Advanced Luminescent Materials for Technological Applications

This thesis focusses on the study of several luminescent materials and investigates some related technological applications. It is made of six chapters. Chapter 1 introduces a brief history, basic principles and applications of photoluminescence. Chapter 2 presents the photophysical properties of five benzoheterodiazole dyes. These molecules were incorporated in PMMA- and PDMS-based LSC-PV devices to determine the emission quantum yields, transmission, re-absorption and IPCE properties. DFT calculations were performed to investigate the structures and energy levels of these dyes. Chapter 3 concerns the preparation of a luminescent film to calibrate an ESA satellite that will monitor the fluorescence of terrestrial vegetation. ZnPc was selected as suitable dye to make the film. Ferrocene was selected as quencher to control the emission intensity. An industrial printing technology was used to produce large-area calibration sheets coated with green pigment that simulates the NIR reflectance of green plants in which the ZnPc is embedded. Photophysical properties of a series of alkynyl gold NHC complexes containing naphthalimide chromophore were studied in Chapter 4. All the compounds were studied in solution and solid state. Further investigations were carried out by incorporating these compounds in PMMA matrix to make films. XRD and DFT calculations were made to determine the structures and energy levels of the complexes. In chapter 5 we studied the photophysical properties of star-shaped molecular systems which can operate as molecular motors when attached onto surface, along with those of their related ligands/moieties in tetrahydrofuran solution. The photophysical properties of these molecular systems can show if they are suitable to operate as light-triggered molecular machines. Finally, chapter 6 concerns the photoluminescence behavior of three NHC half-sandwich Ir/Rh metal complexes. The photophysical properties of these compounds were examined in CH2Cl2 solutions and PMMA films. These complexes may prove potential candidates for organic phosphorescent materials.

Design, realization, and characterization of complex reaction networks in dynamic mechanically interlocked molecules

The experimental projects discussed in this thesis are all related to the field of artificial molecular machines, specifically to systems composed of pseudorotaxane and rotaxane architectures. The characterization of the peculiar properties of these mechano-molecules is frequently associated with the analysis and elucidation of complex reaction networks; this latter aspect represents the main focus and central thread tying my thesis work. In each chapter, a specific project is described as summarized below: the focus of the first chapter is the realization and characterization of a prototype model of a photoactivated molecular transporter based on a pseudorotaxane architecture; in the second chapter is reported the design, synthesis, and characterization of a [2]rotaxane endowed with a dibenzylammonium station and a novel photochromic unit that acts as a recognition site for a DB24C8 crown ether macrocycle; in the last chapter is described the synthesis and characterization of a [3]rotaxane in which the relative number of rings and stations can be changed on command.

Electrospun hierarchical artificial muscle for soft robotics applications

Nowadays, one of the most ambitious challenges in soft robotics is the development of actuators capable to achieve performance comparable to skeletal muscles. Scientists have been working for decades, inspired by Nature, to mimic both their complex structure and their perfectly balanced features in terms of linear contraction, force-to-weight ratio, scalability and flexibility. The present Thesis, contextualized within the FET open Horizon 2020 project MAGNIFY, aims to develop a new family of innovative flexible actuators in the field of soft-robotics. For the realization of this actuator, a biomimetic approach has been chosen, drawing inspiration from skeletal muscle. Their hierarchical fibrous structure was mimicked employing the electrospinning technique, while the contraction of sarcomeres was designed employing chains of molecular machines, supramolecular systems capable of performing movements useful to execute specific tasks. The first part deals with the design and production of the basic unit of the artificial muscle, the artificial myofibril, consisting in a novel electrospun core-shell nanofiber, with elastomeric shell and electrically conductive core, coupled with a conductive coating, for the realization of which numerous strategies have been investigated. The second part deals instead with the integration of molecular machines (provided by the project partners) inside these artificial myofibrils, preceded by the study of several model molecules, aimed at simulating the presence of these molecular machines during the initial phases of the project. The last part concerns the realization of an electrospun multiscale hierarchical structure, aimed at reproducing the entire muscle morphology and fibrous organization. These research will be joined together in the near future like the pieces of a puzzle, recreating the artificial actuator most similar to biological muscle ever made, composed of millions of artificial myofibrils, electrically activated in which the nano-scale movement of molecular machines will be incrementally amplified to the macro-scale contraction of the artificial muscle.

Peptidoglycan assembly machines: The Staphylococcus aureus Penicillin-Binding Proteins

Dissertation presented to obtain the Ph.D degree in Biology

Formation of a spatial memory trace : a behavioral, cellular and molecular study

THESIS ABSTRACTThis thesis project was aimed at studying the molecular mechanisms underlying learning and memory formation, in particular as they relate to the metabolic coupling between astrocytes and neurons. For that, changes in the metabolic activity of different mice brain regions after 1 or 9 days of training in an eight-arm radial maze were assessed by (14C) 2-deoxyglucose (2DG) autoradiography. Significant differences in the areas engaged during the behavioral task at day 1 (when animals are confronted for the first time to the learning task) and at day 9 (when animals are highly performing) have been identified. These areas include the hippocampus, the fornix, the parietal cortex, the laterodorsal thalamic nucleus and the mammillary bodies at day 1 ; and the anterior cingulate, the retrosplenial cortex and the dorsal striatum at day 9. Two of these cerebral regions (those presenting the greatest changes at day 1 and day 9: the hippocampus and the retrosplenial cortex, respectively) were microdissected by laser capture microscopy and selected genes related to neuron-glia metabolic coupling, glucose metabolism and synaptic plasticity were analyzed by RT-PCR. 2DG and gene expression analysis were performed at three different times: 1) immediately after the end of the behavioral paradigm, 2) 45 minutes and 3) 6 hours after training. The main goal of this study was the identification of the metabolic adaptations following the learning task. Gene expression results demonstrate that the learning task profoundly modulates the pattern of gene expression in time, meaning that these two cerebral regions with high 2DG signal (hippocampus and retrosplenial cortex) have adapted their metabolic molecular machinery in consequence. Almost all studied genes show a higher expression in the hippocampus at day 1 compared to day 9, while an increased expression was found in the retrosplenial cortex at day 9. We can observe these molecular adaptations with a short delay of 45 minutes after the end of the task. However, 6 hours after training a high gene expression was found at day 9 (compared to day 1) in both regions, suggesting that only one day of training is not sufficient to detect transcriptional modifications several hours after the task. Thus, gene expression data match 2DG results indicating a transfer of information in time (from day 1 to day 9) and in space (from the hippocampus to the retrosplenial cortex), and this at a cellular and a molecular level. Moreover, learning seems to modify the neuron-glia metabolic coupling, since several genes involved in this coupling are induced. These results also suggest a role of glia in neuronal plasticity.RESUME DU TRAVAIL DE THESECe projet de thèse a eu pour but l'étude des mécanismes moléculaires qui sont impliqués dans l'apprentissage et la mémoire et, en particulier, à les mettre en rapport avec le couplage métabolique existant entre les astrocytes et les neurones. Pour cela, des changements de l'activité métabolique dans différentes régions du cerveau des souris après 1 ou 9 jours d'entraînement dans un labyrinthe radial à huit-bras ont été évalués par autoradiographie au 2-désoxyglucose (2DG). Des différences significatives dans les régions engagées pendant la tâche comportementale au jour 1 (quand les animaux sont confrontés pour la première fois à la tâche) et au jour 9 (quand les animaux ont déjà appris) ont été identifiés. Ces régions incluent, au jour 1, l'hippocampe, le fornix, le cortex pariétal, le noyau thalamic laterodorsal et les corps mamillaires; et, au jour 9, le cingulaire antérieur, le cortex retrosplenial et le striatum dorsal. Deux de ces régions cérébrales (celles présentant les plus grands changements à jour 1 et à jour 9: l'hippocampe et le cortex retrosplenial, respectivement) ont été découpées par microdissection au laser et quelques gènes liés au couplage métabolique neurone-glie, au métabolisme du glucose et à la plasticité synaptique ont été analysées par RT-PCR. L'étude 2DG et l'analyse de l'expression de gènes ont été exécutés à trois temps différents: 1) juste après entraînement, 2) 45 minutes et 3) 6 heures après la fin de la tâche. L'objectif principal de cette étude était l'identification des adaptations métaboliques suivant la tâche d'apprentissage. Les résultats de l'expression de gènes démontrent que la tâche d'apprentissage module profondément le profile d'expression des gènes dans le temps, signifiant que ces deux régions cérébrales avec un signal 2DG élevé (l'hippocampe et le cortex retrosplenial) ont adapté leurs « machines moléculaires » en conséquence. Presque tous les gènes étudiés montrent une expression plus élevée dans l'hippocampe au jour 1 comparé au jour 9, alors qu'une expression accrue a été trouvée dans le cortex retrosplenial au jour 9. Nous pouvons observer ces adaptations moléculaires avec un retard court de 45 minutes après la fin de la tâche. Cependant, 6 heures après l'entraînement, une expression de gènes élevée a été trouvée au jour 9 (comparé à jour 1) dans les deux régions, suggérant que seulement un jour d'entraînement ne suffit pas pour détecter des modifications transcriptionelles plusieurs heures après la tâche. Ainsi, les données d'expression de gènes corroborent les résultats 2DG indiquant un transfert d'information dans le temps (de jour 1 à jour 9) et dans l'espace (de l'hippocampe au cortex retrosplenial), et ceci à un niveau cellulaire et moléculaire. D'ailleurs, la tâche d'apprentissage semble modifier le couplage métabolique neurone-glie, puisque de nombreux gènes impliqués dans ce couplage sont induits. Ces observations suggèrent un rôle important de la glie dans les mécanismes de plasticité du système nerveux.

Decoding algorithms using side-effect machines

Bioinformatics applies computers to problems in molecular biology. Previous research has not addressed edit metric decoders. Decoders for quaternary edit metric codes are finding use in bioinformatics problems with applications to DNA. By using side effect machines we hope to be able to provide efficient decoding algorithms for this open problem. Two ideas for decoding algorithms are presented and examined. Both decoders use Side Effect Machines(SEMs) which are generalizations of finite state automata. Single Classifier Machines(SCMs) use a single side effect machine to classify all words within a code. Locking Side Effect Machines(LSEMs) use multiple side effect machines to create a tree structure of subclassification. The goal is to examine these techniques and provide new decoders for existing codes. Presented are ideas for best practices for the creation of these two types of new edit metric decoders.

Généalogie des résistances cinématographiques : le cas de trois machines autopoïétiques (Eisenstein, Syberberg, Welles)

Notre thèse s’attache à définir certains rapports possibles entre le cinéma et le politique ou, plus précisément, entre le cinéma et le concept pratique de résistance. Il existe une théorie de la résistance, soit sous la forme d’une cartographie du pouvoir moléculaire (M. Foucault), soit sous la forme d’une analytique de la résistance (F. Proust). Il existe également une théorie de la résistance cinématographique, soit comme sociologie ou histoire de l’action politique par le cinéma (cinéma de la Résistance, cinéma militant), soit comme une sémiotique des formes et des genres marginaux (cinéma expérimental, cinéma des avant-gardes). Suivant une direction tracée par Serge Daney et Gilles Deleuze, nous croyons qu’il faut poser autrement le problème : si « le politique est affaire de perception », alors la résistance est d’abord une invention de visibilité et d’audibilité. En ce sens, la résistance cinématographique est une exploration de ce que peut le cinéma, tant d’un point de vue esthétique, éthique, que politique. D’où notre hypothèse, qui est double : d’une part, cette invention de visibilité, cette création de potentiel n’est peut-être possible qu’en passant par un ré-enchaînement anachronique d’une déformation plastique, narrative et audiovisuelle à une autre, c’est-à-dire d’une survivance à une autre. D’autre part, nous croyons qu’une forme esthétique est en soi matière politique et manière de politique, et qu’elle implique également une éthique venant brasser notre propre subjectivité (de cinéaste, de spectateur, de citoyen, etc.). Or, pour saisir cette invention de visibilité inhérente au cinéma, il faut en passer par une généalogie de certaines alliances théoriques et pratiques parmi les plus importantes. Le cas de trois machines autopoïétiques nous intéressera tout particulièrement, trois constellations d’œuvres et de pensées qui débordent le nom propre des « Auteurs » convoqués : Eisenstein et les résistances méthodologiques ; Syberberg et les résistances plastiques ou audiovisuelles (à travers la seule figure du hors-champ) ; Welles et les résistances esth/éthico-politiques. Ainsi, une telle chaîne généalogique nous permettra de mieux mesurer l’efficace de ces résistances, mais sans désir de systématisation ni constitution d’une Théorie de la résistance cinématographique.

The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64's biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought.

Molecular epidemiology of Staphylococcus aureus isolates at different sites in the milk producing dairy farms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epidemiologia molecular dos Staphylococcus aureus isolados em diferentes pontos do fluxograma de produção do leite

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

DNA block copolymers – from micelles to machines

DNA block copolymer, a new class of hybrid material composed of a synthetic polymer and an oligodeoxynucleotide segment, owns unique properties which can not be achieved by only one of the two polymers. Among amphiphilic DNA block copolymers, DNA-b-polypropylene oxide (PPO) was chosen as a model system, because PPO is biocompatible and has a Tg < 0 °C. Both properties might be essential for future applications in living systems. During my PhD study, I focused on the properties and the structures of DNA-b-PPO molecules. First, DNA-b-PPO micelles were studied by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). In order to control the size of micelles without re-synthesis, micelles were incubated with template-independent DNA polymerase TdT and deoxynucleotide triphosphates in reaction buffer solution. By carrying out ex-situ experiments, the growth of micelles was visualized by imaging in liquid with AFM. Complementary measurements with FCS and polyacrylamide gel electrophoresis (PAGE) confirmed the increase in size. Furthermore, the growing process was studied with AFM in-situ at 37 °C. Hereby the growth of individual micelles could be observed. In contrast to ex-situ reactions, the growth of micelles adsorbed on mica surface for in-situ experiments terminated about one hour after the reaction was initiated. Two reasons were identified for the termination: (i) block of catalytic sites by interaction with the substrate and (ii) reduced exchange of molecules between micelles and the liquid environment. In addition, a geometrical model for AFM imaging was developed which allowed deriving the average number of mononucleotides added to DNA-b-PPO molecules in dependence on the enzymatic reaction time (chapter 3). Second, a prototype of a macroscopic DNA machine made of DNA-b-PPO was investigated. As DNA-b-PPO molecules were amphiphilic, they could form a monolayer at the air-water interface. Using a Langmuir film balance, the energy released owing to DNA hybridization was converted into macroscopic movements of the barriers in the Langmuir trough. A specially adapted Langmuir trough was build to exchange the subphase without changing the water level significantly. Upon exchanging the subphase with complementary DNA containing buffer solution, an increase of lateral pressure was observed which could be attributed to hybridization of single stranded DNA-b-PPO. The pressure versus area/molecule isotherms were recorded before and after hybridization. I also carried out a series of control experiments, in order to identify the best conditions of realizing a DNA machine with DNA-b-PPO. To relate the lateral pressure with molecular structures, Langmuir Blodgett (LB) films were transferred to highly ordered pyrolytic graphite (HOPG) and mica substrates at different pressures. These films were then investigated with AFM (chapter 4). At last, this thesis includes studies of DNA and DNA block copolymer assemblies with AFM, which were performed in cooperation with different group of the Sonderforschungsbereich 625 “From Single Molecules to Nanoscopically Structured Materials”. AFM was proven to be an important method to confirm the formation of multiblock copolymers and DNA networks (chapter 5).

Molecular motors-based micro- and nano-biocomputation devices

Protein molecular motors, which are natural nano-machines that convert the chemical energy into mechanical work for cellular motion, muscle contraction and cell division, have been integrated in the last decade in primitive nanodevices based on the motility of nano-biological objects in micro- and nano-fabricated structures. However, the motility of microorganisms powered by molecular motors has not been similarly exploited. Moreover, among the proposed devices based on molecular motors, i.e., nanosensors, nano-mechanical devices and nano-imaging devices, biocomputation devices are conspicuously missing. The present contribution discusses, based on the present state of the art nano- and micro-fabrication, the comparative advantages and disadvantages of using nano- and micro-biological objects in future computation devices. (c) 2006 Elsevier B.V. All rights reserved.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.