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In dieser Arbeit wurde die Methode der breitbandigen fs-zeitaufgelösten Fluoreszenzaufkonversionsspektroskopie (FLUPS) weiterentwickelt und vollständig theoretisch beschrieben, was anhand des Vergleichs von vorhergesagten und experimentell bestimmten photometrischen Korrekturfunktionen gezeigt werden konnte. Die Methode wurde verwendet, um lokale Fluoreszenzspektren von solvatochromen Sonden in der Nähe bestimmter Matrizes in wässrigen Lösungen zu messen. Aus der Dynamik der Stokes-Verschiebung konnte die Solvatations- bzw. Umgebungsdynamik bestimmt werden. Es wurden mittlere Solvatationszeiten τsolv von 0.57±0.06 für reines Wasser, 2.8±0.2 ps für DNA, 480±30 ps für Phospholipid-Kopfgruppen, 0.71±0.03 ps für ein Peptid (α-Helix) und 0.76±0.03 ps für eine t-Butyl-Gruppe erhalten. Hervorzuheben sind dabei die überraschend schnelle Relaxation nahe des Peptids und die sehr langsame Dynamik nahe der Lipid-Kopfgruppen, welche über 5 Größenordnungen der Zeit beobachtet wurde. Um den Einfluss einer hydrophoben Gruppe auf die Solvatationsdynamik erstmals zu aufzuzeigen, wurden präzise Messungen bei verschiedenen Temperaturen vor-genommen. Zuordnungen dieser Dynamiken zu molekularen Prozessen konnten durch Vergleiche zu MD-Simulationen durchgeführt werden. / The method of broadband fs time-resolved fluorescence upconversion spectroscopy (FLUPS) was further developed and completely theoretically described in this work. This was shown by comparing predicted and measured photometric correction functions. This method was used to obtain local fluorescence spectra of solvatochromic dyes near certain matrices in aqueous solution. From the dynamics of the Stokes-Shift the solvation or environmental dynamics respectively were obtained. Average solvation times τsolv of 0.57±0.06 for bulk water, 2.8±0.2 ps for DNA, 480±30 ps for phospholipid head groups, 0.71±0.03 ps for a peptide (α-helix) and 0.76±0.03 ps for a t-butyl group were obtained. Emphasized are the surprisingly fast dynamics near the peptide and the slow dynamics of the lipid head group region. The latter was observed over 5 orders of magnitude in time. To distinguish the influence a hydrophobic group for the first time, precise measurements at different temperatures were performed. Molecular processes were assigned to the obtained dynamics by comparisons to MD studies.

Betalaínas são alcalóides coloridos e com alta capacidade antioxidante que são encontrados em plantas e fungos. A biossíntese destes produtos naturais baseia-se na conversão enzimática da L-tirosina em ácido betalâmico e na condensação aldimínica deste com aminoácidos. A semissíntese de betalaínas naturais para aprofundar o estudo desta classe de pigmentos estimulou o desenvolvimento de betalaínas artificiais, incluindo derivados funcionais. Uma betalaína cumarínica foi criada para ser usada como sonda fluorescente para marcação de Plasmodium falciparum em glóbulos vermelhos. Esta Tese de Doutorado apresenta a semissíntese e estudo de três betalaínas cumarínicas (cBeets) e uma carboestiril-betalaína (csBeet). Procurou-se estabelecer relações entre as estruturas destes compostos e suas propriedades físico-químicas e fotofísicas como ponto de partida no desenvolvimento de uma nova classe de betalaínas funcionais. São apresentados dados sobre a lipofilicidade, estabilidade frente à hidrólise, potencial redox, absorção de um e dois fótons e fluorescência. Interações intermoleculares destes compostos foram investigadas por medidas de fluorescência em misturas binárias de solventes polares, albumina sérica bovina e micelas reversas de AOT em heptano/água. / Instituto de Química, Universidade de São Paulo, São Paulo, 2017. Betalains are colorful alkaloids with high antioxidant capacity that are found in plants and fungi. The biosynthesis of these natural products is based on the enzymatic conversion of L-tyrosine into betalamic acid and aldimine condensation thereof with amino acids. The semisynthesis of natural betalains improved the knowledge on this class of pigments and stimulated the development of artificial betalains, including functional derivatives. A coumarinic betalain was created to be used as a fluorescent label for Plasmodium falciparum on red blood cells. This Doctoral Thesis presents the semisynthesis and study of three coumarin betalains (cBeets) and one carbostyril betalain (csBeet). It was sought to establish relationships between the structures of these compounds and their physical-chemical and photophysical properties as a starting point in the development of a new class of functional betalains. Data on lipophilicity, hydrolysis stability, redox potential, one- and two-photon absorption and fluorescence are presented. Intermolecular interactions of these compounds were investigated by fluorescence measurements in binary polar solvent mixtures, bovine serum albumin and AOT reverse micelles in heptane/water.

Les brins d’ADN riches en guanine, comme ceux présents à l'extrémité des chromosomes humains, sont capables de s’associer entre eux pour former des structures G-quadruplexes, résultant de l’association de quatre guanines (G-tétrade). Ces structures sont actuellement l’objet d’un intérêt particulier pour le développement de nouvelles thérapies anti-cancéreuses et des applications potentielles pour l’électronique moléculaire. Il n’existe cependant que très peu d’études des propriétés photophysiques des G-quadruplexes. L'objectif de ce travail de thèse est d'étudier l’influence de la structrure des G-quadruplexes sur leurs propriétés photophysiques au moyen de la spectroscopie de fluorescence résolue en temps sur une gamme temporelle allant de la centaine de femtosecondes à la centaine de nanosecondes. Nous avons examiné l’effet de la taille de structures G-quadruplexes tétramoléculaires sur leurs propriétés photophysiques. Nous avons pu montrer que le caractère collectif des états ππ* des guanines est renforcé lorsque le nombre de tétrades augmente et qu’un transfert d'énergie ultra-rapide, en moins de 100 fs a lieu entre ces états. Nous avons ensuite mis en évidence le rôle des cations métalliques situés dans la cavité centrale des quadruplexes dans le processus de désactivation des états excités. En présence de K+, l'émission provient principalement des états délocalisée ππ* des guanines, alors qu’en présence de Na+, l’émission dominée par la contribution d’états excités à caractère de transfert de charge. Enfin, nous avons abordé l'effet de la topologie, en comparant les propriétés photophysiques des G-quadruplexes tétramoléculaires avec celles de structures formées par le repliement d’un simple brin d’ADN. Les différences observées peuvent s’expliquer par la rigidité accrue des structures simple-brins et l'orientation relative différente des tétrades qui détermine la force du couplage électronique entre les bases. / Guanine rich DNA strands have the ability to form four-stranded structures (G-quadruplexes). Their repetitive unit is the G-quartet (tetrad) where each base is connected with two others via four hydrogen bonds. These structures have a crucial role in biological aspect, as targets for anti-cancer therapies, and have great potential for applications in nanotechnology. We studied the electronic excited states of G-quadruplexes using two different techniques, fluorescence up-conversion (FU) and time-correlated single photon counting (TCSPC) , which allow probing the emissive states over six decades of time (from hundred femtoseconds to hundreds of nanoseconds). At first, we examined the effect of the size of tetramolecular G-quadruplexes on their photophysical properties. We have found that the collective behavior of Franck-Condon excited states is enhanced when the number of tetrads increases. For all systems studied, the anisotropy of the G-quadruplex, on the time scale of hundreds of femtoseconds, is lower than that of non-interacting mono-nucleotides in solution. This decrease in anisotropy is associated with an ultrafast energy transfer process between the bases. Then we demonstrated that the metal cations located in the central cavity of quadruplexes also affect their photophysical properties. In the presence of K+, emission arises mainly from delocalized ππ* states (excitons), whereas in the presence of Na+, it is dominated by the contribution of charge transfer excited states. Finally, we studied the effect of conformation, comparing the properties of tetramolecular G-quadruplexes with those formed by folding a single strand (intramolecular G-quadruplexes). We have shown that the conformation of the nano-structures influences the properties of the excited Franck-Condon states as emissive states as well. These effects are attributed to different geometric arrangement of G-tetrads in tetramolecular and intramolecular quadruplexes.

Este estudo reúne os principais resultados de fluorescência estática e resolvida no tempo sobre a interação da porfirina meso-tetrakis (4-metilpiridil), na forma de base livre (TMPyP) e complexada com Zn2+ (ZnTMPyP), com vesículas de fosfolipídio. Adicionalmente foram utilizadas as técnicas de potencial zeta e espalhamento de luz dinâmico (DLS, do inglês \"dynamic light scattering\"). As vesículas de fosfolipídio foram formadas por dois conjuntos de fosfolipídios: saturados e insaturados. O primeiro grupo é formado pela mistura dos fosfolipídios zwiteriônico 1,2-dipalmitoil-sn-glicero-3-fosfocolina (DPPC) e aniônico 1,2-dipalmitoil-sn-3-glicero-[fosfo-rac-(1- glicerol)] (DPPG), a diferentes razões molares. Os estudos utilizando tais sistemas foram realizados abaixo (25oC) e acima (50oC) da temperatura de transição de fase gel-líquido cristalino destes fosfolipídios (~ 41oC). O segundo grupo é formado pela mistura dos fosfolipídios zwiteriônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfocolina (POPC) e aniônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfo(1-rac-glicerol) (POPG). Como a transição de fase destes dois fosfolipídios ocorre a temperaturas negativas, todos os experimentos foram realizados a 25oC (vesículas no estado líquido cristalino). Todos os sistemas foram preparados através do método de extrusão para a obtenção de vesículas grandes unilamelares (LUV, do inglês \"large unilamellar vesicles\"). As análises dos dados de fluorescência indicaram que a atração eletrostática entre os substituíntes (positivamente carregados) das porfirinas TMPyP e ZnTMPyP e o grupo das cabeças polares (camada de Stern) das vesículas de fosfolipídio desempenha um papel fundamental na associação da porfirina. A distribuição da TMPyP entre o meio aquoso (tampão) e as vesículas de fosfolipídio foi evidenciada pela coexistência de um tempo de vida de fluorescência mais curto (~ 5 ns) e outro mais longo (~ 9-11 ns), respectivamente. Baseado nos valores das constantes pré-exponenciais, estudos adicionais mostram que a distribuição acima é afetada pela concentração de sal na solução. Os resultados de supressão de fluorescência com o supressor iodeto de potássio (KI) indicaram que ambas porfirinas estão localizadas, preferencialmente, na região da camada de Stern. Este resultado foi confirmado pelos estudos de potencial zeta e de DLS, os quais mostraram uma neutralização parcial das cargas negativas na superfície das vesículas devido à associação da porfirina. / This study presents time-resolved and steady-state fluorescence results on the interaction of the meso-tetrakis (4-methylpyridil) porphyrin, in free base form (TMPyP), and complexed with Zn2+ (ZnTMPyP), with phospholipid vesicles. Zeta potential and dynamic light scattering (DLS) techniques were also used. Phospholipid vesicles were formed by two phospholipid systems: saturated and unsaturated. The first group is a mixture of zwiterionic dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) and anionic 1,2-dipalmitoyl-sn-3-glycero-[phospho-rac-(1-glycerol)] (DPPG) phospholipids, at different molar ratios. Measurements were performed bellow (25oC) and above (50oC) the main gel-liquid crystalline phase transition temperature (~ 41oC). The second group is constituted by a mixture of zwiterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1-rac-glycerol) (POPG) phospholipids, at different molar ratios. Since the gel-liquid crystalline phase transition of these phospholipids occurs at a very low temperature value, all experiments were performed at 25oC (liquid crystalline state vesicles). All phospholipid systems were prepared through the extrusion method in order to obtain large unilamellar vesicles (LUV). The fluorescence data analyses indicated that the electrostatic attraction between the porphyrin substituents (positively charged) and the polar head groups of the phospholipid vesicles (Stern layer) plays an important role on the porphyrin binding affinity. The distribution of TMPyP between the aqueous medium (buffer) and the phospholipid vesicles was characterized by the coexistence of a shorter (~ 5 ns) and a longer (~ 9-11 ns) fluorescence lifetimes, respectively. Based on the pre- exponential values, additional time-resolved experiments showed a redistribution of the porphyrin at increasing salt concentration. The quenching studies, using potassium iodide (KI) as quencher, indicated that both TMPyP and ZnTMPyP are preferentially located at the Stern layer region. This result is in agreement with the zeta potential and DLS findings, which demonstrated a partial neutralization of the negative charges at the vesicle surface due to the porphyrin association.

An extended Förster theory (EFT), previously derived (L. B.-Å. Johansson et al. J. Chem. Phys., 1996,105) has theoretically been adapted and used in simulations of donor-acceptor energy transfer (DAET), which is a process often referred to as FRET. It was shown that the classical Förster theory is only valid in the initial part of the fluorescence decay. In this thesis an EFT is derived and outlined for electronic energy transport between two fluorescent molecules which are chemically identical, but photophysically non-identical. The energy migration within such asymmetric pairs is partially reversible and therefore referred to as partial donor-donor energy migration (PDDEM). The previously derived model of PDDEM (S. V. Kalinin et al. Spectrochim Acta Part A, 2002,58) is an approximation of the EFT. In particular, the EFT accounts for the time-dependent reorientations as well as the distance that influence the rate of electronic energy migration. The reorientation of the fluorophores transition dipole moments has been simulated using Brownian dynamics. As a result, the related “k2-problem” has been solved. The EFT of PDDEM has also been studied regarding the effect of PDDEM on experimental observables e.g. quantum yield of fluorescence and steady-state anisotropies

Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

Τα φωτοβολταϊκά στοιχεία με ευαισθητοποίηση χρωστικής (Dye Sensitized Solar Cells, DSSCs) κίνησαν το ενδιαφέρον της επιστημονικής κοινότητας ύστερα από την πρωτότυπη δημοσίευση του 1991 των Grätzel και O' Regan. Προτάθηκαν ως μία φθηνή εναλλακτική λύση σε σύγκριση με τα συμβατικά ηλιακά στοιχεία από άμορφο πυρίτιο (amorphous silicon). Οι κύριοι παράγοντες που οδήγησαν την επιστημονική κοινότητα να στραφεί προς αυτή την κατεύθυνση ήταν η ευκολία σύνθεσης των χρωστικών με σχετικά απλές χημικές διαδικασίες και η λειτουργία των νέων αυτών φωτοβολταϊκών στοιχείων υπό συνθήκες διάχυτου φωτός. Γενικά, ένα τέτοιο φωτοβολταϊκό στοιχείο αποτελείται από μία φωτοάνοδο (photoanode), ένα πορώδες υπόστρωμα από ημιαγώγιμο οξείδιο μετάλλου (metal oxide semiconducting film), μία χρωστική που χρησιμοποιείται ως φωτοευαισθητοποιητής (sensitizer), έναν ηλεκτρολύτη (electrolyte) και ένα αντιηλεκτρόδιο (counter electrode), το οποίο, συνήθως, επικαλύπτεται με ένα λεπτό στρώμα από πλατίνα (Pt). Η κύρια διεργασία που λαμβάνει μέρος σε ένα DSSC, μετά από την απορρόφηση φωτός, είναι μία διεπιφανειακή μεταφορά φορτίου (interfacial electron transfer IET) από την ηλεκτρονιακά διεγερμένη στάθμη της χρωστικής προς τη ζώνη αγωγιμότητας του ημιαγωγού. Η χρονική της διάρκεια είναι της τάξεως των μερικών εκατοντάδων fs και κατατάσσεται στα υπερταχέα φαινόμενα. Ο όρος που έχει επικρατήσει, για τη διεργασία αυτή στα DSSCs, είναι έκχυση ηλεκτρονίων (electron injection) και χρησιμοποιείται στην παρούσα διπλωματική εργασία. Η τεχνική της φασματοσκοπίας φθορισμού χρονικής ανάλυσης με παλμούς διάρκειας μερικών δεκάδων fs, αποτελεί μία από τις πιο αξιόπιστες και άμεσες τεχνικές για την καλύτερη δυνατή καταγραφή υπερταχέων φαινομένων, όπως η έκχυση ηλεκτρονίων. Σκοπός της παρούσας διπλωματικής εργασίας είναι η μελέτη της έκχυσης ηλεκτρονίων με τη χρήση δύο νέων οργανικών χρωστικών, της μορφής D-π-A, ως φωτοευαισθητοποιητές σε DSSCs με την τεχνική αυτή.Στο πρώτο κεφάλαιο πραγματοποιείται μία γενική επισκόπηση των βασικών αρχών που διέπουν τα φωτοβολταϊκά στοιχεία με ευαισθητοποίηση χρωστικής. Αρχικά, γίνεται αναφορά στα μέρη που αποτελούν ένα τέτοιο φωτοβολταϊκό στοιχείο και ακολούθως στα υλικά και στις διεργασίες οι οποίες συμμετέχουν σε ένα ολοκληρωμένο DSSC.Στο δεύτερο κεφάλαιο επιχειρείται, στο πρώτο σκέλος, μία γενική ανασκόπηση της θεωρίας του Markus για τη μεταφορά των ηλεκτρονίων (Markus Theory). Έπειτα, πραγματοποιείται μία αναλυτική επισκόπηση της δυναμικής και κινηματικής των διεργασιών που συντελούνται στα DSSCs. Συνεχίζοντας στο τρίτο κεφάλαιο, παρουσιάζονται πληροφορίες σχετικές με τα υποστρώματα και τις χρωστικές που χρησιμοποιούνται στα DSSCs. Το κεφάλαιο επικεντρώνεται στην περιγραφή των υποστρωμάτων TiO2 και ΖnO, τα οποία αποτελούν τα κύρια υποστρώματα που χρησιμοποιούνται στα DSSCs. Στο δεύτερο σκέλος του κεφαλαίου, πραγματοποιείται αναφορά στις ιδιότητες που οφείλουν να πληρούν οι χρωστικές, για τη χρήση τους στα DSSCs, καθώς και εκτενής ανασκόπηση των χρωστικών, οι οποίες έχουν χρησιμοποιηθεί, μέχρι σήμερα, ως φωτοευαισθητοποιητές. Στο τέταρτο κεφάλαιο παρουσιάζονται οι μηχανισμοί που συμμετέχουν κατά την αποδιέγερση ενός οργανικού μορίου και οι χρονικές κλίμακες, που αυτοί εμφανίζονται (διάγραμμα Jablonski). Επίσης, γίνεται αναφορά στις πληροφορίες που εξάγονται από τα φάσματα σταθερής κατάστασης (steady state spectra) και χρονικής ανάλυσης (time-resolved spectra), καθώς και η μεταξύ τους σύγκριση. Στο πέμπτο κεφάλαιο πραγματοποιείται μία αναλυτική περιγραφή της πειραματικής διάταξης, η οποία χρησιμοποιήθηκε για την εξαγωγή των πειραματικών δεδομένων. Τέλος, στα τελευταία δύο κεφάλαια (πέμπτο και έκτο) περιγράφεται, στο πρώτο, ο φωτοφυσικός χαρακτηρισμός των δύο νέων οργανικών χρωστικών, ΜΖ-173 και ΜΖ-175, της δομής D-π-Α, σε διάλυμα THF και σε στερεό υπόστρωμα TiO2 αντίστοιχα, το οποίο χρησιμοποιήθηκε ως το υπόστρωμα προσρόφησης των χρωστικών. Ακολούθως, μελετήθηκε η δυναμική και η απόδοση της έκχυσης των ηλεκτρονίων από τις χρωστικές αυτές προς το ημιαγώγιμο υπόστρωμα TiO2, με χρήση της τεχνικής της φασματοσκοπίας χρονικής ανάλυσης φθορισμού με παλμούς διάρκειας μερικών δεκάδων fs (femtosecond time resolved fluorescence spectroscopy). Ως δείγμα αναφοράς, για την εύρεση της απόδοσης της έκχυσης των ηλεκτρονίων στη ζώνη αγωγιμότητας του ημιαγωγού, χρησιμοποιήθηκε νανοκρυσταλλικό υπόστρωμα Al2O3. Τέλος, πραγματοποιήθηκε η μελέτη της δυναμικής της έκχυσης των ηλεκτρονίων με τη χρήση του μορίου CDCA, ως συνπροσροφητή στην επιφάνεια των υποστρωμάτων TiO2 και Al2O3, μαζί με χρωστική ΜΖ-173, σε διάφορες συγκεντρώσεις. Αυτή η μελέτη έγινε με σκοπό τη μείωση της συσσωμάτωσης των μορίων της χρωστικής, αφού το μόριο CDCA έχει την ιδιότητα, λόγω της δομής του, να κρατά σε απόσταση τα μόρια της χρωστικής. / Dye-sensitized solar cells (DSSCs) have attracted great scientific interest after the first demonstration of Grätzel and O’Regan in 1991. They were proposed as low cost alternatives to the conventional amorphous silicon solar cells. The key factors which led the scientific community to this direction are the simplicity of their fabrication procedures with mild chemical processes and their operation under ambient conditions of diffused light. Generally, a DSSC consists of a photoanode, a nanostructured metal oxide semiconducting film, a dye sensitizer, an electrolyte and a counter electrode which is usually coated with Pt. The fundamental process that takes place in a DSSC, after the absorption of a photon by the dye, is an interfacial electron transfer (IET) from the dye’s electronically excited state to the semiconductor’s conduction band (CB), taking place within a few hundred femtoseconds. The term which is generally used for this process in DSSCs is electron injection. Ultrafast fluorescence upconversion spectroscopy is one of the most precise and direct techniques for the study and interpretation of such phenomena. The main subject of this master thesis is the presentation of two novel synthesized organic dyes with D-π-A structure and their study as photosensitizers for DSSCs. It is focused on the photophycical properties of these two dyes in solution and on titanium dioxide (TiO2) substrate, which is used as the metal oxide semiconducting film, and especially on the dynamics of electron injection process from the dye’s excited state to the conduction band of the TiO2 with the aforementioned technique. Finally, the electron injection dynamics of one of dyes with coadsorption of co-adsorbers also investigated. This type of molecules can decrease the amount of aggregates penetrating among the dye molecules but on the same time they cause a decrease of the total amount of the adsorbed dye molecules.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal