University of Groningen
Dissecting the temporal dynamics of eukaryotic metabolism in single cells
Takhaveev, Vakil
DOI:
10.33612/diss.119793412
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Publication date: 2020
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Takhaveev, V. (2020). Dissecting the temporal dynamics of eukaryotic metabolism in single cells. University of Groningen. https://doi.org/10.33612/diss.119793412
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Nederlandse samenvatting
Vertaling: Hanna M. TerpstraOnze onderzoeksgroep heeft recentelijk metabole oscillaties in de tijdspanne van één tot enkele uren ontdekt in individuele cellen van Saccharomyces cerevisiae, een eukaryoot modelorganisme. Deze oscillaties zijn waargenomen voor NAD(P)H en ATP en vinden plaats in synchronie met, maar toch onafhankelijk van de celcyclus. Het doel van dit proefschrift is 1) het vaststellen van de onderliggende oorzaak van deze metabole oscillaties in afzonderlijke S. cerevisiae-cellen, alsmede 2) het valideren van eerder beschreven methoden en het ontwikkelen van nieuwe technieken om de temporele dynamica van het metabolisme van S. cerevisiae te onderzoeken op het niveau van individuele cellen.
In Hoofdstuk 2 hebben wij onderzocht wat de metabole oscillaties veroorzaakt. Allereerst hebben we experimenteel aangetoond dat het vermoeden dat metabole oscillaties voortkomen uit stofwisseling van opgeslagen koolhydraten of uit respiratie onjuist is. Vervolgens stelden we de hypothese op dat de metabole oscillaties veroorzaakt worden door temporele scheiding tussen de synthese van de voornaamste componenten van biomassa, zoals eiwitten, lipiden en koolhydraten. Hierdoor verandert de vraag naar specifieke precursors, wat leidt tot een herordening van de fluxen in het centraal koolstof- en energiemetabolisme. Door gebruik te maken van microfluïdica en het dynamisch meten van NAD(P)H in individuele cellen, hebben we een nieuwe, op verstoringen gebaseerde methode ontwikkeld om de metabole activiteit van deze cellen te meten. Op basis van deze en andere, onafhankelijke technieken hebben we ontdekt dat er twee activiteitsgolven van eiwitsynthese zijn per celcyclus. Dit weerspreekt het momenteel heersende inzicht. Lipide- en polysaccharidesynthese pieken daarentegen eenmaal per celcyclus. De biosyntheseactiviteit van deze componenten neemt af ten tijde van de eerste piek in eiwitsyntheseactiviteit, en bereikt een maximum tijdens de tweede piek. Bovendien hebben we aangetoond dat groei, dat wil zeggen toename in omvang van de cel, niet strikt gekoppeld is aan eiwitsyntheseactiviteit gedurende de celcyclus. Groei wordt waarschijnlijk beperkt door de dynamica van lipide- en polysaccharidesynthese. Wij hebben de relatieve, eenheidsloze biosynthesesnelheden, bepaald via onze op verstoring gebaseerde methoden, omgezet in fundamentele, absolute eenheden. Hiervoor hebben we gebruik gemaakt van een nieuw wiskundig model dat de dynamica van celmassa en haar samenstelling gedurende de celcyclus beschrijft. Door de absolute biosynthesesnelheden te introduceren in een uiterst geavanceerd thermodynamisch-stoichiometrisch model van het metabolisme van gist, hebben we de dynamische fluxverdeling van het centraal koolstof- en energiemetabolisme gedurende de celcyclus afgeleid. Om deze fluxverdeling te verifiëren, hebben we experimenteel aangetoond dat de opnamesnelheid van glucose verandert gedurende de celcyclus en piekt rond het slot van de mitose.
In dit hoofdstuk hebben we dus aangetoond dat de biosynthese van verschillende biomassacomponenten inderdaad gescheiden is in de tijd, wat zorgt voor dynamische veranderingen in het centrale metabolisme. Zo hebben we onze kennis over de metabole oscillaties en hun oorsprong verdiept. Bovendien geven wij het eerste grootschalige overzicht van fluxveranderingen in het metabolisme gedurende de celcyclus, zowel voor de belangrijkste biosyntheseprocessen als de kern van het cellulair metabolisme.
In Hoofdstuk 3 streefden we ernaar om een FRET (Förster Resonance Energy Transfer) -sensor voor pyruvaat te introduceren en valideren in gist. Deze -sensor is meermaals met succes toegepast in dierlijke cellen en was daarom een veelbelovend middel om de dynamica van pyruvaat te volgen ten opzichte van de metabole oscillaties. Pyruvaat is een metaboliet die zich bevindt op het snijpunt van talrijke metabole routes en die mogelijk fluxen stuurt. Een sensor voor pyruvaat in gist zou daarom een groot goed zijn voor zowel fundamenteel onderzoek naar metabolisme en de celcyclus, als de biotechnologie, die veel gebruikmaakt van gist als productieorganisme voor relevante moleculen. We hebben een codongeoptimaliseerde versie van de FRET-sensor tot expressie gebracht in S. cerevisiae en hebben deze getest in time-lapse fluorescentiemicroscopie-experimenten waarbij het metabolisme dynamisch werd verstoord. Hoewel we waarnamen dat de sensor een ander signaal geeft na diverse wisselingen van koolstofbron, geeft de sensor de verwachte veranderingen na gerichte verstoring van het pyruvaatmetabolisme niet waarachtig weer. Een onafhankelijke enzymassay doet dit echter wel. Verrassend genoeg geeft de FRET-sensor, die niet reageert op pyruvaat, wel een oscillerend signaal gedurende de celcyclus. Dit kan een artefact zijn, dat mogelijk ook voorkomt bij andere FRET-sensoren die gebruikt worden in de context van het celcyclusonderzoek.
In Hoofdstuk 4 hebben we ab initio een synthetische sensor op basis van RNA ontwikkeld voor fructose-1,6-bisfosfaat (FBP); met deze sensor kunnen we de flux door de glycolyse meten. We hebben een grootschalige bibliotheek van fusies van een in vitro geselecteerd FBP-bindend aptamer en een hammerhead ribozyme opgebouwd en deze RNA-moleculen zijn tot expressie gebracht in gistcellen. Deze gistcellen zijn gecultiveerd onder verschillende omstandigheden met uiteenlopende intracellulaire FBP-concentraties. De sensoren die reageerden op verschillen in de FBP-concentratie zijn vervolgens in vivo getest in een high-throughput screening. In deze screening hebben we grote hoeveelheden FACS- (fluorescence-activated cell sorting) en NGS- (next generation sequencing) data geanalyseerd en zodoende enkele kandidaat-FBP-sensoren geïdentificeerd. Deze zijn vervolgens individueel getest, waarbij we hebben bevestigd dat het afgegeven signaal voor bijna alle kandidaten verandert tegelijk met de intracellulaire FBP-concentratie. Interessant genoeg werd zowel positieve als negatieve correlatie gevonden tussen de FBP-concentratie en het door de sensoren afgegeven signaal. Dit wijst erop dat verschillende RNA-moleculen de FBP-concentratie op een andere wijze weergeven. Nadat we de FBP-bindingsplaats van twee van de sensoren muteerden, correleerde het gemeten signaal niet langer met de FBP-concentratie.
Hierdoor kunnen we concluderen dat we twee FBP-sensoren ontwikkeld hebben. Door gebruik te maken van fluorescentiemicroscopie in combinatie met microfluïdica, hebben we gevonden dat één van de FBP-sensoren ook de glycolytische flux rapporteert. Deze sensor laat namelijk onderscheid zien tussen langzaam en niet-delende cellen. Daarnaast verandert het signaal van de sensor na toevoeging van een inhibitor van de glycolyse.
In Hoofdstuk 5 beschrijven wij welk onderzoek, voortbouwend op de kennis uit dit proefschrift, gedaan moet worden. Daarnaast behandelen we toepassingen van in dit proefschrift gegenereerde kennis om in de toekomst na te streven.
Curriculum Vitae
2015 – 2019 Marie Curie PhD student, Molecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, the Netherlands; European research-training network MetaRNAMajor results of this PhD work are presented in this thesis. The supervisor: Prof. Dr. M. Heinemann.
2014 – 2015 MSc student intern, Department of Human Genetics, Leiden University Medical Center, the Netherlands
Specialist (MSc) diploma work: Effects of biological ageing on RNA processing. The supervisors: I. Pulyakhina (now Dr.), Dr. J. Laros, Prof. Sc.Dr. M. Gelfand, Dr. P.A.C 't Hoen (now Prof.).
2011 – 2014 Student intern, Bioinformatics and Structural Biology group, Department of Biokinetics, Belozersky Research Institute of Physico-Chemical Biology, Moscow State University, Russian Federation
Three research projects: Comparative bioinformatic analysis of fungal pyranose 2-oxidases and pyranose dehydrogenases reveals amino acid residues responsible for regioselectivity of monosaccharide oxidation; Characterization of enzyme-substrate interactions in glutaryl acylase from Brevundimonas diminuta by molecular modelling; Bioinformatic analysis of proteins from Mycobacterium tuberculosis complex: new targets for selective inhibition. The supervisors: Dr. D. Suplatov, Prof. Sc.Dr. V. Švedas.
2010 – 2015 Specialist (MSc) (Summa Cum Laude), Faculty of Bioengineering and Bioinformatics, Moscow State University, Russian Federation 2008 – 2010 High school for gifted children, Faculty of Biology and Chemistry, School for Cosmonautics, Zheleznogorsk, Siberia, Russian Federation 1999 – 2008 General school, Ikshurma village, Siberia, Russian Federation
List of publications and manuscripts
1) Takhaveev, V., Özsezen, S., Chillet, M., Litsios, A., Chen, H., Papagiannakis, A., & Heinemann, M. Temporal segregation among biosynthetic processes is responsible for metabolic oscillations during eukaryotic cell cycle. Manuscript in preparation.2) Takhaveev, V., Łazarewicz, N., Bonsing-Vedelaar, S., & Heinemann, M. A pyruvate FRET-sensor used in animal cells does not function in Saccharomyces cerevisiae but exposes an oscillatory behavior of metabolism. Manuscript in preparation.
3) Ortega, A.D. #, Takhaveev, V. #, Bonsing-Vedelaar, S., Long, Y., Mestre-Farràs,
N., Incarnato, D., Ersoy, F., Olsen, L.F., Mayer, G., & Heinemann, M. (2020). A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux. Under review in Nature Chemical Biology.
4) Monteiro, F.#, Hubmann, G.#, Takhaveev, V., Vedelaar, S., Norder, J., Hekelaar,
J., ... & Heinemann, M. (2019). Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor. Molecular Systems Biology, 15(12).
5) Leupold, S.#, Hubmann, G.#, Litsios, A., Meinema, A.C., Takhaveev, V.,
Papagiannakis, A., ... & Heinemann, M. (2019). Saccharomyces cerevisiae goes through distinct metabolic phases during its replicative lifespan. Elife, 8, e41046.
6) Takhaveev, V., & Heinemann, M. (2018). Metabolic heterogeneity in clonal microbial populations. Current opinion in microbiology, 45, 30-38.
7) Filer, D., Thompson, M. A., Takhaveev, V., Dobson, A. J., Kotronaki, I., Green, J. W., ... & Alic, N. (2017). RNA polymerase III limits longevity downstream of TORC1. Nature, 552(7684), 263.
8) Pulyakhina, I.#, Takhaveev, V.#, Vermaat, M., van Iterson, M., Gelfand,
M.S., Laros, J.F.J., ’t Hoen, P.A.C., BIOS consortium, LifeLines, & Leiden Longevity Study (2015). Aging is associated with increased incidence of alternative splicing. Pulyakhina, I., 2016, Doctoral Thesis, Leiden University, ISBN: 9789461826459 9) Suplatov, D., Kirilin, E., Arbatsky, M., Takhaveev, V., & Švedas, V. (2014). pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families. Nucleic acids research, 42(W1), W344-W349.
10) Suplatov, D., Kirilin, E., Takhaveev, V., & Švedas, V. (2014). Zebra: a web server for bioinformatic analysis of diverse protein families. Journal of Biomolecular Structure and Dynamics, 32(11), 1752-1758.
Conference participation
1) Talk, Yeasterday 2019, 17th June 2019, Groningen, the Netherlands2) Talk, 19th International Conference on Systems Biology, 30th October 2018,
Lyon, France
3) Poster, Dutch Biophysics, 1-2nd October 2018, Veldhoven, the Netherlands
– the best poster award, 1st prize
4) Talk, MetaRNA Mini Symposium, 2nd July 2018, London, the UK
5) Poster, Yeasterday 2018, 7th June 2018, Delft, the Netherlands
6) Poster, 25th Annual GBB Symposium, 29th August 2017, Groningen, the
Netherlands – the best poster award, 1st prize
7) Poster, 24th Annual GBB Symposium, 1st September 2016, Groningen, the
Netherlands – the best poster award, 1st prize
Science-communication pitches
1) 3 Minute Thesis Competition, University of Groningen, 14th March 2018,Groningen, the Netherlands – the winner
2) National final, Famelab 2017, 3rd May 2017, Utrecht, the Netherlands
– a national finalist
3) Regional heat, Famelab 2017, 9th March 2017, Groningen, the Netherlands
– the winner
Research grants
NWO Open Competition XS grant. The 8-month postdoc project “Do cancer cells have a thermodynamic Achilles’ heel?” V. Takhaveev, M. Heinemann (submitted by M. Heinemann). Announced on 13th December 2019.Daily supervision of students
1) Arriën Symon Rauh, bachelor’s student, August 20162) Cas Cornet, bachelor’s student, September 2016 – July 2017 3) Hanna Terpstra, master’s student, February – July 2017
4) Marije Been, master’s student, February – July 2017 (partial supervision) 5) Natalia Łazarewicz, master’s student, September 2017 – February 2018 6) Marten Chaillet, master’s student, March – September 2018
7) Eglė Kybartaitė, Erasmus+ master’s student, July – September 2018 8) Michelle Scharte, master’s student, December 2018 – July 2019
Acknowledgements
Regarding this bookI am grateful to Prof. Dr. Matthias Heinemann without whose guidance, inspiration and help this book would be impossible. I am very much obliged to the co-supervisor and members of the assessment committee Prof. Dr. Peter van Haastert, Prof. Dr. Bas Teusink, Prof. Dr. Ida van der Klei and Prof. Dr. Bert Poolman for spending their time and energy on the careful inspection of this thesis. I also thank members of the examination committee for going through this work and thinking about it.
Regarding my work, leisure and development
I am an incredibly lucky man because I have been always surrounded by exceptional people. It would take me many volumes to give a comprehensive description to whom and why I have been grateful. Not to bore everybody with a very long reading, in the following text, I will try to briefly mention only some of the exceptional people who inspired me and helped to finish this PhD. If you, dear reader, do not find yourself here, please contact me, and I will express you my gratitude personally. Matthias, a.k.a. Prof. Dr. Matthias Heinemann, I remember I wrote in the motivation letter that I would like to have a boss from whom I would learn a lot in both professional and personal dimensions. After more than four years of interacting with you daily, I say that you were the best match for me. I thank you for giving me the most interesting projects with huge potential. I thank you for stimulating my creativity, giving the immense freedom to implement it and simultaneously suggesting how to get the most out of experiments, analysis and writing. I thank you for being sincerely excited about my results and conclusions, it greatly motivated me to do more. I thank you for being available virtually 24/7. I thank you for teaching me a lot about the visualisation of complex data, you are extremely good in this! I thank you for your countless advices regarding academic writing, presentations, design of scientific studies, career in academia etc. You are an extraordinary person, which made my PhD a very special time of my life with a lot of nice memories. There is a feature of your personality that inspires me most: it is your remarkable proactivity, tireless desire and ability to improve yourself and your environment – I would like to be like you in this regard. I wish you many scientific breakthroughs, a pleasant group of colleagues, life without worries and every-day happiness.
Andreas, a.k.a. Dr. Andreas Milias-Argeitis, I am grateful to you for numerous advices regarding statistics and data analysis. Gaussian process regression that you encouraged me to use has been one of the most interesting and powerful statistical tools in my research. I greatly appreciate scientific discussions on cell cycle, signalling and metabolism in which you participated as I learnt a lot from them. You have
encyclopaedic knowledge, which makes it incredibly interesting to talk with you. There is a flame of pure scientific curiosity in your eyes, and it has an inspiring effect. Álvaro, a.k.a. Dr. Álvaro D. Ortega, I thank you immensely for the opportunity to work together on the RNA-sensor project that I find scientifically beautiful and relevant for many applications. You designed this cool project and put a lot of effort and time in it to make everything work, which I more than admire. Working with you, I learnt much with respect to science, visualisation and data analysis. It makes me sad that there was a period of schism between us regarding this work, it was heart-breaking especially because I like you a lot. Thanks for solving this. I am so much happy that we managed to submit our work. I respect your honesty, directness and integrity. Thanks for interesting talks, dinners, drinks when you were living in Groningen and when I came to Madrid.
Serdar, a.k.a. Serdar Özsezen, it has been a great pleasure to talk to you almost every day of my PhD. Discussing our science, writing a code together, philosophizing on political issues, complaining to each other, dining, travelling to MetaRNA meetings, your learning Russian – I have been enjoying many moments of our mutual experience. Thanks for finding time and energy to work on our project when you were looking for a new position. I am convinced that you will manage to finish your book very soon, and that we will submit our paper in a short time. I wish you good luck in your future, in which I hope we will often meet!
Silke, a.k.a. Silke Bonsing-Vedelaar, thanks for being a very good colleague. I love the data of your pyruvate measurements. I appreciate that you always want to improve, discuss any problem openly and carefully listen to others.
Hanna, a.k.a. Hanna Marije Terpstra, you were a wonderful student, and now you are a very helpful and funny colleague. Thanks a lot for your beautiful and purist Dutch translations of my thesis summary.
Natalia, a.k.a. Natalia Łazarewicz, thanks for being one of the best students ever! You not only worked very hard, were very organized, quadruple-checked all your strains, but also made me laugh almost non-stop, you are amazing.
Paranymph Luc-Alban, a.k.a. Luc-Alban P.E. Vuillemenot, I fell in love with you immediately when we started discussing Harry Potter during your first visit to apply for PhD. I have been enjoying so much being with you in the lab, in our office, while having numerous coffees, while recalling all words of French origin that I know etc. You are funny and very supportive; your checking me in those moments when I was sad was the most potent remedy for me to get back my normal good mood, thanks. Your positive effect on me is done even not by words, but by you sarcastic smiles and the aura of tenderness that you radiate. I am sure you will make your demanding FRET-sensor working, at least more useful than mine, and find something novel and exiting in TOR. Alexane, thanks for your always very funny company, take care of Luc.
Paranymph Mattia, a.k.a. Mattia “Enrico” Rovetta, for me, it would already be worth joining MSB group only for the sake of meeting you there three years later. I love your light-heartedness, optimism and open-mildness (although you are a milder libertarian than me). It is a gift to me that I can discuss with you anything: science, work, controversial social topics, things that might be considered disgusting by some other people. Thanks a lot for your sincere curiosity in my projects, for really getting into them, advising me and always supporting me, especially in the last year! I wish you many interesting findings and eureka effects in the impressive omics-data that you are working so hard to get.
Тамара, a.k.a. Tamara Kurdyaeva, you are солнышко1. My work and life at large
became so much funnier and brighter when you arrived to Groningen. I love our chats, your contagious laughter, your always dazzling, inventive and colour-balanced style of appearance. I especially thank you for the events and places you brought me to (although, being a boring person, I joined only 1% of all suggested by you). I am grateful to you for your statistical advices to me, with the most important lesson of abandoning comparisons between p-values. I will never forget how you were happily jumping after my performance at the 3MT, and how you brought me a cake with 4.5 candles after my thesis submission. You are a wonderful person, I am so much lucky to share with you these years.
Diego, or “Dee–ye–ga”, a.k.a. Dr. Diego Alonso Martinez, you short stay in our lab and Groningen was an explosion of positive emotions! The rare cases of seeing you nowadays are pure celebrations for me when I forget of all my worries and just have fun. Thanks for support, thanks for becoming Uncle Diego to our daughter-cat, thanks for the unforgettable trip to Russia that we had. I wish you to get to the heights you are striving for but still keep me as your dear friend.
Якопо, a.k.a. Jacopo Frallicciardi, gorgeous, the icon of style, the flamboyant one, bright, unconventional, Jasper Hale (Cullen), царица2, “I am a dancer, I love to
dance”, sophisticated, intelligent etc. – the list of your epithets is very long, which has been giving me immense joy of realizing that I got this once-in-a-lifetime chance of meeting a person like you! God, aside from all these qualities, you are also so kind, modest and very helpful. Thank you for being close to me and Alisa, you are incredible.
Vanessa, a.k.a. Vanessa Clerico Mosina, thanks for bringing beauty and style to our university and Groningen.
Dirk, a.k.a. Prof. Dr. Dirk Jan Slotboom, thanks so much for being a very good boss (inspiring, full of ideas, emotionally competent, funny etc.) and hence – indirectly – a great source of my personal happiness and calmness in the last years. Besides, I am
1 [Solnyshko], lit. a diminutive of sun, used to convey a sense of affection to a person and to highlight
grateful to you for sharing with me your wisdom and coaching me regarding some very interesting questions.
Альберт, a.k.a. Dr. Albert Guskov, first of all, thanks for recommending Alisa to Dirk as well as suggesting her the most interesting project, which became, with your contribution, a tremendous success and, consequently, my happiness. I thank you also for numerous advices that you gave to me, for supporting and cheering me up. Most importantly, Albert, Марина, a.k.a. Marina Guskov, and Leo plus Teo Guskov, thanks for the unforgettable Eurovision parties and dinners!
Валентина, a.k.a. Dr. Valentina Arkhipova, with hard work and persistence, you achieve success in very different things you touch – you inspire me a lot.
I am grateful to the former and current members of GBB that have been making my work and leisure unforgettable. Specifically, in my very own Molecular Systems Biology, I admire the extraordinariness, friendliness and intelligence of everybody: Matthias, Andreas, Álvaro, Serdar, Silke, Luc-Alban, Mattia, Tamara, Diego, Alex, Kuba, Brenda, Hannah, Francisca, Georg, Robson, Tom, Zheng, Yonathan, Ying, Ivan, Fleur, Marta, Renate, Haoqi, Douwe, Xiang, Joana, Paolo, Hanna, Anna, José, Daniele, Hugo, Kerlen, Andriana, Natalia, Marten, Cas, Michelle, Eglė etc.
In my dear lab-in-law Membrane Enzymology, I have been enjoying the company of so many bright people whom I like very much: Jacopo, Vanessa, Dirk, Albert, Valentina, Weronika, Stephan, Gianluca, Alex the Englishman, Emanuela the Ent-wife, Masha, Pavlo, Luca, Patricia, Valeria, Aike, Michele, Gea, Evi, Wojciech, Adi etc.
The neighbor lab Biotechnology added to my PhD the pleasure of talking to the most cheerful people, such as Nikola, Nikolas, Misun, Jeroen, Elvira, Eduardo, Friso, Cynthia, Alejandro etc.
Faculty of bioengineering and bioinformatics, Moscow State University, I am grateful to you for giving me the best higher education in diverse branches of biology, chemistry and mathematics, for developing my English and, most importantly, for acquainting me with so many bright, intelligent and nice people, including my wife and other best friends.
Дима, a.k.a. Dr. Dmitry Suplatov, my first scientific mentor at university, I am more than grateful for the amazing three years in your group. It was thanks to you that I got the first taste of programming and statistics, two fields very interesting to me. Your convincing talks, your style, your ability to make outstanding visual material and your organisational skills have been always an example to me. I do appreciate your generosity to me: with regards to the time you spent teaching me various subjects, three papers in which you made me a co-author for my humble contributions etc. With great pleasure and nostalgia, I remember being your student.
Ира, a.k.a. Dr. Irina Pulyakhina, I got a lot of inspiration from you: how to keep my code organised, how to prepare impressive and clear presentations, how to talk in a professional way, how to plan everything, how to organize unforgettable parties, how to be very kind to others etc. Sadly, I am far from reaching your level in these things. Thanks a lot for supporting me during my master internship at LUMC, for guiding my work, for generating ideas and for suffering from the pain of checking my Russian writing. I am so lucky that you were my supervisor and that I know you! (Years after our work together, after having several students myself, I have realised much better how incredibly good you were as a supervisor).
Peter-Bram, a.k.a. Prof. Dr. Peter-Bram 't Hoen, my master internship at LUMC under your guidance was an extremely valuable and unique experience that, I am sure, not so many students are fortunate to have. There was plenty of creative thinking, long coding, frequent presenting and productive interacting with numerous colleagues. Thanks a lot for the project that was so relevant and exciting! Thanks a lot for finding funds for my stay in the Netherlands; your efforts to make my internship possible have had many positive consequences in my life that I am becoming more and more conscious about. For me, you remain one of the best examples of a true scientist – very critical and careful to details. I do wish you a lot of interesting discoveries in future.
Yahya, a.k.a Dr. Seyed Yahya Anvar, I admire your style of making scientific plots, I want to get to your level. Thanks a lot for the inspiring and motivating words that you have told me several times, they pop up in my mind quite often.
Михаил Сергеевич, a.k.a. Prof. Sc.Dr. Mikhail Gelfand, while taking your bioinformatics lectures and seminars as well as discussing with you my master thesis, I was always amazed how fast you grasp diverse ideas and give unexpected feedback. I want to be like you in this.
Елена Николаевна, a.k.a. Prof. Sc.Dr. Elena Temereva, I was impressed by your invertebrate zoology lectures in my first semester at university. They gave me this excitement by the diversity of Nature which drives me to discover something new – for example, in (much less flamboyant) molecular biology.
Анастасия Владимировна, a.k.a. Dr. Anastasia Proshkina, I enjoyed your lectures and seminars on calculus and differential equations. They upgraded my brain, now I would like to return the intellectual power that I had when taking your course. Thanks for inspiring of how to describe complex material in the simplest way and of how to be very pedantic when it comes to maths.
Спасибо всем учителям Икшурминской начальной и средней школы, а также Школы космонавтики за то, что внесли большой вклад в мое развитие3.
Екатерина Викторовна Селезова, боже мой, как же здорово, что я учился химии у вас! Ваши теоретические уроки и зачеты были прекрасны. Я вообще сомневаюсь, что есть ещё такая школа в мире, где можно было бы проводить столько разнообразных интересных опытов, включая взрывы газов, удары молотком по врывающейся соли, реакции с серебром, вонючие реакции и пр. пр. Спасибо за очень большую химическую подготовку и ваш энтузиазм4. Юрий Валерьевич Прокофьев, вы так сильно простимулировали моё изучение биологии. С вами я узнал много вещей, о которых мне ни в Московском, ни в нидерландских университетах не говорили. Спасибо за ваши уроки и интересные классные активности5. Татьяна Михайловна Жукова, мне очень нравились ваши уроки по литературе и наши беседы. Спасибо, что не забываете обо мне и поддерживаете.6 Мамочка, Людмила Николаевна Косолапова, спасибо большое за твою любовь, поддержку и веру в меня. Спасибо за то, что, когда я рос, ты окружила меня абсолютной свободой и дала очень большой заряд креативности, нестандартности и бунтарства, который влияет на меня каждый день. Спасибо за то, что ты научила любить творчество, всё красивое, людей, природу, – ты самая лучшая мама, которую только можно пожелать. Лиза и Андрей, спасибо за то, что вы следите за мамой7. Я очень благодарен своей бабуле, Антониде Николаевне Георгиевой, за её безграничную любовь и заботу обо мне. Интерес к естественным наукам, литературе и философствованию я перенял от нее в ходе наших бесчисленных бесед в моем прекрасном детстве. Отдельное спасибо бабуле за то, что она научила меня думать математически. Благодаря ее урокам по геометрии я
4 Ekaterina Selezova, my God, it is so cool that I learnt chemistry from you! Your theoretical lessons and
exams were amazing. To be honest, I doubt that there is yet another school in this world where it is possible to perform so many different exiting experiments, including gas explosions, hammering an explosive salt, reactions with silver, stinking reactions and many others. Thanks for the broad chemical education and your enthusiasm
5 Yuri Prokofiev, you greatly stimulated my studying biology. In your lessons, I learnt many phenomena
that were taught to me neither at the Moscow State University nor at my Dutch universities. Thanks for your lessons and extracurricular activities.
6 Tatiana Zhukova, I loved your literature lessons and our conversations. Thanks that you don’t forget
about me and support me.
7 Mom, Lyudmila Kosolapova, thank you so much for your love, support and believing in me. Thanks
for that when I was growing up you surrounded me with absolute freedom and gave a big momentum of creativity, extraordinariness and rebelliousness that influence me every day. Thanks for teaching me how to like art, everything beautiful, people, nature, – you are the best mom that one could wish. Liza and Andrei, thanks for taking care of our mom.
получаю огромное удовольствие от строгой логики. Моё интеллектуальное и эмоциональное развитие во многом определено талантом моей бабули8. Папа, Ахмат Габдулхатович Тахавеев, спасибо за твою поддержку и стимулирование к достижениям (хоть оно и вызвано твоим татарским тщеславием – но именно оно вращает планету вокруг своей оси). Так здорово, что я могу обсуждать с тобой политику, историю и религию9. Мои прекрасные Гараевы: тёща Марина Александровна, тесть Алмаз Анварович и шурин Ильдарчик, – вы интересные и милые люди, я очень рад, что вы появились у меня в жизни. Father-in-law Almaz, your crazy example inspires me to believe in myself and to start new things. Ильдар, спасибо, что все эти годы, пока я живу за границей, ты просветлял меня относительно всего, что касается современной русскоязычной молодежной культуры. Если не было бы тебя, то я стал бы скучным эмигрантом, который культурно застыл в прошлом, чего я очень боюсь10.
Николай, a.k.a. Dr. Nikolai Kondratev, I immensely grateful to this universe that brought us together at university almost ten years ago. Thanks for discussing all my problems, stupid worries, events and successes as well as culture, science, politics and all other exiting and boring things in this world – it has been every day, and I want it to continue like this. I estimated that we had more than 230 Skype/FaceTime video calls during our PhDs, which, I believe, is our most important achievement! My love, support and desire to see you happy are with you wherever you are.
Долгор, a.k.a. Dolgor Buyantueva, I feel so happy and special that you are always very glad to see us. I greatly enjoy your company, you bring peace to my mind (Baikal effect). Thanks a lot for your kindness and friendship!
Ирочка, a.k.a. Irina Garanina, and Оля, a.k.a. Olga Shadrina, it is pleasant to realize that I have such funny, intelligent and sarcastic friends like you, with whom we shared so much of our university years; I love you both.
8 I am immensely grateful to my granny Antonida Georgieva for her limitless love and care. My interest
in natural sciences, literature and philosophizing originated from numerous conversations that I had with granny during my wonderful childhood. I want to thank her especially for teaching me how to think in an analytical way. Thanks to granny’s geometry lessons, I get a huge pleasure from formal logic. My intellectual and emotional development were largely determined by the talent of granny.
9 Papa, Akhmat Takhaveev, thanks for your support and stimulating me to achieve more (although this
stimulating is caused by your Tatar vanity – but vanity, in the end, rotates the Earth around its axis). It is amazing that two of us can discuss politics, history and religion.
10 My beautiful Garaevs: mother-in-law Marina, father-in-law Almaz and brother-in-law Ildar – you are
very interesting and cute people, I am very glad that you appeared in my life. Almaz, your crazy example inspires me to believe in myself and to start new things. Ildar, thanks that in all these years of me living abroad you’ve been enlightening me regarding everything related to modern Russian-speaking youth culture. Without you, I would have become a boring emigrant who is culturally frozen in the past, which
Дорогая и любимая моя Алисочка, a.k.a. Alisa “Diamond” Garaeva, I don’t see my life without you – you witnessed how everything around me broke apart when you left me for conferences or internships. Without you, I would have gone mad, become very sad and miserable. I like your sincerity, natural happiness, passion, and the fact that you are full of all kinds of emotions and feelings – they make our life exciting and rich. You are very beautiful, and it makes every day of my life worth living already in the first moments of the morning when I delight in seeing you. Thanks for supporting and inspiring me! Your believing in me is priceless. I admire your hard work and desire to develop. I think we will make it to become if not Eurovision winners then certainly at least presidents and Nobel Prize laureates. Я очень тебя люблю.
