University of Groningen Ex vivo fibrosis research: 5 mm closer to human studies Bigaeva, Emiliia

(1)

University of Groningen

Ex vivo fibrosis research: 5 mm closer to human studies

Bigaeva, Emiliia

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date:

2019

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

Bigaeva, E. (2019). Ex vivo fibrosis research: 5 mm closer to human studies. University of Groningen.

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

10

ABBREVIATIONS

AUTHOR AFFILIATIONS

CURRICULUM VITAE

LIST OF PUBLICATIONS

ACKNOWLEDGEMENTS

(3)

ABBREVIATIONS CHAPTER 10

10 ABBREVIATIONS

3D Three-dimensional

3R Refinement, reduction and replacement

ADAMTS A disintegrin and metalloproteinase with thrombospondin motifs ADF Advanced Dulbecco’s Modified Eagle Medium F-12

ALD Alcoholic liver disease ALK Activin-like kinase ANOVA Analysis of variance a-SMA Alpha-smooth muscle actin

ATP Adenosine triphosphate

BMP Bone morphogenic protein

BSA Bovine serum albumin

CCl4 Carbon tetrachloride

CD Crohn’s disease

cDNA Complementary deoxyribonucleic acid (DNA) CKD Chronic kidney disease

COL1A1 Collagen type I alpha 1

COPD Chronic obstructive pulmonary disease CSF1R Colony stimulating factor 1 receptor CTGF Connective tissue growth factor DAMPs Damage-associated molecular patterns DAPI 4',6-diamidino-2-phenylindole

DEG Differentially expressed gene DKD Diabetic kidney disease

DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide

ECM Extracellular matrix EGF Epithelial growth factor EGR1 Early growth response 1

ELISA Enzyme-linked immunosorbent assay EMT Epithelial-to-mesenchymal transition EndoMT Endothelial-to-mesenchymal transition ERK Extracellular signal-regulated kinase ESCs Embryonic stem cells

ESRD End-stage renal disease

FBS Fetal bovine serum

FC Fold change

FDR False discovery rate FGF Fibroblast growth factor FGFR Fibroblast growth factor receptor FLT3 Fms related tyrosine kinase 3

FN1 Fibronectin 1

FSGS Focal segmental glomerulosclerosis

GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFR Glomerular filtration rate

GTPase Guanosine triphosphatase H&E Hematoxylin and eosin HCC Hepatocellular carcinoma HIF1a Hypoxia-inducible factor 1 alpha HIOs Human intestinal organoids HMGB1 High mobility group box 1 HRFs Human renal fibroblasts HSCs Human stellate cells HSP47 Heat shock protein 47

IG Immunoglobulin

IL Interleukin

IPA Ingenuity pathway analysis IPF Idiopathic pulmonary fibrosis IPKB Ingenuity pathways knowledge base iPSCs Induced pluripotent stem cells

JAK1 Janus kinase 1

JNK c-Jun N-terminal kinase KHB Krebs-Henseleit buffer LOXL Lysyl oxidase-like enzyme

LPS Lipopolysaccharide

LXR Liver X receptor

MAPK Mitogen-activated protein kinase MCP Monocyte chemoattractant protein M-CSF Macrophage colony stimulating factor Mdr2KO Multidrug resistance protein 2 knockout (mice) MFI Mean fluorescent intensity

MMP Metalloproteinase

MPGN Membranoproliferative glomerulonephritis mRNA Messenger ribonucleic acid (RNA)

MSCs Mesenchymal stem cells

(4)

ABBREVIATIONS CHAPTER 10

10

mTOR Mechanistic target of rapamycin

NAFLD Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis

NF-kB Nuclear factor kappa light chain enhancer of activated B cells NGS Next generation sequencing

NOX Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase NRF2 Nuclear factor erythroid 2-related factor 2

OPG Osteoprotegerin

OPN Osteopontin

PAI Plasminogen activator inhibitor PAS Periodic acid Schiff

PC Principal component

PCA Principal component analysis PCIS Precision-cut intestinal slices PCKS Precision-cut kidney slices PCLS Precision-cut liver slices

PCOLCE Procollagen C-endopeptidase enhancer PCTS Precision-cut tissue slices

PDAC Pancreatic ductal adenocarcinoma PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor

PI3K/AKT phosphatidylinositol 3-kinase and Akt/Protein kinase B PLOD2 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PPAR Peroxisome proliferator-activated receptor

PSR Picro-Sirius Red

PVP Polyvinylpyrrolidone

RAAS Renin angiotensin aldosterone system RNA-Seq RNA sequencing

ROCK Rho-associated kinase ROS Reactive oxygen species

RPKM Reads per kilobase of transcript per million mapped reads

RT Room temperature

RTK Receptor tyrosine kinase

RT-qPCR Real-time quantitative polymerase chain reaction

RXR Retinoid X receptor

SAA1 Serum Amyloid A1

SD Standard deviation

SEM Standard error of mean

STAT3 Signal transducer and activator of transcription 3

TAK1 Transforming growth factor-associated kinase 1 TCE 2,2,2-trichloroethanol

TF Transcription factor

TGFb Transforming growth factor beta TGFbR Transforming growth factor beta receptor TIMP Tissue inhibitor of metalloproteinase TKI Tyrosine kinase inhibitor

TLDA Taqman low-density array TLR Toll-like receptor

TNF Tumor necrosis factor

TRAF6 Tumor necrosis factor receptor-associated factor 6

UC Ulcerative colitis

UUO Unilateral ureteral obstruction

UW University of Wisconsin (organ preservation solution) VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

WME Williams’ medium E

(5)

AUTHOR AFFILIATIONS CHAPTER 10

10 AUTHOR AFFILIATIONS

Bank, Ruud A.

Department of Pathology and Medical Biology, Division of Medical Biology

University Medical Center Groningen University of Groningen

The Netherlands

de Jong, Koert P.

Department of Hepato-Pancreatico-Biliary Surgery and Liver Transplantation University Medical Center Groningen University of Groningen

The Netherlands

Biel, Carin

Department of Pharmaceutical Technology and Biopharmacy

University of Groningen The Netherlands

Gore, Emilia

Boersema, Miriam

Jensen, Michael S.

Department of Clinical Medicine Aarhus University

Denmark

Bomers, Jordy J.M.

Department of Pharmaceutical Technology and Biopharmacy University of Groningen The Netherlands PROdermpath, Labor für Dermatohistopathology Vreden, Germany

Mutsaers, Henricus A.M.

Denmark

de Graaf, Inge A.M.

Department of Pharmacokinetics, Toxicology and Targeting

Leliveld, Anna M.

Department of Urology

The Netherlands

de Jong, Amos J.

Nicklin, Paul

Research Beyond Borders

Boehringer Ingelheim Pharma GmbH & Co. KG Biberach an der Riss, Germany

de Jong, Igle J.

Department of Urology

The Netherlands

Nørregaard, Rikke

Denmark

Oldenburger, Anouk

Cardiometabolic Disease Research

Seelen, Marc A.

Department of Internal Medicine, Division of Nephrology

The Netherlands

Olinga, Peter

Simon, Eric

Computational Biology

Oosterhuis, Dorenda

Stribos, Elisabeth G.D.

Piersma, Bram

The Netherlands

van Goor, Harry

Department of Pathology and Medical Biology, Division of Pathology

The Netherlands

Puerta Cavanzo, Nataly

The Netherlands

Wollin, Lutz

Respiratory Diseases

Rippmann, Jörg F.

Cardiometabolic Disease Research

Zwick, Matthias

Computational Biology

Boehringer Ingelheim Pharma GmbH & Co. KG Biberach an der Riss, German

Schlepütz, Marco

Respiratory Diseases

(6)

CURRICULUM VITAE CHAPTER 10

10 CURRICULUM VITAE

Emilia Bigaeva was born on May 23d_{, 1990 in Saint-Petersburg, Russia. She completed her secondary}

education at the Saint-Petersburg Gymnasium № 526, with special focus on biological sciences. From 2007 to 2012, she attended the Saint-Petersburg State Chemical Pharmaceutical Academy (later renamed to University), specializing in Pharmacy. During the Bachelor, she became particularly interested in toxicology, and towards the end of her studies, worked in a public pharmacy. After obtaining Bachelor’s degree in 2012, Emilia moved to Groningen, The Netherlands, to enter the Master program, Medical Pharmaceutical Sciences, at the University of Groningen. She conducted her Master’s research at the Department of Pharmacokinetics, Toxicology and Targeting under the mentorship of prof. Geny Groothuis. Under the supervision of prof. Angela Casini, she completed her first research project investigating biological properties of fluorescent metal-based complexes as potential anticancer agents. For her second research project, Emilia spent 6 months as a guest researcher in the group of prof. Dusica Maysinger at the McGill University in Montreal, Canada. She completed the project that aimed to evaluate the developmental toxicity of nanoparticles in murine limb bud culture. After obtaining her Master’s degree in 2014, she decided to continue in pharmaceutical research and joined the group of prof. Peter Olinga at the Department of Pharmaceutical Technology and Biopharmacy, University of Groningen. In this project, together with Emilia Gore, she investigated the use of animal and human precision-cut tissue slices for the development of antifibrotic compounds. Next to her research, Emilia was a member of the Groningen Graduate Interest Network and a member of the organizing committee of the 2015 PhD Day of the University of Groningen. After obtaining PhD degree, Emilia will continue her career in pharmaceutical/biomedical sciences.

(7)

LIST OF PUBLICATIONS CHAPTER 10

10 LIST OF PUBLICATIONS

Published

E. Bigaeva, J.J.M. Bomers, C. Biel, H.A.M. Mutsaers, I.A.M. de Graaf, M. Boersema, P. Olinga. Growth

factors of stem cell niche extend the life-span of precision-cut intestinal slices in culture: A proof-of-concept study. Toxicology in Vitro, 59, 312-321 (2019)

T. Luangmonkong, S. Suriguga, E. Bigaeva, M. Boersema, D. Oosterhuis, K.P. de Jong, D. Schuppan, H.A.M. Mutsaers, P. Olinga. Evaluating the antifibrotic potency of galunisertib in a human ex vivo model of liver fibrosis. British Journal of Pharmacology,174(18):3107-3117 (2017)

E. Bigaeva, E. Doorn, H. Liu, E. Hak. Meta-Analysis on Randomized Controlled Trials of Vaccines with

QS-21 or ISCOMATRIX Adjuvant: Safety and Tolerability. PLoS One,11(5):e0154757 (2016)

E. Bigaeva*_{, F.H. Paradis}*_{, A. Moquin, B.F. Hales, D. Maysinger. Assessment of the developmental}

toxicity of nanoparticles in an ex vivo 3D model, the murine limb bud culture system. Nanotoxicology, 9(6):780-91 (2015)

M. Wenzel, A. de Almeida, E. Bigaeva, P. Kavanagh, M. Picquet, P. Le Gendre, E. Bodio, A. Casini. New Luminescent Polynuclear Metal Complexes with Anticancer Properties: Toward Structure-Activity Relationships. Inorganic Chemistry, 55(5):2544-57 (2016)

M. Wenzel, E. Bigaeva, P. Richard, P. Le Gendre, M. Picquet, A. Casini, E. Bodio. New heteronuclear gold(I)-platinum(II) complexes with cytotoxic properties: are two metals better than one? Journal of

Inorganic Biochemistry, 141,10-16 (2014)

E. Bigaeva, O. Strelova, N. Kotova. Fluorimetric analysis of aqueous solutions of dibazol. Applied Analytical Chemistry, 4(1):26-33 (2013)

*_{these authors share first authorship} †_{these authors share last authorship}

(8)

LIST OF PUBLICATIONS CHAPTER 10

10 Submitted

E. Bigaeva, E. Gore, H.A.M. Mutsaers, D. Oosterhuis, R.A. Bank, M. Boersema, P. Olinga. Exploring

organ-specific features of fibrogenesis using murine precision-cut tissue slices.

E. Bigaeva*_{, E.G.D. Stribos}*_{, H.A.M. Mutsaers, B. Piersma, A.M. Leliveld, I.J. de Jong, R.A. Bank, M.A.}

Seelen, H. van Goor, L. Wollin, P. Olinga, M. Boersema. Inhibition of tyrosine kinase receptor signalling attenuates fibrogenesis in an ex vivo model of human renal fibrosis.

E. Gore, E. Bigaeva, A. Oldenburger, K. Yong Ook, J.F. Rippmann, D. Schuppan, P. Olinga, M. Boersema. PI3K inhibition reduces murine and human liver fibrogenesis in precision-cut liver slices.

E. Bigaeva*_{, E. Gore}*_{, E. Simon, M. Zwick, A. Oldenburger, M. Schlepütz, P. Nicklin, M. Boersema, J.F.}

Rippmann†_{and P. Olinga}†_{. Part I. Exploring organ-specific features of fibrogenesis using murine}

precision-cut tissue slices. Part II. Ex vivo human fibrosis model: characterization of healthy and diseased precision-cut tissue slices by next generation sequencing.

Manuscripts in preparation

E. Bigaeva*_{, N. Puerta Cavanzo}*_{, E.G.D. Stribos, A.J. de Jong, C. Biel, H.A.M. Mutsaers, M.S. Jensen, R.}

Nørregaard, A.M. Leliveld, I.J. de Jong, H. van Goor, M. Boersema, R.A. Bank, P. Olinga. Predictive value of precision-cut kidney slices as an ex vivo screening platform of antifibrotic therapies for human renal fibrosis.

E.Bigaeva*_{, C. Biel}*_{, J.J.M. Bomers, M. Teunis, J.P. ten Klooster, S. Vaessen, P. Olinga. Influence of}

organoid development supplements on the life-span of murine precision-cut tissue slices: a detailed examination

C. Biel*_{, E. Bigaeva}*_{, M.R. Hesse, J.J.M. Bomers, M. Teunis, J.P. ten Klooster, S. Vaessen, P. Olinga.}

Characterization of rat precision-cut intestinal slices in culture: exmination of main intestinal cell types E. Gore, E. Bigaeva, A. Oldenburger, D. Schuppan, M. Boersema, J.F. Rippmann, A. Broermann, P. Olinga. Investigating fibrosis and inflammation in an ex vivo NAFLD murine model.

E. Gore, E. Bigaeva, A. Oldenburger, Y. Ook Kim, J.F. Rippmann, D. Schuppan, M. Boersema, E. Simon†

and P. Olinga†_{. Thorough evaluation of the liver expression of GPNMB (Glycoprotein Nonmetastatic}

Melanoma Protein B) in murine and human liver diseases using precision-cut liver slices.

N. Puerta Cavanzo, E. Bigaeva, M. Boersema, P. Olinga, R.A. Bank. Macromolecular crowding as a tool to screen antifibrotic drugs: the Scar-in-a-Jar system revisited.

K.S.S. Putri, Adhyatmika, Suriguga, T. Luangmonkong, E. Bigaeva, E. Gore, F. van Dijk, Habibie, D. Oosterhuis, D. Schuppan, H.S. Hofker, K.P. de Jong, I.J. de Jong, P. Heukels, W. Timens, W.L.J. Hinrichs, B.N. Melgert, P. Olinga. Osteoprotegerin as a new marker to study early fibrosis in various organs. A. Oldenburger, Y. Liu, M. Fleck, E. Gore, E. Bigaeva, M. Baader, P. Olinga and J.F. Rippmann. Interference with hepatic myofibroblast expansion and fibrosis development by selective FGFR1-3 inhibition. E.G.D. Stribos, E. Bigaeva, S.A. Russel, A.H. Wolters, H.A.M. Mutsaers, M.A. Seelen, H. van Goor, P. Olinga, M. Boersema. Mitochondrial injury, oxidative stress and apoptosis evaluated in precision-cut kidney slices.

(9)

ACKNOWLEDGEMENTS CHAPTER 10

10 ACKNOWLEDGEMENTS

What a journey these last few years have been! I have to say, I do believe in destiny. I believe that everything we go through and everyone we meet during the course of our lives happens for a reason. So, if I think about it, it is actually not that very surprising that on the 27th August 2012 I arrived to Groningen. I decided to give science a chance and enrolled to the Master’s degree program of Medical Pharmaceutical Sciences. Almost 7 years later, here I am, ready to defend my PhD title, surrounded by brilliant people that now are a part of my journey. And I am grateful for the opportunity to write these few pages to thank them.

There is no other person I would like to thank first but my promotor Prof. Peter Olinga. It was September 2012 when the first master course started, and studying in English seemed very terrifying. Dear

Peter, you teaching the Drug Development course was a perfect beginning for this scary adventure! I am

beyond grateful to you for offering me the PhD position and the opportunity to join your research group. You hired not one, but two Emilias (!) for the same project and we never made it easy on you. Thank you for putting up with us these many years. Our meetings were never boring: we talked about science, we solved problems, we argued and you fired me and Emma like a million times. You let us complain about everything and taught us to be wiser and more patient. And I think I am now. Thank you for not allowing us to forget that “there is more to life than just lab work”, for insisting to add to the to-do item “finishing PhD on time” the words “with fun” (although neither of those came true), sending us outside Groningen for more experience and supporting us all the way. You value family above all and you take care of the group as your family. I am very fortunate to had you as my supervisor.

Handling me and Emma required some extra forces. I would like to thank my co-promotors Dr.

Miriam Boersema and Dr. Henricus A.M. Mutsaers for their active involvement in this research project.

Dear Miriam, you joined us when we were 2 years in, and I had no idea what to expect of having a postdoc as a second supervisor. I know now that I can’t imagine finishing this thesis without you! You brought so much knowledge and experience, and offered your advice the minute when any of us had a doubt or problem. Even at your new job, you still had time to help. You became our friend, whose opinion we will always value. I wish you and your beautiful family much happiness! (P.S. baby Lynn will always be baby Lynn). Dear Rick, we started to work closely together when I switched to kidney slices. Ever since then, I’ve got my kidney expert, editor and a travel companion! I am very grateful for improving my writing and meticulously correcting all “the” and “a” (such concept doesn’t exist in Russian so I’m particularly bad at articles). In fact, these pages are among the only few that were not edited by you. Our interaction was never uninteresting: you wear cool T-shirts, you have fascinating choice of music and a beer app on your phone. We had a great time in Chicago attending ASN conference (those insane breakfasts and suchi I’ll never forget) and you introduced me to the wonderful Aarhus. Much success in your future career and I hope little Colin will take after his father!

As I am a proud member of the Department of Pharmaceutical Technology and Biopharmacy, I would like to extend my deepest gratitude to Prof. Erik Frijlink. Dear Erik, from the first moments on, I always felt welcome in this department. You created a strong team that actually combines two very different research groups, and yet, everyone is surrounded by very friendly atmosphere. You seem to have endless energy and enthusiasm, and you manage million things at once. The fact that you successfully administrate the department and the Institute of Pharmacy says that you are an exceptional leader, so rule them all!

Like they say, behind every successful man there is a strong woman. In this department, it is Sonja

Graver. Dear Sonja, all this time you’ve been making our lives so much easier, taking care of everything we

might need! Thank you for having all the answers all the time.

I would like to sincerely thank the members of the reading committee, Prof. Jan-Luuk Hillebrands,

Prof. Klaas Nico Faber and Prof. Wladimiro Jiménez, for taking the time to read and evaluate this thesis.

Thank you for sharing this moment with me!

I think this is the moment when I have to thank Emilia Gore. Have I told you that I’ve never met anyone with my name before and I was quite proud of my unique (turns out, unique only in Russia) name? Then I arrive to Groningen just to find out that I’ll be doing my masters with a girl with my name! It was quite frustrating to be honest. Now that I’m over it (truly), I want to thank you for adopting your new (nick)name Emma at that first day 7 years ago when we met. Ever since, we’ve spent an insane amount of time together, it is almost unhealthy. Maybe it was for the best that Peter didn’t allow us to sit in the same office. We got our MSc degree on a same day, 4th September 2014, and now both of us are graduating on 12th July 2019. We shared this PhD project, shared all the same problems, countless meetings and presentations, weekends at work, and we celebrated our successes together. Emma, I’ve considered to place your picture here to remember your face forever, but luckily this idea quickly faded away. Jokes aside, I couldn’t wish for a better friend, colleague and a paranymph. Somehow “partner in crime” sounded too cliché, and I think accomplice fits us better. Thank you for sticking with me, because doing this PhD together was so much easier and so much more fun! We can talk for hours, share information (Nia’s words :p) and you are always my help hotline. It is a fact that we disagree with each other more often that we agree, but Iet me tell you that for a Russian, it’s a definition for a great friend :p. And if it happens that we fight, thank God there is Peter to flip a coin. We still don’t know what our near future looks like, but I’m sure we will be great friends forever!

I am lucky to be surrounded by so many wonderful colleagues, because nothing makes a day at work better than seeing their familiar friendly faces, and a cake at the coffee break of course. First and foremost, I would very much like to thank all members of Peter’s group: Bao Tung Pham, Dorenda Oosterhuis, Carin

Biel, Gerian Prins, Isabel Stribos, Jasper van Praagh, Konstanze Gier, Kurnia Sari, Louise van Wijk, Mitchel Ruigrok, Raditya Iswanda, Ruby Karsten, Suriguga, Theerut (Jo) Luangmonkong, Tobias van Haaften and Yvette Jansen. Guys, it’s been fantastic to work by your side and learn from you! Along the

ways, I’ve shared an office with most of you, because let’s face it, our office is the best.

Dorenda, my forever officemate, I have to say I’m always a bit jealous when people call our office “Dorenda’s

office” because I’ve been there for such a long time (haha). You are a heart (and hands and legs) of this group;

we wouldn’t survive in the lab without you. Everything seems so easy when you take care of things, no matter if it is merging the slicelabs, setting up a new staining lab or organizing Peter’s group event. You are a great listener, and always ready to help. I loved sharing the office with you and your perfectly beautiful face! I wish you, Teun and the girls all the great happiness! My “slice girls”, Isabel and Gerian, having you as my close friends was the best part of my “Dutch experience”! Thank you both for helping me with translating the summary, especially considering my last-minute fashion. Isabel, I consider the time when we worked together the best time of my PhD. We became friends so quickly and effortlessly that I got “kidney slice fever” instantaneously. You taught me a lot on the subject and we were a great, very efficient team in the

(10)

10

lab (especially when it comes to pipetting PCR plates :p). We had tons of fun outside work too, together

with Emma, Gerian and Su we created some precious memories. I’m happy that even after you moved away from Groningen, we keep our “slice girls group” alive and still do things, and I think next time Ed passes by the Netherlands, we should definitely go! I can’t wait to see the new house of you and Tjalling, and please visit Antarctica soon. I wish you great success in your specialty training because the world is about to get the best specialist! Gerian, you are special in many ways, and because we are different, we are close. Your social skills are beyond control, and you are a definition of “unusual working hours”. Sometimes I receive a message from you past midnight about things in the lab, another time you post a picture at 4 am with a capture “sunrise at umcg”. You keep weird things under your desk, you wear skirts in winter and you talk about bunnies all-the-time. And you will do everything for a friend, even if it’s inconvenient for you, and I can always rely on you. Thank you for that and thank you for laughing at my proposition #9, it shows that you are my person! Please take care of yourself, not that me and Emma will stop keeping an eye on you :p

When I just started my PhD, I got not one, but two best teachers –Jo and Radit. I must have been exhausting back then and asked you a million or two of questions, but you always treated me with patience and kindness, and I am truly grateful. Jo, it was my great pleasure to work with you. You are a talented researcher, hardworking and always cheerful! You taught me everything I know about Western blot and I tried to pass on that knowledge the best I could. We had lots of fun times with our small group, going to the PhD weekends, getting lost on Borkum island, having dinners together. Thank you for all the nice memories and I wish you and Gift all the best! Radit, thank you for helping me with countless mice experiments and taking your time to help me master intestinal slices. We often talked about religion and you made us see and understand a lot of things. You and Nia are perfect for each other, and we still once in a while talk about your magnificent wedding. Nia, we started our PhD projects at about the same time and it was truly joyful to learn together and share the lab. You were always ready to help even when you were the one who needed help in the lab. We travelled as far as Keystone in Colorado for a conference, where we tried very hard to keep up with the Dutch on skis. I don’t know who of the two of us fell into snow piles more, but it was definitely fun! Thank you for your eternal kindness. I’m looking forward celebrating your and Radit’s PhD degrees and I wish a lifetime of happiness to both of you and little Salman! Su, you brought a different flavour of Chinese and Mongolian culture to our group, and we spent great time together. We worked a lot in the lab and you were a constant member of “working on a weekend team”. You finished your PhD before me and Emma and I could never beat you in table tennis, but I forgive you for that :p Thank you for encouraging me to do a paining course. Now you are about to start a new adventure – being a mom, and I wish you, Naqin and the baby best of health and happiness!

Mitchel, you make our days at work that much more fabulous! I am inspired by your dedication;

I enjoy all our conversations and I would never be able to find a guy better dressed than you. I hope you will publish, what is it like tenth (?), paper, submit your thesis before the end of PhD contract, proving that the impossible is possible! Mitchel, shantay you stay :p Louise, you were by far not an ordinary technician: you came, you conquered (our hearts) and sadly you left. And you wrote The slicelab manual! It was great sharing an office with you and talking about all the random things. I hope you enjoy your new job and congratulations on the upcoming wedding! Yvette, we didn’t have the chance to work together, but it is great to see friendly and enthusiastic people like you around. I hope you will have best time working in

Peter’s group. Konstanze, my new officemate, you joined our group less than a year ago, but you fitted right in, ever so seamlessly! I was in the last months of writing this thesis when we got acquainted, and you offered me a lot of moral support. It is always fun watching new people getting to know the slicing world. You were very excited about slices at first, going around “this is so interesting”, “what else can we do” and I thought to myself “Koni in phase I”. With time you changed to “something is weird” (that was “Koni in phase

II”), and later I finally heard “f** slices!” and I said “welcome to the group” :). I hope your project with slices will

go smoothly and I wish you much success. Tobias and Jasper, it was really great to have you around, share office time, coffee time, candy time and above all, our not-so-much-love for intestinal slices. I was writing my proposition #8 thinking of both of you. I hope we will see each other soon (and by soon I mean 12th July)! Carin, you just started your PhD and with that much skill and dedication you will be finished with your project very soon, maybe you can even beat Mitchel’s record :p. I’m happy that I could teach you a thing or two in the lab, and in return, you helped me so much! We just published our paper and hopefully two more will follow! If that’s not a great start of the PhD, then I don’t know what is. Keep your enthusiasm but be aware what the proposition #8 says.

My warmest wishes and great appreciation go to my colleagues from the Formulation group:

Anko Eissens, Caroline Visser, Doetie Gjaltema, Duong Nguyen, Floris and Niels Grasmeijer, Herman Woerdenbag, Jan Ettema, Khan Nguyen, Kim Swieringa-Blake, Marinella Visser, Paul Hagedoorn, Rick Heida and Wouter Hinrichs. You create an atmosphere of a team, and every birthday cake we share

and lab outing we go feels like one big family getting together. Thank you for that! I was lucky to get to know many now former PhDs of PTB department: Anne Lexmond, Gaurav Kanojia, Jasmin Tomar,

Maarten Mensink, Marcel Hoppentocht, Naomi Teekamp, Philip Born and Wouter Tonnis. It is always

a nice occasion when we get to see each other and catch up on things. I address my special thanks to my generation of PhDs: Annemarie Broesder, Imco Sibum, Max Beugeling, Renee van der Kooij and Yu

Tian. Guys, I’m happy we never talk about slices. Yet, we got to do so much fun stuff together: canoeing,

sailing, escape rooms, board game nights, drinks at Noorderzon, parties, stuffing our faces with delicious food, you name it! I’m very grateful to all of you for making my PhD journey so wonderful. Imco, I dedicate pages 30, 142 and 220 of this book to you. Thank you for always having hope that I will join the Friday drinks (I promise, I will sometime soon!) and please wear your russian shirt!

I would also like to thank the members of Pharmacokinetics, Toxicology and Targeting department. My sincere appreciation goes to Prof. Angela Casini, Dr. Anna Salvati, Prof. Barbro Melgert, Prof. Geny

Groothuis and Dr. Inge de Graaf for all the help and support you showed me in these past years. Dear Geny,

thank you for being a great mentor during my master’s and for welcoming me at your department. Without your thoughtful guidance and encouragement, I definitely wouldn’t be where I am now. Dear Angela, you impacted my life so much, it goes beyond words. You are a brilliant researcher and a second mom to all of your students. You introduced me to an amazing person Sabrina, who leaves in Montreal, and with whom I regret I lost contact. Thank you for being a role model, for all the love, care and support you gave me when you supervised my master project and long after that! I hope we never lose sight of each other. I would like to extend my gratitude to Eduard Post, Marina de Jager and Jan Visser, who would always-always come to the rescue. I appreciate all the help and assistance in the lab!

(11)

10

I would also like to thank my PhD colleagues (current and former) at PTT department: Adhyatmika, Amirah

Adlia, Carian Boorsma, Fransien van Dijk, Ming Li, Natalia Estrada, Roberta Bartucci, Suresh Vatakuti, Viktoriia Starokozhko and Xiaoyu Niu. In the times we shared lab and meetings you added to the positive

working vibe.

I never thought I would enjoy teaching, but by my fourth year of PhD I’ve met and supervised several excellent students from bachelor to masterprojects. Rense Koops, Joris Reijneker, Jitske

Hoogland, Taha Sen, Jordy Bomers and Amos de Jong, thank you for showing interest in my project and

contributing to this thesis. We had our frustrations and successes, and I grew (my patience :p) and learnt together with you. Jordy and Amos, my hardworking helpers, each of you spent 6 months in the lab with me, and you’ve set a very high standard for all the other students to come in this group!

This thesis is a result of collaboration with many excellent researchers I had a pleasure to work with during my PhD. This research wouldn’t be possible without our strong connection with University Medical Center Groningen (UMCG) and I would like to express my profound gratitude to Prof. Ruud A. Bank and

Nataly Puerta Cavanzo, Prof. Jan-Luuk Hillebrands, Prof. Harry van Goor and Marian Bulthuis from the

Department of Pathology and Medical Biology. Thank you for your continuous cooperation, help and input I could always rely on. I sincerely thank the excellent surgeons at UMCG, especially Koert P. de Jong, Anna

M. Leliveld and Igle J. de Jong, for cooperation in providing human tissue from the donors.

As part of my research, I had a chance to visit Mainz on several occasions. I would very much like to thank Prof. Detlef Schuppan for providing the opportunity to perform experimental work at his laboratory and hosting our annual project meetings. It was always a pleasure to receive your feedback and to meet your research group. Dear Berit, thank you for arranging everything we could have possibly needed for sometimes last-minute visits, and for your great patience! Dear Yong Ook, we always received a warm welcome every time we came to Mainz. Thank you for taking care of animals for our experiments. It is unfortunate that our in vivo experiments didn’t work, but I’m happy I got a chance to work with you.

This project wouldn’t be possible without our collaborators at Boehringer Ingelheim. I would like to thank Anouk Oldenburger, Jörg Rippmann, Paul Nicklin, Eric Simon, Marco Schlepütz, Matthias

Zwick and Lutz Wollin for giving us so much of your time and attention and contributing to so many

beautiful papers! At our meetings you always provided a new angle to our research, you supplied us with interesting compounds and you welcomed us in Biberach. Thank you for the great experience!

I also had a chance to work with Stefan Vaessen, Jean Paul ten Klooster and Marc Teunis from Hogeschool Utrecht. Thank you for our beautiful collaboration and providing the organoid medium (a.k.a. “excellent stuff”). I enjoyed our every meeting because you always brought positive energy, your enthusiasm and Marc :p. I really tried to include one more chapter to this thesis, one of our co-authored papers, but I decided not to kill the self-esteem of my fellow PhD colleagues. The two publications are in order!

One of truly spontaneous and very joyful contacts I had in my PhD was with Prof. Rikke

Nørregaard at Aarhus University in Denmark. Dear Rikke, thank you for welcoming me in your lab and

introducing to your great research team. It felt like we had an instant connection and I hope we will stay in touch! I would also like to thank Michael S. Jensen for the kind assistance for my experiments in Aarhus. It was really great to see you in Groningen in our lab last December.

I cannot imagine my life in Groningen without my second family: Susana, Manuel, Emma and

Octavio. We’ve met 7 years ago and you’ve been my support and inspiration ever since, you’ve seen me

in my worst and my best, and I love you the most. Our countless evenings we spent together, amazing dinners, movie nights, birthdays, parties, volleyball – we shared it all and more! Thank you for everything and I’m sure a lifetime of friendship is ahead of us. My dear Susana, my beautiful paranymph, it is hard to express with words how much I appreciate you sharing this day with me. You created a family and a home in Groningen together with Edwin, and more so, you created a life: my heart will forever belong to little Aiden! I wish you all the great happiness. Manuel and Octavio, even thought you moved away from Groningen, we will always find our ways back to each other!

I am lucky to have my closest friends from Saint-Petersburg, no matter how big the distance is between us. My dear Lena and Anna, we’ve known each other our whole lives and you are a big part of me. I’m sorry for not calling you more often, but I love and miss both of you very much! My dear Zhenya, it’s impossible to come home to Saint-Petersburg and not run to eat sushi with you. You’ve given me so much support in difficult times and we laughed our lungs out in happy times. Thank you for sticking with me for the last 12 years, and I wish you and Anton eternal love and joy! Girls, we will see each other soon!

I would have never been who I am and where I am now without my family. Your unconditional love, your faith in me opened all the doors in the world. I can do anything as long as I have you by my side. Мои любимые Андрей и Эля, с тех пор как вы переехали в Питер, мы стали ближе и больше общаться, что приносит мне много счастья! Я желаю вам путешествовать еще больше и увидеть весь мир. Кто знает, может вы когда-нибудь доберетесь до Гронингена :р Даша, мы конечно постоянно ссорились когда были детьми и ты мне больше нравишься когда мы не живем вместе (хаха), лучшей сестры ни у кого нет и я тебя очень сильно люблю! Спасибо за все радости и горести, и за Илью, который стал незаменимым членом нашей семьи и так превосходно нас дополняет. Мои любимые бабуля и дедуля, вы моя лучшая опора и поддержка. Всю мою жизнь вы были рядышком и я посвящаю эту книгу вам; я вас безмерно люблю и всегда думаю о вас! Мама, это самая сложная часть всей книги, потому что нет слов чтобы выразить всю мою любовь, нежность и благодарность тебе и папе. Вы всегда были моим примером, делали все возможное и невозможное для меня и Даши. Ты самая сильная, и я верю, что папа всегда с нами рядом. Бесконечно люблю вас! И конечно же, спасибо нашему главному члену семьи – коту Нельсону!

My dear Thomas, you are my best partner in life and a never-ending source of happiness. I always tell you that it was destiny that brought us together (after all, you were literally the first person I’ve met in Groningen). We’ve been through so much, we take care of each other and we are about to celebrate our 7 years anniversary. Love you with all my heart.

Thank you,

Emilia

(12)