University of Groningen
Development of genetic manipulation tools in Macrostomum lignano for dissection of
molecular mechanisms of regeneration
Wudarski, Jakub
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Publication date: 2019
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Wudarski, J. (2019). Development of genetic manipulation tools in Macrostomum lignano for dissection of molecular mechanisms of regeneration. Rijksuniversiteit Groningen.
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APPENDICES
Nederlandse Samenvatting
Acknowledgements
CV
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NEDERLANDSE SAMENVATTING
Deze thesis is gecentreerd rondom de ontwikkeling en het gebruik van methoden voor de genetische manipulatie van de platworm Macrostomum lignano. Wij presenteren hoe de toegang
to transgene technieken het onderzoek in het veld van platworm biologie mogelijk maken. We hebben ook gebruik gemaakt van deze ontwikkelde methoden om biologische vraagstukken op te lossen rondom de reactie op stress veroorzaakt door temperatuurschok, alsmede de verschillen in de transcriptomische profielen tussen de neoblast en kiembaan cellen.
Hoofdstuk 2 bevat een algemene introductie tot het model organisme wat gebruikt word in deze thesis. Het is een literatuur onderzoek met als de doel de lezer te informeren in de belangrijkste onderwerpen en de nomenclatur die gebruikt word in de opvolgende hoofdstukken. Naast het presenteren van de basis van de ontwikkeling, voorplanting en de regeneratie van Macrostomum lignano, geeft hoofdstuk 2 ook een overzicht hoe onderzoeksvelden
als; stamcell onderzoek, regeneratie, verjonging en onderzoek naar genetisch onderhoud de voordelen kunnen ondervinden van het gebruik van M. lignano als modelorganisme.
Hoofdstuk 3 presenteerd de ontwikkeling en de implementatie van de transgene technieken voor Macrostomum lignano. Hier word de moleculaire gereedschapskist voor
de genetische manipulatie van M. lignano geintroduceerd waarop verder uitgewerkt en
getest word in de opvolgende hoofdstukken. De microinjectie methode die gebruikt word is robuust, reproduceerbaar en betrouwbaar. Tot op de dag van vandaag is het de enige methode die beschikbaar is om stabiele transgene platwormen te creëren. Dit hoofdstuk presenteerd ook de meest recente genoomassemblage van M. lignano alsmede de bijgewerkte
transcriptoomassemblage en genoom annotatie.
Hoofdstuk 4 verkent de mogelijkheid om het piggyBac transposon systeem te gebruiken om
de stabiele integratie van gewenste DNA fragmenten te faciliteren. Er ligt een sterke nadruk op de vooruitgang die deze methode bied ten opzichte van de methode die gepresenteerd word in hoofdstuk 3. Hierin worden ook de nieuwe mogelijkheden voor de toekomstige ontwikkeling van de technieken voor de genetische manipulatie van M. lignano verkend.
Hoofdstuk 5 is geconcentreerd op hoe de temperatuur de basis eigenschappen van
Macrostomum lignano beinvloed, voornamelijk ontwikkeling, voortplanting en regeneratie.
Er word ook uitgelicht hoe transgene dieren gebruikt kunnen worden om de hitteschok geactiveerde stress reactie en regeneratie snelheid in kaart te brengen. Tot slot laat dit hoofdstuk zien hoe de manipulatie van de temperatuur toekomstige experimenten met
Macrostomum lignano kan vergemakkelijken.
Hoofdstuk 6 presenteerd hoe de weefsel specifieke expressie van fluorescerende eiwitten gebruikt kan worden om betrouwbaare informatie te verkrijgen over de genexpressie. De specificiteit van de techniek maakt het mogelijk om een meer detailleerde kenschets te maken van de platworm neoblasten door ze meer precies te scheiden van de kiemlijn. Dit hoofdstuk presenteerd de praktische toepassing van de beschikbare transgene lijnen en hoe de gegevens die verzameld zijn met M. lignano een meerwaarde hebben aan huidige bevindingen en de weg
zullen wijzen voor toekomstig onderzoek.
Het werk wat gepresenteerd word in deze thesis opent de weg voor nieuwe mogelijkheden in de velden van platworm biologie, stam cell onderzoek en verouderingsonderzoek. Een van de belangrijkste factoren om in gedachte te houden is dat de methode die gepresenteerd word voornamelijk een basis principe is. Ondanks dat de afleveringsmethode verfijnt en geoptimaliseerd is, kunnen de methoden voor de genetische manipulatie nog steeds verbeterd en uitgebreid worden. Een voor de hand liggende eerste stap is het in gebruik nemen van de CRISPR/Cas9 systeem. Hierdooor kunnen precieze genetische manipulatie, knock-in- mutaties en knock-out mutaties plaatsvinden waardoor het scala aan mogelijkheden voor de genetische manipulatie van Macrostomum lignano compleet zal zijn.
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ACKNOWLEDGEMENTS
This thesis is the effect of a long journey I started in 2012. It would not be possible without the aid and good will of many people to whom I owe my deepest gratitude. I am pretty sure I will forget someone, so I want to apologize for that immediately and move on to the long list of fabulous people I had the privilege to work with.
First of all I want to thank my boss – prof. Berezikov, Zhenya. Thank you so much for accepting me as your PhD student, I am still amazed that I was able to get this position! I really hope that I met your expectations and that this thesis proves your trust in my abilities was not ill-placed. I am very grateful for your guidance, both scientific and organizational. I am in constant admiration of the way you are tackling the most important matters of running your lab and I regard you as ‘the best PI I have ever met’! I can safely say that you are my role model and that you have inspired me in many ways. I want to wish you all the best in any future endeavors and I hope we will work together more!
Second I want to give my gratitude to my committee members, prof. Nollen, prof. Reggiori and prof. Tijsterman, who accepted the time consuming task of evaluating my work. Thank you very much for your effort and I hope that reading my thesis was not a waste of your precious time.
Next I want to thank my co-promotor – prof. Guryev. Dr. Victor thank you for accepting this task and making sure that what I wrote was accurate. I want to also thank you for your good advice throughout all my years as a PhD especially during the Molecular Biology Tools seminar and the NGS course. Thank you also for our joined lab retreats that will remain one of my most fond memories from the PhD times!
I have started my PhD in ERIBA as one of the first and by now I consider myself a bit of a ‘Van Wilder’ of the institute (google if you don’t get the movie reference). Throughout the years in ERIBA I have met many fantastic people and I want to at least try to put them all over here to save the good memories. So, without any further ado:
Edyta! Thank you so much for being here and agreeing to be my paranymph! I am honored by the sole fact that you will bother coming here for my defense! Thank you for the discussions, (both scientific and not-so-much) and all the help I got from you. The fact that we still keep in touch proves how much you mean to me. Jeszcze raz dziękuję za wszystko!
Tristan, my second paranymph and my friend! Me Brudah! Thanks to you I kno da wae! I am extremely happy that chose ERIBA for you PhD and that I have met you! Thanks to you coffee corner became a lively place and PhD committee became a synonym of ‘fun activities’. I think the whole institute owes you for that! Thank you for agreeing on the paranymph task. It’s another one I will throw into the basket
named ‘I owe you’. Thanks for being there and I wish you all the best! Remember that you can call me at any time and I will heed the call.
Seka! My best procrastination partner! I don’t know if you will manage to come for the defense, but if you will we’ll gonna celebrate! The amazing thing is, that our chats have actually evolved into pushing the neogenin project you started. I think this is the prime example how much non-forced discussions in a scientific institute can do. I really wish to thank you for everything (all the alms) and I hope we will continue to keep in contact.
I guess now is the time for my lab members, so here goes nothing:
Magda, I don’t know if you will get this book, but thank you for pushing me to apply for the position and for helping out along. Our ways may have parted but I will remember all the good times we had.
Stijntje! Thank you very much for your help and support, besides, it was on your birthday party I met Zhenya, so double thanks for that. Talking to you is always a well spent time. BTW congrats to you and Sara on your engagement! I wish you all the best.
Lisa, Margriet and Frank. If it was not for you and your hard work this thesis would not have been possible, so great thanks for your support.
Dr. Daniel and Dr. Philipp – thank you for teaching me molecular biology, I think I managed to put your lessons to good use.
Dan, Turan, Imre – thank you for starting the project and paving the way towards what became my thesis.
Joscha, Katja, Lera – thank you for all the discussions we had.
All the students I had – Raphael, Gwenny, Arno, Filipa, Polina – thank you for teaching me patience and some organizational skills. I hope you have learned something useful during your time with me as a supervisor.
Finally Kirill! Kiriusza, мой друг, you thought me so many things I can’t even name. You are a fantastic person and a great scientist, I am really sorry I didn’t manage to finish before your departure, but we will meet again, hopefully soon. I wish you and your fantastic wife Liza all the best.
I was very fortunate to do my PhD in a newly opened institute and ERIBA proved to be an amazing place to work in, a place where people with different expertise come together and lear from each other. For these opportunities I wanted to first thank all the PI’s: Cor, Michael, Maria, Marco, Floris, Peter, Judith, Christian and Liesbeth. Next, the fantastic administration team: Henk, Nina, Annet and Sylvia, it’s amazing how efficient and helpful you people are! Finally all the post docs, technicians and PhD students that I had contact with. I am really grateful for the lively discussions we had, so thanks to: Klaske, Bjorn, Sara (Russo), Martti, Celine, Sara (Merzouk), Anton, Inge, Ilke, Bora, Judith, Arthur, Diana, Danielle, Sonia, Jennefer, Thamar,
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Nynke, Katya, Justyna, Georges, Paula, Irina, Laura, Wytse, Erik, Johannes, Marije, Genia, Eike, Mathilde, Tobias, David, Leen, Anita, Renee, Jas, Alejandra, Simone, Christine, Hidde, Britt, Gertrude, Evert-Jan, Yin Fai, Ronald, Roland, Xin Xuan. If I missed someone, please forgive me!
There are of course people to whom I owe a special thanks, so I need to give them some more acknowledgement space:
Clemence. Thank you very much for your support. I am very happy we have met and I wish you fantastic results with your newly acquired grant!
Quentin – It’s been a while since we last saw each-other, but the legend of Quentin ‘Me Gusta!’ still lives in ERIBA and I think it will never die!
Eniko, thank you for staying in ERIBA and making this institute a better place. Nothing makes me more happy than tightening good old Polish-Hungarian friendship! Best of luck for you and Tristan!
Gloria, I know we are in a constant argument, but I must admit that this cat-and-dog relationship is one that I really enjoy. All the best with you PhD, I am positive you will nail it, and remember that the sculpture outside of ERIBA is representing chromosomes!
Sanjana, I am very happy for you, to finally start your own PhD. I am sure you will make for a great scientist! Thank you for your stories and bringing some literature discussion into the building!
Satya! Dr. P. You made me realize that talking about difficult philosophical topics can help one in organizing their thoughts around scientific projects and for that I owe you a great deal of gratitude. I truly hope we will have an occasion to meet again one day, sit down with a cup of god tea and continue to talk about the foundations of the universe.
The Pelican keep team. Paco and Mark (and Tristan obv.), thank you very much for helping me out with many things, including having me as a guest for quite some time. I hope we will keep in touch.
Lenya! Thank you so much for being here and helping everyone with improving their scientific thinking. You are one of the most recognizable characters in the building and I am glad I had the opportunity to meet you.
Thank you to all the good people I have met in Groningen: Maciek, Marysia and Lenka, Łukasz, Ewa, Milenka, Mieszko and Dobruś, Ana and Iker. You made this time much more enjoyable!
Czas na szybką zmianę języka, więc…
Janek, nawet nie wiem jak cię podpisać, tak czy siak wiesz, że podziękowania są tutaj niezbędne, także za to, że chciałeś przejrzeć tę pracę i wytknąć parę błędów.
Pajo, Paula, Jaś i Olga, dziękuję, że cały czas możemy się widzieć i od czasu do czasu pogadać.
Krzysiu Kałużny, dzięki za liczne pogadanki i wspólne spotkania. Specjalne podziękowanie dal Jacka i Woźniaków!
Mama i Tata, dziękuję za wsparcie i pomoc we wszystkim co robię. Bez was nie byłbym w stanie ukończyć tego doktoratu.
Gisio i Jula, dziękuję za pomoc z tekstem oraz dziećmi.
Buko i Szymi (i wasze rodzinki), dzięki, że jesteście i że cały czas jesteśmy w kontakcie. Na koniec najważniejsze podziękowania dla mojej najlepszej żony Kingi oraz wspaniałych dzieci Wandzi, Wawrzyńca i Tereski. Dzięki wam to wszystko było możliwe i chyba warte wszelkich poświęceń.
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CURRICULUM VITAE
Personal Data:
Name: Jakub Jozafat Wudarski
Date and place of birth: January 1986, Torun, Poland Nationality: Polish
Education: 2010 – 2011
Postgraduate education training Forensic science Toxicology Center for Forensic Science, University of Warsaw
2007 – 2009
MSc in Biotechnology Warsaw University 2004 – 2007
BSc in Biotechnology
Nicolaus Copernicus University, Torun Scientific Education
2012 – 2018 PhD Candidate
European Research Institute for the Biology of Ageing, University Medical Centre Groningen Grants
2013
PUBLICATIONS
J. Wudarski, K. Ustyantsev, L. Glazenburg, and E. Berezikov, “Influence of temperature on the development , reproduction and regeneration in the flatworm model organism Macrostomum lignano,” bioRxiv 389478, pp. 1–8, 2018.
S. Mouton, J. Wudarski, M. Grudniewska, and E. Berezikov, “The regenerative flatworm Macrostomum lignano , a model organism with high experimental potential,” Int. J. Dev. Biol., vol. 558, pp. 551–558, 2018.
J. Wudarski, D. Simanov, K. Ustyantsev, K. de Mulder, M. Grelling, M. Grudniewska, F. Beltman, L. Glazenburg, T. Demircan, J. Wunderer, W. Qi, D. B. Vizoso, P. M. Weissert, D. Olivieri, S. Mouton, V. Guryev, A. Aboobaker, L. Schärer, P. Ladurner, and E. Berezikov, “Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano,” Nat. Commun., vol. 8, no. 1, p. 2120, 2017.
C. Chichioco-Hernandez, J. Wudarski, L. Gevaert, and L. Verschaeve, “Evaluation of cytotoxicity and genotoxicity of some Philippine medicinal plants,” Pharmacogn. Mag., vol. 7, no. 26, p. 171, 2011.
L. Verschaeve, R. Anthonissen, M. Grudniewska, J. Wudarski, L. Gevaert, and A. Maes, “Genotoxicity investigation of ELF-magnetic fields in Salmonella typhimurium with the sensitive SOS-based VITOTOX test,” Bioelectromagnetics, vol. 32, no. 7, pp. 580–584, 2011.
