University of Groningen
Single-molecule fret study on structural dynamics of membrane proteins Aminian Jazi, Atieh
DOI:
10.33612/diss.135802718
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Publication date: 2020
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Aminian Jazi, A. (2020). Single-molecule fret study on structural dynamics of membrane proteins. University of Groningen. https://doi.org/10.33612/diss.135802718
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Chapter 5:
124 Summary and Outlook
The Na+ coupled betaine symporter BetP counteracts hyperosmotic stress and is regulated via an osmosensing C-terminal domain. While the biochemistry of the system is well characterized, structural information is only partially available due to a lacking C-terminal domain in the crystal structure. Thus, both the activation and transport mechanism remain elusive. In this work, we established different single-molecule-based methods to study the conformational dynamics and heterogeneity of BetP in proof-of-principle studies (chapter 2/3) and a detailed mechanistic study of transport activation. Our studies showed that smFRET approach was sussef ully adopted for BetP and we obtained the results that was not yet accompolished by standard strucrtal biology teqniques such as crystal structure method. The established methods will pave the way for future single-molecule studies and enhanced mechanistic understanding of the secondary-active transporter BetP in a near-native biochemistry environment. The thesis contained the following scientific chapters:
Chapter 2, Novel approaches for fluorescence labelling of membrane transporters and isolated domains:
Single-molecule FRET has become a widely used method to investigate protein conformational dynamics and heterogeneity. FRET has provided depth insights into biochemical properties and dynamics of many biomolecular systems. Recent developments of this approach allow investigation of the structural studies in great details. Besides the structural studies, this technique has enabled scientist to visualize dynamic nature and monitor functional properties of biomolecules of interest that are hidden under ensemble measurement in the biological time-dependent interactions with high spatial and temporal resolution. However, one the main experimental challenges in FRET technique is photostability of FRET fluorophores with main driving force of their transient excursions to dark states (blinking) and irreversible destruction (photobleaching). One of the main factors limiting mechanistic insights are poor photophysical properties of dyes and the need for specific attachment strategies of fluorophore to the biomolecule [7]. These biophysical processes fundamentally hamper dye applicability and have, for a long time, restricted development of advanced microscopy techniques with single
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molecule sensitivity or optical super-resolution <250 nm. In the first part chapter 2, we demonstrated that with using FRET photostabilizer–dye conjugates with intramolecular triplet-state quenching with ‘self-protecting’ properties, prevents photodamage and keeping their applicability for labelling of different biomolecules. Photostabilizer-dye conjugates were applied to study the conformational dynamic of protein by single molecule FRET, revealing an increase in obtained smFRET data quality from labelled biomolecules compared to their non -stabilized parent commercial FRET fluorophores. Additionally, obtained result revealed that despite increase in photostability of used self-healing dyes on protein of interest, conjugated chromophores did not show any interfere with the biochemical structure and function of the protein of interest, such as SBD2 protein from ABC transporters. This non-invasive strategy has clear advantages compared to usual labelling method by preventing photo damage without the use of solution additives to FRET buffer measurement.In second part of chapter 2, I elaborated smFRET method to lable BetP which has a complex structure using different experimental approaches and FRET technique. Site-specific labelling is a crucial step in order to investigate biological systems with smFRET-based single-molecule methods, since it needs to map a relevant reaction coordinate of the system [21]. We aimed to provide an insight into protein conformational changes upon binding of substrates and activation cycle. For proteins, cysteines are artificially introduced at non-conserved and solvent-exposed positions as anchor points for the fluorescent labels. While this strategy has various problems that deal with the molecular biology and preparation of the protein mutants (and their functionality), it is further complicated in multi-subunit proteins such as BetP since one cysteine (in one subunit) appears multiple times in the complex; thus multiple labelling sites are created. Designing an appropriate strategy for site-directed mutagenesis and further conjugation of fluorophores in multi-subunit proteins such as BetP enabled us to obtain more insights into mechanistic understanding of protein.
Chapter 3, Caging and Photoactivation in Single-molecule Förster-resonance energy transfer Experiments: Caged organic fluorophores are established tools for localization-based super-resolution imaging [1]. The photoactivation mechanism of caged fluorophores relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available
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caged dyes with ON switching of the fluorescent signal by UV light. In chapter 3, experimental approaches were established to use photochemical uncaging of cyanine fluorophores and caged rhodamine dyes for single-molecule Förster resonance energy transfer (smFRET) experiments. The obtained results revealed that using Caged dyes enable temporal separation and sorting of multiple intramolecular donor-acceptor pairs in solution-based smFRET photoactivation. Thus, both a potent donor and acceptor pair have to be identified, where the two requirements, i.e, efficient caging and photoactivation, are fulfilled. While the presented experiments are of proof -of-principle character, they demonstrate our approach to visualize complex biochemical species such as multi-subunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented in this work. The caged FRET result revealed that chemical caging and UV-reactivation allows temporal uncoupling of convoluted fluorescence signals from e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. In addition, we also used caging without UV to remove unwanted “overlabelled” species in smFRET experiments with the homotrimeric membrane transporter BetP. Such a strategy is already widely used in localization -based super-resolution microscopy (PALM, STORM and PAINT) [1][2][3]. We consequently think that caged FRET methodology relates to other multi-ruler techniques in a similar way as stochastic super-resolution techniques (STORM/PALM) compares to targeted readout (STED/RESOLFT) [1][4][5]. This idea might be useful to distinguish multi-scale smFRET-based approaches such as photo-switchable FRET and caged FRET (temporal signal separation) from combinations of different rulers, e.g., PIFE-FRET [8] [9], PET-FRET [6] or farFRET [7](spatial signal separation).
We finally outline other possible future applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion -based smFRET techniques due to required low concentrations of fluorescently labelled biomolecules. The latter observations have interesting implications for application of photoactivation FRET on different biomolecular structures for future investigations.
Chapter 4, Conformational states and dynamics of the regulatory C-terminal domain of BetP: In the framework of chapter 4, we have performed a detailed study on the regulatory role of the
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C-terminal domain of BetP protein using smFRET. The Na+ coupled betaine symporter BetP counteracts hyperosmotic stress and is regulated via an osmosensing C-terminal domain. Here, we assessed potassium-induced conformational changes of the C-terminal domain by employing Förster-resonance energy transfer (FRET) in combination with single -molecule detection – an approach that allowed us to elucidate the structure and functional role of C-terminal domain. For this reason, we first developed a direct approach to characterize the structural arrangement of BetP in different biochemical environments with homotrimeric cysteine mutants in the C -terminal helix. We observed major differences in the conformational states in polymeric Amphipols compared to detergent environment. Strikingly, the observed differences and specifically the state identified in Amphipol likely reflects the native cellular state of the C -terminal domain of BetP. Finally, we investigated the effect of potassium and osmo-activated conditions on the conformational states of BetP. We demonstrate that BetP senses and rearranges the C-terminal helices in response to increasing concentration of K+ within an optimal protein/Amphipol complex that potenitilay could represente the native protein state. This helical conformation is likely to differ from the X-ray crystal structure of BetP that was previously obtained in a detergent environment. Our studies in chapter 4, also showed that neither sodium nor lithium have a comparable effect on the conformational state of the protein. In addition, our findings ascribe a new role to potassium by promoting conformational changes that are relevant during for the BetP activation cycle to promote the substrate-bound closed state.
The results described in chapter 4, were consistent with functional analysis and structural studies of BetP in great details in both Amphipol and detergent. The information provides novel insights into stress-regulation of membrane transport processes with visualizing conformational changes within the BetP trimer and provided further insights into the biochemical function of BetP at inactive and active conditions.
In the outlook, we propose potential directions for future studies with respect to available biochemical experimental methods and computational modelling to improve the precision and accuracy of proposed models for role of BetP C-terminal domain.
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REFRENCES CHAPTER 5:
[1] Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J., and Hess, H. F. (2006) Imaging intracellular fluorescent proteins at nanometer resolution, Science 313, 1642-1645.
[2] Rust, M. J., Bates, M., and Zhuang, X. (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (storm), Nat Methods 3.
[3] Sharonov, A., and Hochstrasser, R. M. (2006) Wide-field subdiffraction imaging by accumulated binding of diffusing probes, Proc. Natl. Acad. Sci. U S A 103, 18911-18916.
[4] Hell, S. W. (2007) Far-field optical nanoscopy, Science 316, 1153-1158.
[5] Stefan, W. H., Steffen, J. S., Mark, B., Xiaowei, Z., Rainer, H., Martin, J. B., Joerg, B., Gleb, S., Harald, H., Philip, T., Alf, H., Stefan, J., Ilaria, T., Laurent, C., Brahim, L., Helge, E., Simon, J. D., Christian, E., David, K., Katrin, I. W., Giuseppe, V., Marco, C., Alberto, D., and Thorben, C. (2015) The 2015 super-resolution microscopy roadmap, Journal of Physics D: Applied Physics 48, 443001. [6] Haenni, D., Zosel, F., Reymond, L., Nettels, D., and Schuler, B. (2013) Intramolecular Distances and
Dynamics from the Combined Photon Statistics of Single-Molecule FRET and Photoinduced Electron Transfer, Journal of Physical Chemistry B 117, 13015-13028.
[7] Krainer, G., Hartmann, A., and Schlierf, M. (2015) farFRET: Extending the Range in Single-Molecule FRET Experiments beyond 10 nm, Nano letters 15, 5826-5829.
[8] Ploetz, E., Lerner, E., Husada, F., Roelfs, M., Chung, S., Hohlbein, J., Weiss, S., and Cordes, T. (2016) Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers, Scientific Reports 6, 33257.
[9] Lerner, E., Ploetz, E., Hohlbein, J., Cordes, T., and Weiss, S. (2016) A Quantitative Theoretical Framework For PIFE-FRET, The journal of physical chemistry. B 26, 6401-6410.
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Samenvatting
De Na+ -gekoppelde betaïne symporter BetP van Corynebacterium glutamicum gaat hyperosmotische stress tegen en wordt gereguleerd via een osmodetecterend C-terminaal domein. Hoewel de biochemie van het systeem goed is gekarakteriseerd, is structurele informatie slechts gedeeltelijk beschikbaar, vanwege een ontbrekend C-terminaal domein in de kristalstructuur. Dus zowel het activerings- als transportmechanisme blijven onopgelost. We hebben verschillende enkele molecuul technieken ontwikkeld om de conformationele dynamiek en heterogeniteit van BetP te bestuderen in proof-of-principle studies (hoofdstuk 2/3) en een gedetailleerde mechanistische studie van transportactivatie. Onze studies en ontwikkelde methoden zullen de weg vrijmaken voor toekomstige enkele molecuul studies en een verbeterd mechanistisch begrip van de secundaire actieve transporter BetP. Het proefschrift bevat de
volgende wetenschappelijke hoofdstukken:
Hoofdstuk 2, Nieuwe benaderingen voor fluorescent labelen van membraantransporters en geïsoleerde domeinen: FRET is een veelgebruikte methode geworden om, in combinat ie met detectie van een enkele moleculen, eiwitconformationele dynamiek e n heterogeniteit te onderzoeken. Een van de belangrijkste factoren die mechanistische inzichten beperken, zijn slechte fotofysische eigenschappen van fluorescente labels en de behoefte aan specifieke koppelingsstrategieën van fluorofoor aan het biomolecuul [7]. Locatiespecifieke labeling is een cruciale stap om biologische systemen te onderzoeken met op enkele molecuul FRET gebaseerde methoden, omdat deze een relevante reactiecoördinaat van het systeem in kaart moet brengen [21]. Voor eiwitten worden cysteïnen kunstmatig geïntroduceerd op niet-geconserveerde en aan oplosmiddel blootgestelde posities als ankerpunten voor de fluorescerende labels. Hoewel deze strategie verschillende problemen heeft die te maken hebben met de moleculaire biologie en de bereiding van de eiwitmutanten (en hun functionaliteit), is het verder gecompliceerd in multi-subeenheid eiwitten zoals BetP omdat één cysteïne (in één multi-subeenheid) meerdere keren in het complex voorkomt; er worden dus meerdere labelsites aangemaakt. Het ontwerpen van een geschikte strategie voor plaatsgerichte mutagenese en verdere conjugatie van fluoroforen met geoptimaliseerde fotofysische eigenschappen voor FRET zal het mogelijk maken om me er mechanistische inzichten te verkrijgen. Hoofdstuk 2 van dit proefschrift presenteert pogingen om
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homo- en heterotrimere cysteïne-constructen van het membraaneiwit BetP te labelen voor smFRET-studies. Tot slot laat ik het labelen zien van ABC-transporterdomeinen in het aminozuurimporteur GlnPQ als testsystemen voor karakterisering van conjugaten van fotostabilisator en kleurstof. Hiermee heb ik methoden ontwikkeld om het natriumgekoppelde Betaine-eiwit BetP-eiwit mechanistisch te bestuderen. De langetermijndoelen van onze onderzoeken waren het volgen van katalytische bewegingen van BetP tijdens substraattransport
en transportregulatie via de c-terminale domeinen en kalium.
Hoofdstuk 3, Uitschakelen en fotoactivering in enkele-molecuul Förster-resonantie energieoverdracht Experimenten: Uitgeschakelde organische fluoroforen zijn gangbare hulpmiddelen voor op lokalisatie gebaseerde superresolutie microscopie. Het fotoactiveringsmechanisme van uitgeschakelde fluoroforen berust op omkeerbare deactivering van standaard organische fluoroforen door chemische reductie of commercieel verkrijgbare uitgeschakelde kleurstoffen met aanschakelen van het fluorescentiesignaal door UV -licht. In hoofdstuk 3 werden experimentele methoden opgezet om fotochemisch aanschakelen van cyaanfluoroforen en uitgeschakelde rhodamine kleurstoffen te gebruiken voor experimenten met één molecuul Förster resonantie energieoverdracht (smFRET). De verkregen resultaten suggereerden dat het gebruik van uitgeschakelde kleurstoffen temporele scheiding en sortering van meerdere intramoleculaire donor-acceptor paren in oplossing-gebaseerde smFRET mogelijk maakt. Hiervoor moesten zowel een krachtig donor- als acceptorpaar worden geïdentificeerd, waarbij aan de twee vereisten, d.w.z. efficiënte uitschake ling en fotoactivatie, is voldaan. Hoewel de gepresenteerde experimenten een proof-of-principlekarakter hebben, demonstreren ze onze aanpak om complexe biochemische entiteiten zoals multi-subeenheid eiwitten of nucleïnezuren met meer dan twee fluorescerende labels te visualiseren. Proof-of-principle experimenten en een karakterisering van het aanschakelen-proces in het confocale volume worden in dit werk gepresenteerd. Deze onthullen dat chemisch uitschakelen en UV -reactivering een tijdelijke ontkoppeling mogelijk maken van ingewikkelde fluorescentiesignalen van bijvoorbeeld meerdere spectraal vergelijkbare donor- of acceptormoleculen gekoppeld aan nucleïnezuren. We gebruikten ook uitschakelen zonder UV-reactivering om ongewenste "overlappende" soorten te verwijderen in smFRET-experimenten met de homotrimere membraantransporter BetP. Een
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dergelijke strategie wordt al veel gebruikt in op lokalisatie gebaseerde superresolutiemicroscopie (PALM, STORM en PAINT) [1] [2] [3]. Wij denken daarom dat de uitschakel FRET-methode op een vergelijkbare manier betrekking heeft op andere multi-liniaaltechnieken als stochastische superresolutietechnieken (STORM / PALM) in vergelijking met gerichte uitlezing (STED / RESOLFT) [1] [4] [5]. Dit idee kan nuttig zijn om op meerdere schaal gebaseerde benaderingen op basis van smFRET zoals foto-schakelbare FRET en uitschakel FRET (tijdsgebaseerde signaalscheiding) te onderscheiden van combinaties van verschillende linialen, bijvoorbeeld PIFE-FRET [8] [9], PET-FRET [6]. of farPET-FRET [7]. (ruimtelijke signaalscheiding). We schetsten ook andere mogelijke toekomstige toepassingen van de uitschakel FRET-methodologie, zoals de studie van zwakke biochemische interacties, die anders onmogelijk zijn met op diffusie gebaseerde smFRET-technieken vanwege de vereiste lage concentraties van fluorescent gelabelde biomoleculen. De laatste observaties hebben interessante implicaties voor de toepassing van fotoactivatie -FRET op
verschillende biomoleculaire structuren in toekomstig onderzoek.
Hoofdstuk 4, Conformationele toestanden en dynamiek van het regulatoire C-terminale domein van BetP: In het kader van hoofdstuk 4 hebben we een gedetailleerde studie uitgevoerd naar de regulerende rol van het Cterminale domein van BetPeiwit met behulp van smFRET. De Na + -gekoppelde betaïne symporter BetP van Corynebacterium glutamicum gaat hyperosmotische stress tegen en wordt gereguleerd via een osmodetecterend C-terminaal domein. Hoewel de biochemie van het systeem goed is gekarakteriseerd, is structurele informatie slechts gedeeltelijk beschikbaar, vanwege een ontbrekend C-terminaal domein in de kristalstructuur. Hier hebben we kalium-geïnduceerde conformationele veranderingen van het C-terminale domein beoordeeld door gebruik te maken van Förster-resonantie energieoverdracht (FRET) in combinatie met single-molecule detectie - een strategie die ons in staat stelde de structuur en functionele rol van het -helische C-terminale domein op te helderen. Hiervoor hebben we eerst een directe methode ontwikkeld om de structurele rangschikking van BetP in verschillende biochemische omgevingen met homotrimere cysteïnemutanten in de C-terminale helix te karakteriseren. We hebben grote verschillen waargenomen in de conformationele toestanden in polymere amfipolen in vergelijking met zeepstoffen. Opvallend is dat de waargenomen
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verschillen, en in het bijzonder de in amfifiel geïdentificeerde toestand, waarschijnlijk de oorspronkelijke cellulaire toestand van het C-terminale domein van BetP weerspiegelt. Tot slot hebben we het effect van kalium- en osmo-geactiveerde aandoeningen op de conformationele staten van BetP onderzocht. We tonen aan dat BetP de C-terminale helices detecteert en herschikt als reactie op de toenemende concentratie van K+ binnen een optimaal eiwit / amfipol-complex dat de oorspronkelijke eiwittoestand vertegenwoordigt. Deze helische conformatie zal waarschijnlijk verschillen van de röntgenkristalstructuur van BetP die eerder werd verkregen in een zeepomgeving. Onze studies tonen ook aan dat noch natrium noch lithium een vergelijkbaar effect hebben op de conformationele toestand van het eiwit. Onze bevindingen wijzen een nieuwe rol toe aan kalium door het bevorderen van conformationele veranderingen die relevant zijn tijdens de BetP-activatiecyclus om de substraatgebonden gesloten toestand te bevorderen.
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Acknowledgments
My PhD journey has come to an end. First of all, undertaking this PhD has been a truly life-changing and exciting experience for me and it would not have been possible to do without the support and guidance that I received from my academic supervisors, my family and friends.
First, I would like to thank my supervisor Prof. Dr. Thorben Cordes. This work would not have been possible without your support, enthusiasm and guidance in the course of this project. We had long and fruitful discussions about the scientific methods used in this thesis. We were not always had the same way of thinking but at the end we were aligned to achieve good results. I greatly appreciate the support received through the collaborative works. And thank you for the help and all advices to finalize this thesis. I clearly remember your positive feedbacks to any of my successful experimental steps in laboratory and Microscopy.
I gratefully acknowledge Prof. Dr. Christine Ziegler and her research team in Germany. I would like to thank you for all your support, encouragement, your knowledge and the opportunity you gave me to come to Regensburg and become part of your research team. Working in an interdisciplinary field requires science, expertise and mobilities between universities and researchers. Your attitude is admirable, and I could benefit from all the experimental knowledge with regards to the system I was investigating. Thinking about my PhD, one of the nice st moments that I remember was the moment I showed you one of the first successful result obtained in your biochemistry lab and your support and motivation during the days when I was beginning my training in Regensburg. I also would like to acknowledge Barbara for all her help and support in the lab.
Dear Prof. Dr. Bert Poolman, you are a great inspiration to me, and I believe many others, I would like to express my gratitude because of your scientific knowledge and your perception on both experimental and fundamental aspects of research. Thank you for your valuable input.
Dear Prof.Dr. Wouter Roos, you joined Groningen university in my last stage of PhD, and you brought your new work and scientific atmosphere to the team. Thanks for all the support and encouragement that are prerequisite for a team’s success.
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There is no way to express how much it means to me to have my wonderful friends around me.
Melissa, when we first met in our office at Zernike, I could not imagine we could share a lot of experiences from the journey. We had a lot of nice coffee and chit-chat in our good and hard moments during PhD. You thought me that difficult moments are learning points and that all difficult times build determination and inner strength at the end. Thanks for the support you gave me in my journey!
Giorgos and Evelyne, I would like to thank you both for all insightful scientific discussions and advices. You are great scientists. Giorgos I still remember the time that I came to lab to ask a short question and you started with saying “Ati moment” and nobody could predict how long this one moment could take but in any case, you helped me’ Thank you that was all precious.
Kostas, you always had (I hope you still have) a smart sense of humor! This brought cheers and hope to atmosphere. I remember your words when you saw my face stuck on writing or data analysis saying ‘Do not take life too seriously. You will never get out of it alive!’
Jochem, thanks for the advice and also the nice Dutch translation of the summary of my thesis. I hope one day my Dutch language skills would be in the level that I could translate it without google translator, probably in a second life!
I would like to say thank you to all other team members and to the people from enzymology group which were also part of my research group during my PhD. I start with my office mates. Florence and Pedro (thanks for all moments and time we share in the lab and office ),Guus (I believe your angry mails made a lot of improvements in the lab), Sourav, Yusran, Sarah, Alex, Jasper, Samaneh, Marjin, Nikos, Victor, Ruslan and Jan Peter.
I would like to thank all others which helped me during this journey and honestly, I spent most of my time with them. Dear Centrifuge, Cell Incubator, Microscopy and AKTA, I spent a lot of time with each of you while we had many ups and downs and eventually ended up in nice results
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I would also like to thank all my Thermo Fisher Scientific colleagues in Groningen and Ein dhoven for helping me to start a new chapter in my life. We shared really unique experiments in the UPS team and GMP environment and now incredible technology Cryo-EM Microcopy.
Suruchi, the first time we met at a potluck party I never imagined you become one of my close friends in Groningen. We spent lovely moments and had lots of adventures together - the time we spent during UMCG hospital coffee time and I still remember the night we went on a spontaneous trip and ended up in strange ceremony out of town. We shared hours and hours talking, painting and cooking together with Jethro. I miss you so much.
I stayed with Tashfeen and Aisha for more than 3 years in a nice home. Thank you all and also Prof.Dr. Petra Rudolf and Dear Valerio. I thank you both for all the supports and encouragements. It was very much appreciated for me to feel like home when I first arrived to Groningen. Dear Petra you made me believe more that the society needs strong women scientists!
Jethro, I really would like to express my kind gratitude to you! You made Groningen a different city for me. We spent a lot of time on weekend coffee, cooking and unique photography slots. That was an amazing time.
Dear Farsi Reading team members, that was a unique time and great pleasure for me during all those Sundays we spent together to read a novel or piece of your new story together. Dear Azadeh and Soheil, Pooya and Neda, Saideh and Mohammad Reza, thank you for all cheerful moments. Azadeh, I always admire your positive attitude and great energy. Solmaz, you also made Groningen a different city for me, all the nice time we spent to discover cafes and bars in this small town. And all the Friday and weekend nights we spent together, I truly miss those moments and nights I could skip to your cozy and beautiful flat, running away from a nasty mouse in my flat. I clearly remember that (mid)night I came to you socked in rain and crying because of the mouse in my room. Time flies!
Dear Meysam! You always have been a good friend for me! I remember my last days in Groningen trying to wrap up my research and you were triggered new ideas with your curiosity and your
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fundamental questions about my work. In addition, I believe Antelectory team is not only a channel but also it is a novel thinking style with a lot of potential!
Dr. Somaye, I really miss your calm and nice attitude towards life. I still remember our unforgettable Prague trip with Omid. Laya, you were there for me when I needed someone for late chat or all the long working days and nights which we spent at Zernike. We shared a lot of time and experiences together. Dear Zahra, I always admire your strong spirit and your happy attitude towards life which you brought to our weekend breakfast chats together with Laya.
I came to Eindhoven more than a year ago! Sadjad, we met for first time in the Thermo Fisher Canteen and I would have never imagined we can come that close. I learnt a lot from you. I have been learning from ups and downs we have been facing. You have such a kind heart and you were there for me when I needed you. I would like to say thank you for all the memorable moments we shared together. We will grow together and will become different people during this journey.
Dear Mina, we met each other at the University of Eindhoven for our literature nights. Your energy, motivation and hard work is such an inspiration. I feel you understand my thoughts without me saying a word. Dear Adabi session’s friends - Mina, Payam, Elham, Amir and Moojtaba - we shared a lot of nice and fruitful discussions every week in the last year, thanks for making such an event happening. Let’s keep the wheel rolling!
Dear Yoones, my advisor Kebereh buddy, you have always been like a brother to support me giving all unpractical advices! Thanks! To all my other Eindhoven friends, Parisa and Masoud, Ramineh and Parsa, Shaya and Pegah (I always miss our enriched discussions and joyful moments).
A heartfelt thanks to my family.
Words cannot express how grateful I am to you Mom. I grew up and seeing the world away from home, I realized how you raised me strong and independent. I know I couldn’t be at your side at difficult times. I would not be the same person as I am today without you and your support.
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Madarjoon, you taught me to stand up for my rights and raised me to be strong and determined to accomplish my goals and dreams.
Dear Dad, my childhood was full of joyful moments filled with our nice discussions, your stories, book readings and puzzle competitions we had. I would not trade any of those moments we spent together, and you might not be aware of how all of those moments changed my attitude and my character and made me who I am today! I remember one of the bedtime stories you were telling me, small black fish, I still can remember it line by line. You introduced me the first glance of this new world! Book and literature. To my parent I would like to write this Rumi poems for both of you: ”Do you know what you are? You are a mirror reflecting a noble face. This universe is not outside of you. Look inside yourself; everything that you want, you are already that.”
Dear grandmas (Mamanjoon) Ferdos and Zahra, I would like to express my love and happiness for having you in my life. I admire your positive attitude and great love and encouragements! Thanks for all memorable moments of my childhood. I always feel both Mamani and Aghajoon near but UNHEARD and UNSEEN in my life. Rest in peace!
Mehdi, my dear brother, I always remember this saying that “brothers and sisters are children of the same parents, each of whom is perfectly normal until they get together”. I remember our lovely moments together, we grew up and learnt together, we fought and failed together. I am grateful that I have your support and I wish I could spend more time with you. Dear Jana, you have a beautiful soul and kind heart. I am so happy for you and Mehdi, and no word s can express my love to our little beautiful Leyli. During the last few months I was watching her videos again and again. Leyli’s brought the true color of happiness to our lives with coming to our world.
At the end, I would like to dedicate this work, to women in science who spend their life on science to solve fundamental questions of life with all the barriers and to first prove that” there is no gender in science!” and then “Everything is theoretically impossible, until it is done”.
Atieh
