The volatile metabolome and microbiome in pulmonary and gastro-intestinal disease - Addendum

UvA-DARE (Digital Academic Repository)

The volatile metabolome and microbiome in pulmonary and gastro-intestinal

disease

van der Schee, M.P.C.

Publication date

2015

Document Version

Final published version

Link to publication

Citation for published version (APA):

van der Schee, M. P. C. (2015). The volatile metabolome and microbiome in pulmonary and

gastro-intestinal disease.

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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), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Addendum

Nederlandse Samenvatting

Vanaf het moment van de bevruchting reageert ons lichaam-in-wording op de gebeurtenissen in onze omgeving. Ons genetisch materiaal, het DNA, codeert daarvoor de basis. Alle informatie hierin noemen we gezamenlijk het genotype. Ons DNA wordt door het lichaam, afhankelijk van de omstandigheden, omgeschreven naar RNA, vertaald naar eiwitten en afgebroken tot metabolieten die we uitscheiden via onze urine, ontlasting, zweet en adem. Het uiteindelijke resultaat van de combinatie van onze omgevingsinvloeden met ons DNA is ons fenotype, alle observeerbare eigenschappen van ons lijf; hoe we er uit zien, hoe wij reageren als persoon en hoe ons lijf van binnen reageert op de omgeving. In reactie op die omgeving streeft ons lichaam continu naar dynamische stabiliteit; meeveren met de omgeving, maar wel binnen bepaalde grenzen. Als die grens overschreden wordt kan het lichaam niet meer optimaal functioneren en kunnen we ziek worden. Om ziekte en gezondheid goed te begrijpen is het dus belangrijk om een goed begrip te hebben van hoe dit systeem werkt. De laatste jaren zijn er grote ontwikkelingen geweest op het gebied van de biologie en technologie waardoor het steeds beter mogelijk is om in 1 keer naar al het DNA, RNA, eiwit of metabolieten in een biologisch monster te kijken. In hoofdstuk 1 omschrijf ik hoe met zulke ‘omics’ technieken het in potentie mogelijk is om een gedetailleerd beeld te krijgen van het functioneren van een cel, orgaan of lijf. De onderzoeken in dit proefschrift richten zich voornamelijk op 1 van dat soort technieken. Ik heb me gericht op het analyseren van de vluchtige metabolieten, de zogeheten vluchtige organische componenten of VOCs. Deze VOCs zijn stoffen die wij elke dag met onze eigen neus ervaren als geur. Net als onze neus een patroon van deze VOCs kan detecteren doen wij dat met een electronische neus of eNose. Dit apparaat analyseert deze VOCs en geeft ze weer als een patroon of ‘geurprofiel’. Dit betekend dus ook dat we niet exact weten welke stoffen er in een mengsel zitten. Om dat wel te weten te komen zijn complexe laboratorium technieken nodig die minder geschikt zijn dan de eNose voor gebruik bij de patiënt.

Diagnose van ziekte

Allereerst hebben we onderzocht of mensen met en zonder infectie andere VOCs uitademen. In hoofdstuk 2 stelden we vast dat een eNose met 90% zekerheid kan ruiken of patiënten met een chemokuur een schimmelinfectie (invasieve Aspergillose) hebben. Dit is belangrijk omdat dit misschien vroege detectie van ziekte mogelijk maakt. Op die manier zou behandeling eerder kunnen worden gestart en kunnen mogelijk levens worden gered.

In hoofdstuk 3 laten we vervolgens zien dat een eNose redelijk goed onderscheid kan maken tussen gezonde mensen en patiënten met mesothelioom, een met asbest geassocieerde longkanker. We lieten ook zien dat patiënten met kanker, andere VOCs uitademden dan patiënten met asbest blootstelling maar zonder kanker. Dat laatste is belangrijk omdat de VOCs die onderscheiden tussen gezond en mesothelioom anders bleken te zijn dan diegene die onderscheid maken tussen asbest blootstelling en mesothelioom. Hieruit blijk een belangrijk principe voor alle ‘omics’ studies; verricht experimenten zoveel mogelijk in de klinische setting waarin de test ook gebruikt gaat worden.

In hoofdstuk 4 laten we zien dat de adem van patiënten met longkanker anders ruikt dan die van controles zonder longkanker. Door met een bronchoscoop in de long lucht weg te zuigen konden we ook vaststellen dat een deel van deze stoffen van de plek van de tumor zelf kwamen en andere stoffen waarschijnlijk van de reactie van het lichaam op de tumor komen. Dit maakt het waarschijnlijk dat andere soorten kanker ook via de longen kunnen worden vastgesteld, iets wat inderdaad door anderen ook is aangetoond.

Ziekte fenotypering en monitoring

Hoofdstuk 5 laat zien dat een eNose met 88% zekerheid kan voorspellen of een

patiënt met astma wel of niet reageert op steroïden (prednison). Dit is belangrijk omdat artsen alleen met ingewikkelde technieken kunnen voorspellen of benauwde patiënten met astma goed reageren op steroïden. Uit dit onderzoek bleek ook dat VOCs verschilde tussen stabiele en benauwde patiënten en gecorreleerd was met ziekte activiteit in de long (sputum eosinofielen). Dat opent mogelijk de deur naar het non invasief monitoren van de ziekte activiteit van astma patiënten.

Aangezien een meting van VOCs met de eNose niet belastend is voor patiënten hebben we de test ook onderzocht bij kinderen. Allereerst hebben we in

hoofdstuk 6 gekeken naar de uitgeademde lucht van patiënten met taaislijmziekte

(CF) en PCD; een ziekte van de trilharen in de long, die op CF lijkt. Bestuderen van deze ziektes met de eNose is vooral interessant voor monitoring zodat er vroeg

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behandeld kan worden bij een acute verergering van klachten. Uit dit onderzoek bleek dat kinderen met en zonder acute klachten maar matig uit elkaar konden worden gehouden. Het is op basis van de resultaten niet duidelijk of dit komt doordat er te weinig patiënten met een exacerbatie waren of doordat de eNose dit niet kan meten. Wat wel uit het onderzoek bleek is dat kinderen met taaislijmziekte en PCD andere VOCs uitademen. Dat is een interessant gegeven omdat deze ziekte in de kliniek op elkaar lijken maar toch een andere oorzaak hebben. De vluchtige metabolieten lijken dat verschil in ziekte activiteit dus goed weer te geven. In hoofdstuk 7 zijn wij de eerste die een eNose gebruiken om de VOCs van ontlasting te analyseren. In dit onderzoek tonen we aan dat de ontlasting van kinderen met de ziekte van Crohn en Colitis Ulcerosa anders ruiken dan die van gezonde kinderen. Dit verschil was zo sterk dat dit met 85 tot 100% zekerheid lukte, al moet dit nog in een nieuwe groep kinderen worden bevestigd. Net als bij astma zagen we nu ook dat er verschil was tussen de VOCs van de ontlasting bij actieve ziekte en bij rustige ziekte. We vonden echter ook dat de ontlasting van gezonde kinderen in diezelfde periode net zoveel was veranderd van geur. Ondanks deze verandering bleef die wel duidelijk verschillen van zieke kinderen. Het is daarom niet duidelijk uit dit onderzoek of we met de VOCs in de ontlasting de ziekte activiteit kunnen monitoren. Om dat te onderzoeken zijn we een nieuwe, langdurigere studie gestart.

Voorspellen van ziekte

In hoofdstuk 8 hebben we de eerste stap gezet om te kijken of de eNose kan voorspellen of jonge benauwde kinderen later astma krijgen. Daarvoor hebben we een groot cohort opgezet, de EUROPA-studie, waaraan 1200 kinderen uit Amsterdam en omgeving deelnemen. Door deze kinderen nauwkeurig te onderzoeken konden we vaststellen dat die kinderen die een 10 keer zo hoge kans hebben om astma te krijgen, andere VOCs uitademen dan kinderen zonder dat verhoogde risico. Opvallend genoeg bleek dat zowel tijdens een benauwdheidsaanval, als weken daarna te zijn, als de kinderen weer beter waren. Mogelijk meet de eNose de chronische ontsteking waar kinderen last van hebben die later mogelijk astma krijgen. Dat zou uiteindelijk kunnen betekenen dat we op jonge leeftijd kunnen vaststellen wie er wel of geen astma krijgt, iets

duidelijk dat het niet bij je dragen van de juiste bacteriën een risico vormt voor de ontwikkeling van astma. Daarom heb ik in hoofdstuk 9 met een nieuwe techniek, ontwikkeld in de VU, gekeken naar welke bacteriën in de neus van de kinderen uit de EUROPA-studie zitten. We merkten dat kinderen met een hoge kans om astma te ontwikkelen minder verschillende bacteriën in hun neus hadden, maar dat die wel in hogere hoeveelheden aanwezig waren. We zagen ook dat een deel van deze verschillen bleven nadat de klachten van de kinderen waren overgegaan. In de eerste plaats sterkt ons dat in het idee dat een deel van de VOCs gelinkt zijn aan het microbioom omdat analyse met de eNose een vergelijkbaar patroon liet zien. Ook betekend het dat we mogelijk bacteriesoorten kunnen vaststellen die jonge kinderen zouden kunnen beschermen tegen de ontwikkeling van astma. Dat is iets wat we de komende jaren zullen onderzoeken binnen de EUROPA-studie.

Samenvatting en toekomstbeeld

In hoofdstuk 10 laten we zien dat het mogelijk is om VOCs tot 14 dagen op te slaan op zogeheten Tenax buizen om ze op 1 centrale plek te kunnen analyseren. Dat is een stap die mogelijk bijdraagt aan de ontwikkeling van nieuwe eNose apparatuur doordat monsters van over de hele wereld op 1 plek kunnen worden geanalyseerd. Dit is een principe wat inmiddels in meerder onderzoeken is toegepast.

Uit de verkennende studies in dit proefschrift blijkt duidelijk de potentie van VOC analyse voor de geneeskunde. Het onderzoek tot nu toe op het gebied van de long en op het gebied van darmkanker worden in hoofdstuk 11 en 12 samengevat. Samen met het onderzoek in dit proefschrift blijkt wel dat er nog veel uitdagingen zijn voordat een eNose toepasbaar is in de kliniek; er moeten duidelijke afspraken komen hoe we de monsters analyseren zodat onderzoeken goed vergelijkbaar zijn. Ook zijn er grote stappen nodig op technisch gebied om de electronische neus gevoeliger, betrouwbaarder en specifieker te maken voor bepaalde ziekten. Uiteindelijk is het waarschijnlijk dat specifieke apparaten voor specifieke toepassingen worden ontwikkeld. Om dit mogelijk te maken is het noodzakelijk om met Gas Chromatografie en Massa Spectrometrie in het lab te kijken welke VOCs er precies in een adem of ontlastingmonster zitten.

In hoofdstuk 13 bespreek ik welke algemene lessen over ‘omics’ onderzoeken geleerd kunnen worden op basis van dit proefschrift. Allereerst is het erg belangrijk om alle uitkomsten met strenge statistische testen te onderzoeken en te controleren in een groep nieuwe patiënten. Daarnaast is het belangrijk om te beoordelen, bijvoorbeeld met GC-MS, of de gevonden stoffen een logisch verband hebben met de ziekte die je onderzoekt. Daarom is het zaak om te proberen ook de functie van een stof te betrekken in de analyse, naast alleen maar de aanwezigheid. De aanwezigheid van een bacterie vertelt ons namelijk niet

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direct wat de activiteit van die bacterie is. Ten slotte is het erg belangrijk om die populatie te onderzoeken waarin de test ook daadwerkelijk zal worden toegepast zodat vals negatieve en vals positieve resultaten worden voorkomen.

Als al deze adviezen worden opgevolgd en er investeringen zijn om de techniek verder te ontwikkelen zijn er veel kansen voor de eNose en ‘ omics’ analyse in het algemeen. Het combineren van meerder technieken, zoals VOC en microbioom analyse, kan veel extra inzichten opleveren. Naast aanwezigheid kunnen we dan mogelijk ook de functie van bacteriën op niet invasieve wijze meten. De systeembiologie probeert uiteindelijk al dit soort ‘omics’ analyses op steeds meer niveaus met elkaar te verbinden. Hierdoor kunnen we steeds gedetailleerder inzicht krijgen in hoe ons lichaam functioneert en wat er mis gaat bij ziekte. Uiteindelijk kan dat bijdragen aan het individualiseren van behandeling bij patiënten zodat die maximaal effectief is en minimale bijwerkingen heeft. Dit is een enorme uitdaging om te onderzoeken en een relatief onontgonnen veld. In de komend jaren hoop ik actief te blijven bijdragen aan de kennis op dit gebied.

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Contribution Statement

Chapter 1 Wrote the manuscript

Chapter 2 Helped design the study, conducted study procedures, analyzed the data and and co-authored the manuscript.

Chapter 3 Analyzed the data and co-authored the manuscript

Chapter 4 Designed the study, conducted study procedures and wrote the

manuscript

Chapter 5 Analysed the data and wrote the manuscript

Chapter 6 Designed the study, monitored study procedurs, analyzed the data and co-authored the manuscript

Chapter 7 Designed the study, monitored study procedures, analyzed data and co-authored the manuscript

Chapter 8 Designed the study, conducted study procedures, analyzed data and wrote the manuscript

Chapter 9 Designed the study, conducted study procedures, analyzed data and wrote the manuscript

Chapter 10 Designed the study, conducted study procedures, analyzed data and wrote the manuscript

Chapter 11 Wrote the manuscript

Chapter 12 Co-authored the manuscript

Acknowledgments

It’s a rainy Sunday in Amsterdam when I sit down to write the acknowledgment section of my thesis. To me there are two reasons I’ve been postponing to write this most challenging section; it is the most commonly read part of any thesis and once you start naming and faming individuals you are bound to forget someone. To me there are two basis ways to work around this issue: Just thank everyone without naming anyone specifically. As this may be considered the ‘chicken’s way out’ I’ll try and do it the other way. Attempt to thank everyone relevant to this thesis personally, despite the obvious risk of forgetting some key individuals such as yourself!

Therefore I’ll start by devoting some words to you as a ‘monument for the unknown soldier’. Thank you especially for all the effort you put into my personal growth, the data collecting, number crunching, good atmosphere, laughs, fun, support and helping me realize this thesis.

When coming to think of how I got to this point I feel its foundations were laid out at a very young age. I definitely need to thank my parents Andre and Astrid as well as my sisters Celine and Tamara for providing me with an environment in which I was able to develop my interest for knowledge and science in general. I don’t feel there would be that many parents that realized the perfect birthday gift for this 8 years old boy was an encyclopedia I could read from Aaron to Zundap. Thank you also for putting up with my interest in how things work, which more than once expressed itself as taking toys, household equipment, furniture etcetera apart. Unfortunately putting it back together was often more challenging.... Why was I always stuck with this one screw or bolt after putting it back together? Coming to think of it it’s a good thing I have no surgical ambitions.... Despite your worrying as parents whether I’m on the right career track (happens to me to!) I know you are proud of where I am now. The next major step resulting in my current position was my encounter with Peter. I just finished the first year of med school and was disappointed. Medicine appeared to me to be to ‘soft’ and non-scientific. Sometimes it felt I was just learning to memorize diagnosis, treatment and monitoring protocols without truly learning about the underlying pathophysiological fundaments. I actually considered quitting to study something totally different, such as technical physics. But all that changed drastically when this amazingly enthusiastic professor gave a lecture on asthma pathophysiology and novel diagnostic tools such as exhaled nitric oxide. I immediately knew it: this is why I started studying medicine! Scribbling down Peters e-mail address in my lecture notes and deciding to e-mail him was, in hindsight, one of the most life changing decisions I ever made.

Within a week I started a research project at the department of pulmonology in the LUMC spending all the hours between my lectures (and those of ‘boring’ lectures) working to develop my science skill set. Thank you Peter for grabbing me from the school benches and supercharging my development by stimulating me to combine medicine with biomedical sciences for more in depth pathophysiological knowledge. You did so again more recently by convincing me to finish my MD. It still helps me every day to see the science in clinic and the clinic in science. Thank you especially for providing me with a safe environment to fail in. I remember a meeting during which I told you I found out I was on a wild goose chase the last month. While I was frustrated about the lost time and the further delay this would give to the project Peter calmly stated: I saw this one coming weeks ago. At first this increased my frustration. Why would you not warn me? You could have prevented this! It took me a day to realize that nothing is a better teacher than failing to achieve something. It is the way we deal with such set back that ultimately makes us grow and outperform ourselves. It however takes a very talented mentor to foresee this, not intervene and help someone to grow from this experience personally. Eventually I did overcome the challenge resulting in a chapter of this thesis, something which was extremely rewarding. There have been more instances afterwards (small and big) during which you kindly pointed me to other ways I could have handled a situation which helped me to grow professionally and personally.

I also like to thank you for providing an environment in which everyone was considered to be peers. The moment I joined your group in Leiden 8 years ago I felt I was able to speak my mind, discuss things based on scientific arguments without ever being confronted with arguments based on authority. I also should mention this spoiled me to some extent. I didn’t come to realize how rare such an environment was in a hospital setting until I was confronted with the hierarchy present in many clinical departments. At the past ERS I saw Peter speak once more and he hasn’t lost his potential to draw in an audience and make them part of the scientific adventure he is unfolding. This will always be an example for me how to present your findings. I hope we still get to learn a lot from each other in the coming years, but I’m sure we will. Thank you for being a mentor and helping to define who I am and where I’m headed.

At the department I learned how to set up a proper experiment from Robert Schot and Dirk van de Plas, smelling apples, oranges (and beers) under every possible condition to work out the best way to analyze breath. Thank you both for the scientific discussions we had which helped me to develop both my critical thinking and enthusiasm for science. It is unfortunate our roads separated when I moved to Amsterdam together with Peter.

In Amsterdam I started my fulltime research career opening up a whole new avenue of possibilities. There are so many people at the AMC and VUmc that contributed to my development:

Wim, I’d like to thank you specifically for the unrelenting confidence you expressed in my capabilities and judgment. This helped me grow considerably over the course of my PhD. I’m very proud of the EUROPA-cohort we’ve jointly built. I’m very grateful that you helped to convince so many others of the relevance of our research line. This has proven to be instrumental to the current and future success of the study. I’m sure I would not have come this far without it.

Dear Eric, you are one of the instigators of the EUROPA-study. Without your considerable support in the grant writing process for the lung foundation the study would have never existed. Irrespective of your distance from the AMC your contribution to the study has always been profound. I was always able to tap into your extensive clinical knowledge and experience. Furthermore you’ve been extremely supportive on a personal level when I had trouble seeing the way forward. I’ve come to appreciate you as a colleague and friend and hope to be collaborating extensively on future adventures.

Aline, thank you for your contributions to the thesis even when this was hard for you. I’ve always greatly valued your clinical judgment and your scientific experience. This has often helped to redirect the study in directions we had not considered previously. I’m sure we’ll work on many more projects in the future, both here and abroad.

Dear Lies I’m grateful we got to meet each other as you taught me so much about allergy. I’m glad we got to work together more extensively during the past few months, which will surely result in some nice papers and new insights. I hope to be in contact even after your departure from the AMC as I really value you as a person.

Mister Anders! Our roads crossed every now and then but every encounter has been valuable. By far your biggest contribution was to introduce me to Tim de

Meij thereby initiating a novel research line that makes up half of this thesis. Please continue to provide such nudges to people around you! How “anders” would have things been without this!

Dear Barbara, Tamara, Rene, Marianne and everyone from the experimental immunology lab. Thank you for planning for the unplannable nature of the EUROPA-study. Your help has been instrumental to its success and the future possibilities of the study. You have all provided me with your advice many times which has been invaluable in managing the EUROPA-study and its succession. I’d also like to thank all the people of the various other labs that contributed to the studies in this thesis; virology, bacteriology, clinical chemistry etcetera.

Saeeda and Erica thank you for your constructive attitude towards the demanding EUROPA-study, you always help to create the best situations possible for our study patients despite the high demands on time and space. I’m excited to work with such a talented team on collecting the primary outcome for our study!

Many thanks to all those people who helped facilitate the writing of this thesis: Jamal thank you for dealing with the complex study finances and helping us realize our research aspirations. Marijke Fransen, we haven’t met in person but thank you for your amazing work on the lay-out of the thesis. Bureau promotiezaken, thank you for guiding a ‘lost’ researcher through the logistics of a PhD defense.

Of course the many direct colleagues I had during the past years have been essential to this thesis. Firstly those people that accommodated me as a junior research in their midst: thank you Niki for sharing your clinical insight, drive and pioneering mentality. Thank you Mart, David, Marijke, Marieke, Selma and Sulaiman for taking me up in to your group, the ‘rookie’ I was. Thank you Viviana for sharing so much good times in our ‘fishbowl’ on F5. It’s unfortunate we are so far away from each other. Ariane thanks for your open personality and willingness to contribute your experience to any discussion. Koen de Heer, thank you for opening up new research avenues together. Dear Peter Kunst and Lisette Venekamp, thanks for supporting me with your pioneering mentality in this research field. Liesbeth, thank you for the steering role you have had over the years and for accommodating pediatric

invaluable contribution to any research project. I’m sure we’ll cross roads many times on our paths forward.

Dear Paul, our engineer amongst doctors. It’s impressive what you have built over the years. It must have been challenging to find your way and pioneer this new field but you did so extremely well. You helped determine and shape the future of our joint research line and elevated it to a whole new level. Besides this you are an extremely welcoming person who always is willing to help others. I’m happy to work on these projects together with you. You have also contributed many talented new colleagues and interns.

And of course all the other colleagues I returned to after my internships. I’m thankful for the way you all absorbed me into your group. The way you have all supported me during the past year during personal and professional challenges was heartwarming. I’m proud we got to construct a working atmosphere together in which we could provide each other with honest and constructive criticism. This has been instrumental to acquiring the lung foundation personal grant. Marlous, it is nice to have another biomedical scientist in the room. I admire your commitment to the group process and atmosphere. Marije, thank you for always being ready to help me with your advice and for sharing your warm personality. Pieter-Paul and Guus thank you for bringing so much fun to the department. A visit to a conference isn’t the same without you two! Rianne I’m honestly impressed by your talents and hope to see much more of them in the future. It’s good to have you on board of the EUROPA-study! Julia, our thermoplastic lady (that’s a compliment!), thank you for your support and interest in my endeavors. Nora thank you for keeping the cohort together. Your contribution is extremely valuable to keep the EUROPA-study running, no one knows the children better than you! Thank you Henny, Maria and Jacqueline for your logistic support for the studies in this thesis. I wouldn’t have been able to send 13.000 letters without your help. Thank you Anne-Marie for contributing to the EUROPA-study whilst I was doing my internships, this helped to get us to where we are now! I’d also like to thank all those students that contributed and continue to contribute to the various studies in this thesis: Sarah, Romy, Tessa, Martine, Marloes, Robin, Eva en Emilie. Dear Nine, thank you for joining the EUROPA-study however briefly it was. It is unfortunate we were unable to keep you on board.

Dear Simone you hold a special place amongst colleagues and in my heart. We’ve known each other since we both started our research in Leiden. You have been my moral compass so many times and I’m very happy we got to meet. Thank you for believing in the ‘good’ in me even when my actions unintentionally suggested

otherwise. I am eternally grateful for the way you guarded the fortress whilst I was in my internship, this must not have been easy at times. Please continue to amaze me with your talent and your lust for live. Aichaichai!

Niels, I’m honored you are the next PhD student of the EUROPA-study. During the past months you have already drastically improved the way the EUROPA-study follow-up will be constructed. It is a good feeling to have the study in your capable hands. I hope this will be the kick-off for a longstanding cooperation.

Even though I’ve only met you two years ago we’ve extremely rapidly build an impressive flatografy empire. Tim and Nanne, it is an enormous pleasure and honor to work with you. The can-do-mentality, the friendship, the enthusiasm, the lame jokes, the media exposure, the unconventional and result driven approaches are a pleasure to be working with. I’m extremely proud of what we have build and all the future adventures that await. I’m sure we will continue to push each other out of our comfort zone to make sure we keep learning. Thank you for your unlimited confidence and support. Nanne, thank you for your capabilities as a study initiator and for always being able to bring the right people together at the right time. Tim thank you specifically for being my paranimf, for sharing a great time in Madrid and Israel and for being so genuinely supportive and interested in me. I’m looking forward to pushing this forward together with you guys BAM!

Dries, I’m excited to be able to collaborate with you on the exciting field of microbiome. I’m sincerely impressed by the way you juggle a clinical and scientific career so successfully. ISpro is in impressive feat of work, with huge potential for the future. Thank you, Linda, Anat, Paul and the rest of the ISpro team for your willingness to cooperate on the many studies we collaboratively run. I hope to be able to learn much more from you and your talented team over the next years by integrating the volatile metabolomics and the microbiome analysis.

Dear Robin, my stay with you still holds a special place in my memory. I was very happy to be able to contribute to your beautifully conducted study. I still use many scientific concepts you taught me on a daily basis. Besides this refreshing professional experience I’m very grateful to have got to know you as a person.

our roads will cross once more in the future. Dad Brockway! How are Rachel, Daniel and Thomas doing? Amit and me still quote Daniel often, wearing dads shoes and case; “Off to work!”. Thank you for accommodating us in your house as if we were family. It was truly our home away from home. We hope to be fortunate enough to visit once more in the nearby future. Hopefully one day we’ll be able to repay the favor and accommodate you in Amsterdam. Our door is always open. Ben, we have only recently met but I’m thrilled to start working together in the near future. I feel your approach to science will help me gain many new insight and farther my research career. Thank you sincerely for your confidence to date. Dear Koos, I can hardly exaggerate your contribution to my statistical knowledge over the past years. Thank you for your patience explaining (if necessary over and over) how I should handle my data to obtain the most robust results. I’m honored to have you in my committee and hope to be able to pick your brain many times over the coming years.

Dear professor van Goudoever, over the past year our research interests have began to overlap. I’m really excited to see in what ways we could improve the early diagnosis and management of NEC. Thank you for the confidence in this research line and for being part of my committee.

Dear professor van Leeuwen, we have only briefly met but I’m happy to have you aboard the committee to provide a sound technical background for the defense. Dear Professor Savelkoul, thank you for your genuine interest in my research background. I’m excited to see in what ways we can be complementary. Thank you for being part of the defense committee.

Dear professor Frey, I’m honored you took the effort to travel to Amsterdam for my thesis defense. I’m an admirer of your work and hope to learn from your clinical and scientific expertise in a collaborative VOC project.

Dear dr Pijnenburg, dear Marielle, thank you for attending the defense. Your work on FENO and pediatric cohorts has always been an inspiration and example for me. I hope to be able to collaborate on a joint Dutch cohort in the future. Dear family; Hans, Jacqueline, Dimitri, Micha, Hanneke, Rob, Marcel, Casper, Jolanda, Richard, Marjolein, Marion, grandma, grandpa thank you for your continuous interest in the things I do even if you don’t understand half of it!

Dearest Karel, Dina and Itamar, I’m very grateful I’ve met you all. Itamar, you are always supportive and understanding and have much more capabilities than you dare to admit to yourself! Dina, you are such a warm personality and know just how to support me at the right times and confront me with what I’m trying to hide when it’s necessary. Karel I’m truly impressed by your approach to life and your commitment to the ones you care about. This will always be an example for me. Chère David, I know it’s not the truth but I feel we’ve just met for the first time. This was a life changing moment for me. I’m happy to have found you and your lovely family and hope you will be a part of my life from now on. Dear Oriane, we haven’t met yet but I’m sure we will soon, I hope this will be just as rewarding. My dear friends Monika, Diederik, Marina, Henny, Wendy, Rik and Loes, Victoria, Zeen, thank you all so much for putting up with my overly enthusiastic stories, the ups and downs of research and my never ending search for ‘the next step’. I feel fortunate to have known most of you for many years. Please continue to provide me with such joy, comfort, midnight snacks and failed recipes for many years to come.

All those many people from the DenkTank, thank you profoundly for sharing that amazing period with me, for helping me to grow faster than I’ve ever grown (the muscles still acke!) and continuing to inspire me with all your adventures. You are a special lot and I’m sure our interaction is much more sustainable than any supply chain!

Gerben, Frank and Lisa, thank you for providing me with such enormous musical joy over the past years! The best way to blow off some steam is to play with you guys. I hope we continue to play for a long time with as much joy and enthusiasm as we have so far. In exchange I’ll try and do my best to not hit anyone’s face with my guitar, not to have to ‘ artistic’ timing and not to write songs that are impossible to sing to. Wow how do you guys put up with this…..? And Lisa, be warned I will chase to that other end of the world!

contribution of sponsors to our studies. Thank you TEVA pharma, Merck Sharp & Dohme Corp, Pfizer Inc., Otago Respiratory Research Trust, Stichting PCD Belangengroep, Falk Benelux BV and Biomerieux. I particularly want to thank the Dutch Lung Foundation for providing a grant that helped found the EUROPA-study and for expressing confidence in me by providing a personal grant to farther my research career and continue to contribute to the health of lung patients.

I like to express my warmest gratitude to all thousands of patients and their parents participating in the studies in this thesis. Without your altruistic contribution there would be no scientific progress. It is the most valuable asset for any clinical researcher and I hope to continue working with the EUROPA-study data for many years.

Finally, dearest Amit, there is no way I could think of to thank you that would do justice to the way you support me in everything I do. This thesis has your name written all over it and is as much your work as it is mine. You have this amazing ability to stimulate me to reach the goals I want to achieve whilst, at the same time, you make sure that I don’t outrun myself. I feel we’ve grown in the past 9 years to understand each other on so many levels. Especially when times were trying during the past two years you were able to help me sort my thoughts. You have sincerely surprised me with what we can achieve together. I’m extremely blessed to be on this road together with you, this adds the sparkle to every single day. Vovie shez li, ani ohev otach tamid.

Curriculum Vitae

Marc was born in Pont Audemer, France on 28 january 1986. He attended school in the Netherlands after moving there at a young age. After graduating Cum Laude from the Katholieke Scholengemeenschap Hoofdorp High School he attended med school at the University Leiden. During the second year Marc initiated his first research project at the department of Respiratory Medicine headed by Professor Peter Sterk. He focused on pioneering exhaled breath analysis by electronic nose. He has ever since been part of that research department, ultimately

culminating in this thesis. During medschool Marc also completed a degree in Biomedical Sciences. After completing these two degrees Marc travelled around the world for 6 months.

Upon returning he moved to the University of Amsterdam, together with professor Sterk. Here he initiated the EUROPA-study, a 1200 infants prospective cohort study, investigating early signs of developing asthma. Professor Wim van Aalderen supervised this pediatric study together with professor Sterk as the promoters of Marc. Besides running the EUROPA-study Marc remained very active in several exhaled biomarker studies for which he won several prizes. He conducted a field study into Tuberculosis diagnosis by exhaled breath in Bangladesh and contributed his experience to the research team of professor Robin Taylor in Dunedin, New Zealand.

After two years of fulltime research Marc completed his MD degree, scoring an average 8,5 out of 10 at the VU Medical Centre Amsterdam. In this period Marc was selected as a participant of the Dutch National ThinkTank. During this 6 month ThinkTank project he expanded his skill set to include management, team building and business aspects. He devised a novel market model enabling a low-cost transition of a fully unsustainable bulk produce to a fully sustainable one. This has been implement by a major Dutch supermarket.

During his MD training Marc initiated a research line into fecal volatile diagnostics together with dr Nanne de Boer and dr Tim de Meij. In March 2014 Marc obtained his Medical Degree and returned to fulltime research. Thusfar he has published 17 peer-reviewed papers. Marc was recently awarded a prestigious Lung Foundation personal grant to investigate possible therapeutic options of the microbiome in the prevention of astma. He will run this project in close collaboration with professor Benjamin Marsland, Lausanne and professor Malcolm Sears from the Canadian CHILD-cohort study.

Marc lives in Amsterdam together with his girlfriend Amit Vos.

List of Publications

1. van der Schee MP, Budding AE, Poort L, Hashimoto S, Sprikkelman AB,

Haarman EG, van Aalderen WM, Savelkoul PHM, Sterk PJ. Increased microbial abundance and decreased diversity in preschool children at risk for asthma. submitted.

2. van der Schee MP, Paff T, Brinkman P, van Aalderen WM, Haarman EG, Sterk PJ. Breathomics in Lung Disease. Chest. 2015 accepted.

3. van der Schee MP, Hashimoto S, Schuurman AC, Repelaer van Driel JS,

Adriaens N, van Amelsfoort RM, Snoeren T, Regenboog M, Sprikkelman AB, Haarman EG, van Aalderen WM, Sterk PJ. Altered exhaled biomarker profiles in children during and after rhinovirus-induced wheeze. Eur Respir J. 2014 Oct 16.

4. Niemarkt HJ, de Meij TG, van de Velde ME, van der Schee MP, van Goudoever JB, Kramer BW, Andriessen P, de Boer NK. Necrotizing Enterocolitis: A Clinical Review on Diagnostic Biomarkers and the Role of the Intestinal Microbiota. Inflamm Bowel Dis. 2014 Sep 29.

5. de Meij TG, de Boer NK, Benninga MA, Lentferink YE, de Groot EF, van de Velde ME, van Bodegraven AA, van der Schee MP. Faecal gas analysis by electronic nose as novel, non-invasive method for assessment of active and quiescent paediatric inflammatory bowel disease: Proof of principle study. J Crohns Colitis. 2014 Sep 22.

6. de Boer NK, de Meij TG, Oort FA, Ben Larbi I, Mulder CJ, van Bodegraven AA,

van der Schee MP. The scent of colorectal cancer: detection by volatile organic

compound analysis. Clin Gastroenterol Hepatol. 2014 Jul;12(7):1085-9.

7. Wagener AH, Yick CY, Brinkman P, van der Schee MP, Fens N, Sterk PJ. Toward composite molecular signatures in the phenotyping of asthma. Ann Am Thorac Soc. 2013 Dec;10 Suppl:S197-205.

can discriminate colorectal carcinoma and advanced adenomas by fecal volatile biomarker analysis: proof of principle study. Int J Cancer. 2014 Mar 1;134(5):1132-8.

10. Fens N, van der Schee MP, Brinkman P, Sterk PJ. Exhaled breath analysis by electronic nose in airways disease. Established issues and key questions. Clin Exp Allergy. 2013 Jul;43(7):705-15.

11. de Heer K, van der Schee MP, Zwinderman K, van den Berk IA, Visser CE, van Oers R, Sterk PJ. Electronic nose technology for detection of invasive pulmonary aspergillosis in prolonged chemotherapy-induced neutropenia: a proof-of-principle study. J Clin Microbiol. 2013 May;51(5):1490-5.

12. van der Schee MP, Paff T, Daniels JM, Pals G, Postmus PE, Sterk PJ, Haarman EG. Exhaled molecular profiles in the assessment of cystic fibrosis and primary ciliary dyskinesia. J Cyst Fibros. 2013 Sep;12(5):454-60.

13. van der Schee MP, Fens N, Brinkman P, Bos LD, Angelo MD, Nijsen TM, Raabe R, Knobel HH, Vink TJ, Sterk PJ. Effect of transportation and storage using sorbent tubes of exhaled breath samples on diagnostic accuracy of electronic nose analysis. J Breath Res. 2013 Mar;7(1):016002.

14. Dragonieri S, van der Schee MP, Massaro T, Schiavulli N, Brinkman P, Pinca A, Carratú P, Spanevello A, Resta O, Musti M, Sterk PJ. An electronic nose distinguishes exhaled breath of patients with Malignant Pleural Mesothelioma from controls. Lung Cancer. 2012 Mar;75(3):326-31.

15. Fens N, Roldaan AC, van der Schee MP, Boksem RJ, Zwinderman AH, Bel EH, Sterk PJ. External validation of exhaled breath profiling using an electronic nose in the discrimination of asthma with fixed airways obstruction and chronic obstructive pulmonary disease. Clin Exp Allergy. 2011 Oct;41(10):1371-8.

16. van der Schee MP, Venekamp LN, Kunst PW. The scent of cancer. Ann Intern

Med. 2010 Dec 7;153(11):767.

17. Lazar Z, Fens N, van der Maten J, van der Schee MP, Wagener AH, de Nijs SB, Dijkers E, Sterk PJ. Electronic nose breathprints are independent of acute changes in airway caliber in asthma. Sensors (Basel). 2010;10(10):9127-38.

18. Fens N, Zwinderman AH, van der Schee MP, de Nijs SB, Dijkers E, Roldaan AC, Cheung D, Bel EH, Sterk PJ. Exhaled breath profiling enables discrimination of chronic obstructive pulmonary disease and asthma. Am J Respir Crit Care Med. 2009 Dec 1;180(11):1076-82.

19. Dragonieri S, Annema JT, Schot R, van der Schee MP, Spanevello A, Carratú P, Resta O, Rabe KF, Sterk PJ. An electronic nose in the discrimination of patients with non-small cell lung cancer and COPD. Lung Cancer. 2009 May;64(2):166-70.

Phd Portfolio AMC Graduate School

Summary of PhD training, teaching and parameters of esteem

PhD supervisor: Prof. dr. P.J. Sterk, Prof. dr. W.M.C. van Aalderen

Year Workload ECTS

1.PhD training

General courses 2012 0.9

• BROK-course

Specific courses

• Proteomics, mass spectrometry and protein research 2012 2.0

• Systems Medicine 2014 2.0

Seminars, workshops and master classes

• Microbiome masterclass by Martin Blaser 2013 1.0

• Weekly department seminars 2009-2015 5.0

• Postgraduate course pediatric asthma 2013 1.0

• Masterclass Medical Business 2013 0.8

Presentations (oral and poster)

• Longdagen, Utrecht, Identification of atopy amongst wheezing

infants via electronic nose 2014 0.5

• ERS, Munich, invited speaker, upper airway metabolomics 2014 0.5 • ERS, Munich, Identification of atopy amongst wheezing infants

via electronic nose 2014 0.5

• ATS, San Diego, Symptomatic Rhinovirus infections in pre-school wheezers are associated with decreased microbial abundance in upper airways .

2014 0.5

• ATS, San Diego, Association of specific IgE measurements

with pre-school wheeze and its persistence. 2014 0.5

• Kindersymposium, Amsterdam, Symptomatic Rhinovirus infections in pre-school wheezers are associated with decreased microbial abundance in upper airways.

2014 0.5

• Kindersymposium, Amsterdam, Association of specific IgE

measurements with pre-school wheeze and its persistence. 2014 0.5 • ERS, Amsterdam, Clinical And Epidemiological Predictors

Of Physician Confirmed Wheeze In An Unselected Cohort Of infants (EUROPA-Study)

2014 0.5

• ATS, San Francisco, Clinical and Epidemiological Determinants

Year Workload ECTS

• ATS, New Orleans, Diagnostic value of exhaled breath analysis

in tuberculosis. 2012 0.5

• ERS, Amsterdam, Parent administered questionnaire captures

presence and severity of doctor confirmed wheeze in infants. 2011 0.5 • ISOEN, New York, Towards a multi-centre approach for breath

metabolomics; viability of discriminative potential after adsorption, storage and desorption of exhaled air samples

2011 0.5

• ATS, Denver, Reliability Of ‘Doctor’s Confirmed Wheeze’ In

Infants: Validation In The EUROPA Cohort 2011 0.5

• ATS, Denver, Added Value Of Exhaled Breath Analysis To Viral Profiling And Atopy In The Phenotyping Of Infants With Confirmed Wheeze.

2011 0.5

• EKZ, Amsterdam, Added Value Of Exhaled Breath Analysis To Viral Profiling And Atopy In The Phenotyping Of Infants With Confirmed Wheeze.

2011 0.5

• ATS, Bronchoscopic air-sampling by electronic nose for

molecular assessment of lung cancer 2010 0.5

• ERS, Barcelona, Assessment of virus-induced wheeze by exhaled breath molecular profiling in pre-school children,the EUROPA-study.

2010 0.5

• ERS, Barcelona, Volatile Organic Compounds Assessment of

Asthma in Children 2010 0.5

• ATS, San Diego, Exhaled breath molecular profiles by electronic nose can discriminate doctor-confirmed wheezy infants from controls

2009 0.5

• ATS, San Diego, Electronic Nose sensor responses are

repeatable when measuring a stable VOC-mixture 2009 0.5

(Inter)national conferences

• Longdagen 2013-2014 0,8

• Netherlands Respiratory Society 2010-2014 1,9

• European Respiratory Society 2009-2014 7,5

• American Thoracic Society 2009-2014 8,8

• Amsterdam Kindersymposium 2012-2014 1,0 Other • Journal club 2009-2015 8,0 PhD P or tfolio

Year Workload ECTS

2. Teaching

Lecturing

• Scientifc writing course 2011 3.5

Supervising scientific internships

• Anne Marie, exhaled VOCs in asthmatic children 2012-2013 3.0 • Lies Hulshof, effects of allergy on exhaled VOCs 2014-2015 2.0 • Emilie de Groot, temporal wheeze patterns 2014-2015 2.0 • Hellen Buijze, VOC based tuberculosis diagnostics 2011 3.0 • Marloes van den Ouweland, IgE and allergy in wheezy infants 2014-2015 2.0 • Martine Regenboog, reproducibility of auscultation 2010 3.0 • Nine Repelaer van Driel, exhaled VOCs in Cystic Fibrosis 2010 3.5 • Robin Raabe, reproducibility of VOC measurements 2009 3.0 • Romy van Amelsfoort, exhaled VOCs in wheezy infants 2009 3.0 • Sarah Bever, Pediatric Respiratory Control Questionnaire 2011 2.5 • Tessa Snoeren, VOC based characterization of wheezy infants 2011 3.0

Year

3. Parameters of esteem

Grants

• Lung foundation personal grant 2014

• Lung foundation grant 2008

• TEVA research grant 2010

• Dutch Lung foundation, International Felllowship Grant 2011

• Biomerieux research grant 2012

• NRS Science Award 2012

Awards and prizes

• Best of Pediatric Science abstract, American Thoracic Society 2014

• MLDS science award 2014

• American Thoracis Society, Assembly on Microbiology, Best Abstract 2012 • American Thoracic Society, Assembly on Pediatrics, Best Abstract 2011

• Kindersymposium Best abstract awards 2011

• American Thoracic Society, Assembly on Clinical Problems, Best Abstract 2010 • American Thoracic Society, International Trainee, Best Abstract 2010