen verder geëvalueerd te worden voordat ze in de kliniek kunnen worden toegepast. Uitbreiding van onderzoek naar andere glycaanklassen – bijvoorbeeld de O-glycanen of de glycosylaminoglycanen – alsmede combinatie van verschillende omics-technologieën zal leiden tot een beter begrip van kanker gerelateerde glycobiologie en de regulatie van de glycosylering, omdat aangetoond is dat glycosylering door zowel metabole veranderingen als door transcriptiefactoren wordt beïnvloed. In Hoofdstuk 7 wordt de functie van glycanen vanuit klinisch perspectief besproken en wordt het belang van onze bevindingen en de perspectieven voor toekomstig onderzoek verkend.
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Stephanie Holst was born on the 6th of July 1987 in Kiel, Germany. Her interest in Biology started very early in life and she joined the Bachelor Program ‘Biotechnology and Process Engineering’ at the University of Applied Sciences in Flensburg, Germany in 2006. During her bachelor thesis project at Seracell Stem Cell Technology GmbH, Rostock, Germany, she optimized GMP-compliant quality control workflows using flow cytometry for the more accurate and faster analysis of the vitality of umbilical cord blood stem cells after cryopreservation. Stephanie graduated as a Bachelor of Science in March 2010.
In April 2010 she started the Master of Science program in 'Biotechnology' at the Beuth University of Applied Sciences in Berlin, Germany, from which she graduated cum laude in May 2012. In her research project at the Charité, Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Berlin, Germany, she learned various techniques in biochemistry and molecular biology as well as mass spectrometry of glycans which she successfully applied for the ‘Expression, purification and analysis of glyco-tagged variants of the growth factor erythropoietin’. The work for her master thesis on ‘Cell cycle-dependent studies on the uptake of nanoparticles’ was performed at the Max Planck Institute for Polymer Research, Mainz, Germany, in which she developed and applied new flow cytometry methods to study the uptake behaviors of cancer cells to develop targeted delivery systems for anti-cancer agents.
During her Master studies, Stephanie learned about glycosylation and was immediately enthusiastic.
In June 2012, Stephanie got the opportunity to start her PhD project on ‘Glycomic signatures of colorectal cancer’ in the Glycomics group of the Center for Proteomics and Metabolomics at the Leiden University Medical Center, Leiden, The Netherlands, under the supervision of Prof. André Deelder and Prof. Manfred Wuhrer. In this project she worked on method developments for cellular glycomics and tissue imaging using mass spectrometry and investigated colorectal cancer associated glycosylation changes and their potential for clinical application. In August 2016 she continued as Postdoctoral Researcher in the same group.
L ist O f P ubLiCAtiOns
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1. Holst S, Stavenhagen K, Balog CI, Koeleman CA, McDonnell LM, Mayboroda OA, Verhoeven A, Mesker WE, Tollenaar RA, Deelder AM and Wuhrer M (2013) Investigations on aberrant glycosylation of glycosphingolipids in colorectal cancer tissues using liquid chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). Mol Cell Proteomics 12:3081-93. doi:
10.1074/mcp.M113.030387.
2. Holst S, Wuhrer M and Rombouts Y (2015) Chapter Six - Glycosylation Characteristics of Colorectal Cancer. In: Richard RD and Lauren EB (eds) Advances in Cancer Research, Academic Press, London, UK pp. 203-256.
3. Bladergroen MR, Reiding KR, Hipgrave Ederveen AL, Vreeker GC, Clerc F, Holst S, Bondt A, Wuhrer M and van der Burgt YE (2015) Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification. J Proteome Res 14:4080-6.
doi: 10.1021/acs.jproteome.5b00538.
4. Powers TW, Holst S, Wuhrer M, Mehta AS and Drake RR (2015) Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry.
Biomolecules 5:2554-72. doi: 10.3390/biom5042554.
5. Holst S, Deuss AJ, van Pelt GW, van Vliet SJ, Garcia-Vallejo JJ, Koeleman CA, Deelder AM, Mesker WE, Tollenaar RA, Rombouts Y and Wuhrer M (2016) N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression. Mol Cell Proteomics 15:124-40. doi: 10.1074/mcp.M115.051235.
6. Sonneveld ME, Natunen S, Sainio S, Koeleman CA, Holst S, Dekkers G, Koelewijn J, Partanen J, van der Schoot CE, Wuhrer M and Vidarsson G (2016) Glycosylation pattern of anti-platelet IgG is stable during pregnancy and predicts clinical outcome in alloimmune thrombocytopenia. Br J Haematol 174:310-320, doi:10.1111/bjh.14053.
7. Holst S, Heijs B, de Haan N, van Zeijl RJ, Briaire-de Bruijn IH, van Pelt GW, Mehta AS, Angel PM, Mesker WE, Tollenaar RA, Drake RR, Bovee JV, McDonnell LA and Wuhrer M (2016) Linkage- specific in-situ sialic acid derivatization for N-glycan mass spectrometry imaging of FFPE tissues. Anal Chem. 88:5904-5913, doi:10.1021/acs.analchem.6b00819.
8. Heijs B, Holst S, Briaire-de Bruijn IH, van Pelt GW, de Ru AH, van Veelen PA, Drake RR, Mehta AS, Mesker WE, Tollenaar RA, Bovee JV, Wuhrer M and McDonnell LA (2016) Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section. Anal Chem. 88:7745- 7753, doi:10.1021/acs.analchem.6b01739.
9. Delannoy CP, Rombouts Y, Groux-Degroote S, Holst S, Coddeville B, Harduin-Lepers A, Wuhrer M, Elass-Rochard E and Guerardel Y (2016) Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages. J Proteome Res. doi: 10.1021/acs.jproteome.6b00161.
10. Holst S, van Pelt GW, Mesker WE, Tollenaar RA, Belo AI, van Die I, Rombouts Y and Wuhrer M (2017) High-throughput and high-sensitivity mass spectrometry-based N-glycomics of mammalian cells in High-Throughput Glycomics and Glycoproteomics. Methods in molecular biology 1503:185-196, doi:10.1007/978-1-4939-6493-2_14.
A CknOwLedgements
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I always thought that writing the acknowledgments is the best part of the thesis because I can finally thank all the people who taught me so much, who supported me and my dreams, and who endured a chronically busy person for the last years. And now, here I am, after four years of PhD which went by so fast, many, many people to thank and only 1.5 pages space for that – actually not such an easy task for someone who ‘tends to extensive writing’… but well, let’s try!
Firstly, I would like to express my sincere gratitude to you Manfred, my Promotor and supervisor, for giving me the opportunity to perform my research in your group, for your immense knowledge you’ve shared, your confidence in me as well as your great skill to shorten my too long articles. I came to Leiden due to my interest in glycans (Vielen Dank Herr Prof. Hinderlich) and my work here has proved to be a great choice. I have learned and grew a lot – personally and professionally – in these four years under your guidance and supervision, thank you very much!
My second Promotor, André, thank you for the very helpful discussions and your support for my research and thesis - It was a great pleasure and honor being under your guidance.
Yoann, my Co-Promotor, you are not only a great scientist, but also a great colleague who contributes to a happy and social atmosphere and always makes people smile. I learned a lot from you, especially on the more biological part, and I enjoyed our sometimes very long, but fruitful discussions and social events - thank you for everything!
Next, I also would like to thank my colleagues from the CPM, Parasitology, Department of Surgery, and Department of Pathology as well as all external collaborators and co-authors, and everyone who supported me during my time as a PhD student! I enjoyed a lot working in such an inter-disciplinary environment and to interact with so many different people. I would like to thank all of you for your assistance in the laboratory, solving the weirdest computer and MALDI problems only happening to me, rewarding discussions, and ideas and I apologize that I cannot specify everyone by name.
A special thanks to Carolien and Agnes – without you nothing would be possible in the lab, thank you for all your work and support! Also, I would like to thank Gerhild, Maurice, Dennis and Karli who helped me getting started during my first months with glycan releases, self-made cotton tips and mass spectrometry, as well as Bas and again Karli for support with data analysis and adapting all kinds of parameters and scripts for my ‘weird’ cell line data. To the Glyco-group: thanks for this sweet time together!
Manu, Agnes, and Kathrin, you are great colleagues and have become great friends. I enjoyed working with you and our tea/coffee breaks - you made the rainy times sunnier, thank you! Manu, an extra thanks for you and all your Converis and administration help!
In the last two years of my PhD I embraced a new challenge: mass spectrometry imaging. A new technique, new protocols, and a visit to Charleston where Prof. Rick Drake and Thomas Powers welcomed me at the MUSC. Rick, Thomas, thank you for teaching me and providing me with all knowledge I needed to set up the method at the LUMC and our ongoing collaboration. Of course, I could not have done that alone, and I received a lot of support from the CPM imaging group. Thank you for receiving me so kindly and especially thank you to Bram, Benjamin and Ricardo for patiently explaining to me so much I still needed and need to learn.
Ana, our story is quite special. Starting as collaborators it quickly became clear that our connection is something unique. While our supervisors (thank you Irma for this nice collaboration!) did not appreciate our common passion for overlong articles, working with you is a pleasure and our resulting friendship an even bigger one. I cannot tell you how grateful I am that we met. Collaborators, friends, and as this would not be enough, I am so lucky that you liked to spend your free time helping me with all the InDesign work and patiently adjusting all my changes! Muito obrigada!!!
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Daphne and Julia, as frightening as being alone in a new country is, you two literally danced into my life and made my start here in the Netherlands so much easier and nicer! Thank you for being my friends!
My best friends in Germany, Denise and Inke, many thanks for supporting me in my dreams, for long phone calls and visits, and for not giving up on our friendship even though we are not close in distance!
Lucila, Jorge, Felipe, and Alejo, muchas gracias por recibirme con tanto amor en su familia. Alejo, my second InDesign support and Cover-Designer – a special thanks to you for all your help and creativity!
Mi cariño, Juan, no sé cómo puedo darte las gracias. Encontrarte fue lo mejor que me pudo pasar.
Contigo me siento completa y feliz. Muchas gracias por apoyarme, por estar siempre conmigo cuando te necesito, por amarme y simplemente por ser tú mismo.
Mama und Papa, ich kann Euch nicht sagen wie dankbar ich Euch bin – für alles. Ihr habt mich unterstützt in allem was ich getan habe, habt mir Mut gegeben und an mich geglaubt. Eure Liebe ist immer bei mir, auch wenn ich hunderte Kilometer und 10 Umzüge weit weg bin.
Finally, to all my friends and family, thank you for your patience, understanding, and motivation – without you nothing would be as it is!
