Femtosekundenaufgelöste Ramanspektroskopie ist ein leistungsfähiges Werkzeug, um die Schwingungsentwicklung eines angeregten Chromophors in Echtzeit zu studieren. In dieser Arbeit wurde ein durchstimmbares Ramanspektrometer mit 10 cm-1 spektraler und 50--100 fs zeitlicher Auflösung entwickelt und für eine Anwendung auf flavinbasierte Photorezeptoren optimiert. Es wird der Einfluß der Resonanzbedingungen auf das transientes Ramanspektrum charakterisiert. Die Dynamik des angeregten Zustandes wird zuerst für den Modellphotoschalter Stilben untersucht, ausgehend sowohl vom cis-, als auch vom trans-Isomer. Intensitätsabnahme, spektrale Verschiebung und Bandenverschmälerung liefern Einblicke in die Schwingungsrelaxation des angeregten Chromophors. Wellenpaketbewegung und anharmonische Kopplung werden als Oszillationen beobachtet. Für das "Mutter"-Cyanin 1,1''-Diethyl-2,2''-pyridocyaniniodid wird die Isomerisierung bis in den Grundzustand verfolgt. Ramanspektren des Franck-Condon-Zustandes, des intermediär gebildeten heissen Grundzustandes und der Isomerisierungsprodukte werden erhalten. Als Grundlage für Experimente an Flavoproteinen werden die Eigenschaften des angeregten Flavinchromophors in Lösung untersucht. Transiente Absorptions- und Fluoreszenzexperimente weisen auf den Einfluss dynamischer polarer Solvatation hin. Es werden Ramanspektren des angeregten Zustandes von Flavin aufgenommen und die Schwingungsbanden zugeordnet. Populationsverminderung durch den Ramanimpuls wird als potentielles Artefakt in zeitaufgelösten Messungen identifiziert; der Effekt wird aber auch genutzt, um Wellenpaketbewegung im angeregten Zustand zu markieren. Die Photorezeptormutanten BlrB-L66F und Slr1694-Y8F werden mit transienter Absorption studiert. Dabei wird die Bildung des Signalzustandes und Flavinreduktion durch ein Tryptophan beobachtet. Die Anwendung des entwickelten Ramanspektrometers auf biologische Proben wird in einem ersten Experiment an Glucose Oxidase demonstriert. / Femtosecond stimulated Raman spectroscopy is a powerful tool that allows to study the structural relaxation of an excited chromophore directly in time. In this work a tunable Raman spectrometer with 10 cm-1 spectral and 50-100 fs temporal resolution was developed, and the technique was advanced towards applications to flavin-based proteins. With this device the influence of resonance conditions on the transient Raman spectrum is characterized. Excited-state dynamics is first investigated for the model photoswitch stilbene, starting from both the cis and the trans isomers. Decay, spectral shift, and narrowing of individual bands provide insight into the vibrational relaxation of the excited chromophore. Wavepacket motion and anharmonic coupling is seen as oscillations. Isomerization is followed to the ground state for the "parent" cyanine 1,1''-diethyl-2,2''-pyrido cyanine iodide. From a global analysis, Raman spectra for the Franck-Condon region, the intermediately populated hot ground state, and the isomerization products are obtained. As a basis for experiments on flavoproteins, the excited-state properties of the pure flavin chromophore are studied in solution. Transient absorption and fluorescence experiments suggest an influence of dynamic polar solvation on the electronic properties of the excited state. Raman spectra from the flavin excited state are recorded and the vibrational bands assigned. Population depletion by the Raman pulse is identified as a potential artefact, but the effect is also used to mark wavepacket motion in the excited state. The photoreceptor mutants BlrB-L66F and Slr1694-Y8F are studied by transient absorption; signaling state formation and flavin reduction by a semiconserved tryptophan are seen, respectively. The application of femtosecond Raman spectroscopy to biological samples is demonstrated in a first experiment on glucose oxidase.

No description available.

Esta dissertação consiste em uma abordagem do fenômeno da luz que interesse ao Design da Iluminação. Isso se faz aqui através da leitura da obra de Dan Flavin, artista americano que utilizou a lâmpada fluorescente como material quase que exclusivo em seus trabalhos. Flavin conhece e explora a luz como um fenômeno intangível. Recusa noções preconcebidas de espaço, e busca novas experiências perceptuais e espaciais. Assim, sua obra nos permitiria uma leitura múltipla, levantando diversos aspectos importantes para a atividade do designer da Iluminação. Pesquisar Flavin é uma imersão na luz, na cor, no espaço e na percepção humana. A riqueza visual e conceitual de sua obra nos levaram a explorar o conhecimento que se tem sobre a presença da luz no espaço que complementem a prática do Design. / This essay proposes an approach to the phenomenon of light that involves Lighting Design. The route to this exploration is the work of Dan Flavin, an American artist who used fluorescent lamp as the practically exclusive material of his work. Flavin knows and explores light as an intangible phenomenon. He refuses preconceived notions of space and seeks new perceptual and spacial experiences. As a result, his work allows multiple readings and raises several key aspects for the activity of the Lighting designer. To research Flavin is to immerse oneself in light, color, space and human perception. The visual and conceptual richness of his work led us to explore the knowledge we have of the presence of light in space, which complements the Design practice.

Antibiotic resistance represents a real threat in the modern world. The problem of resistance is brought about by the fast evolution of bacteria, accelerated by misuse and over-prescription of antibiotics and compounded by the decline in the discovery and development of new classes of antibiotics. Consequently, new targets for antibiotics are in high demand. Flavin-dependent thymidylate synthase (FDTS), which is not present in humans and is responsible for the biosynthesis of a DNA building block in several human pathogens (e.g., M. tuberculosis, B. anthracis, H. pylori), is one such novel target. FDTS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to produce 2'-deoxythymidine-5'-monophosphate (dTMP), with N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄fol) serving as the carbon source and a nicotinamide cofactor as the electron source. No efficient inhibitors of FDTS are known, despite high-throughput screening attempts to find them. Intermediate and transition-state mimics are likely to bind the enzyme with greater affinity and hence have a better chance at inhibiting FDTS. Therefore, the understanding of the chemical mechanism of FDTS is critical to the informed design of compounds capable of disrupting its function in bacteria. We utilized various techniques, including chemical trapping of reaction intermediates, substrate isotope exchange and stopped-flow, to investigate the FDTS mechanism and determine what sets it apart from other pyrimidine methylases. We found that at least two different intermediates kinetically accumulate in the FDTS-catalyzed reaction. Both of these intermediates are trapped in acid in the form of 5-hydroxymethyl-dUMP, which has never been isolated in other uracil-methylating enzymes. Under basic conditions, however, the earlier intermediate is converted to a species with an unusual flavin-derived adduct, while the later intermediate is converted to dTMP product. Our experiments also suggest that dUMP is activated for the reaction by the reduced flavin - a substrate activation mechanism distinct from the one employed by the classical pyrimidine-methylating enzymes.

Esta dissertação consiste em uma abordagem do fenômeno da luz que interesse ao Design da Iluminação. Isso se faz aqui através da leitura da obra de Dan Flavin, artista americano que utilizou a lâmpada fluorescente como material quase que exclusivo em seus trabalhos. Flavin conhece e explora a luz como um fenômeno intangível. Recusa noções preconcebidas de espaço, e busca novas experiências perceptuais e espaciais. Assim, sua obra nos permitiria uma leitura múltipla, levantando diversos aspectos importantes para a atividade do designer da Iluminação. Pesquisar Flavin é uma imersão na luz, na cor, no espaço e na percepção humana. A riqueza visual e conceitual de sua obra nos levaram a explorar o conhecimento que se tem sobre a presença da luz no espaço que complementem a prática do Design. / This essay proposes an approach to the phenomenon of light that involves Lighting Design. The route to this exploration is the work of Dan Flavin, an American artist who used fluorescent lamp as the practically exclusive material of his work. Flavin knows and explores light as an intangible phenomenon. He refuses preconceived notions of space and seeks new perceptual and spacial experiences. As a result, his work allows multiple readings and raises several key aspects for the activity of the Lighting designer. To research Flavin is to immerse oneself in light, color, space and human perception. The visual and conceptual richness of his work led us to explore the knowledge we have of the presence of light in space, which complements the Design practice.

In sea urchin development, cell fate specification appears by the 60-cell stage embryo when several embryonic territories are recognized: the small micromeres, the large micromeres which will generate primary mesenchyme cells, the vegetal2 layer that will give rise to pigment cells, immunocytes, and muscle cells, the vegetal1 layer, as well as the oral and aboral ectoderm. A Delta-Notch signaling event is required for the differential specification of mesodermal cells that will give rise to secondary mesenchyme cells (SMCs). SMCs produce four cell types: pigment cells, blastocoelar cells, circumesophageal muscle cells, and coelomic pouch cells. Pigment cells are the first to be specified. During primary invagination at the gastrula stage, eight pigment cell progenitors delaminate from the archenteron into the blastocoel. By the pluteus stage, approximately 30 pigment cells are embedded in the ectoderm. Pigment cells produce echinochrome, a napthoquinone pigment. Previously, several genes in the sea urchin embryo were isolated that are expressed specifically in pigment cell precursors during the blastula stage. The goal of this research was to characterize a subset of these genes, which are highly similar to: the polyketide synthase gene (Pks), a sulfotransferase gene (Sult), three different members of the flavin-containing monooxygenase gene family (Fmo), and the transcription factor glial cells missing (Gcm). Polyketide synthases (PKSs) are a large family of multifunctional proteins mainly found in bacteria, fungi, and plants. They are responsible for the biosynthesis of a variety of polyketide compounds including antibiotics and mycotoxins. In the sea urchin, SpPks is required for echinochrome biosynthesis. Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoproteins mainly found in bacteria, plants, and higher metazoan. They are responsible for catalyzing the oxidation of several compounds including the detoxification of xenobiotics and activation of numerous metabolites. It is known that SpFmo1 is required for echinochrome biosynthesis. Sulfotransferases are found from bacteria through higher eukaryotes. These enzymes catalyze the sulfate conjugation of several substrates resulting in either compound detoxification or bioactivation.

No description available.

No description available.

ABSTRACT Many enzymes require cofactors in order to carry out specific functions. Flavins, which are naturally fluorescent, compose a unique group of redox cofactors because they have the ability to transfer one or two electrons and are therefore found in three different oxidation states. A specific flavin, flavin adenine dinucleotide (FAD), is a crucial cofactor that facilitates electron transfer in many flavoproteins involved in DNA repair, photosynthesis, and regulatory pathways. One example of a FAD-containing DNA repair protein is DNA Photolyase (PL). E. coli PL is a monomeric flavoprotein that facilitates DNA repair via a photoinduced electron transfer reaction. The catalytic cofactor, FAD, transfers an electron to a thymidine dimer lesion, to cleave the cyclobutane ring and restore the DNA strand. Although the mechanism of repair has been partially elucidated by our group, it is still unclear whether or not the electron is transferred directly from the isoalloxazine moiety to the dimer or if the electron hops from the isoalloxazine moiety to the adenine moiety to the dimer. This sequential hopping mechanism should have excited state absorption features for the reduced flavin species, an adenine radical anion, and the semiquinone flavin species. To investigate the mechanistic role of adenine, E. coli PL has been reconstituted with -FAD, an FAD analogue in which the adenine was substituted via chemical means with 1,N6 – ethenoadenine dinucleotide. -FAD was selected due to its ease of synthesis and because its structure changes the thermodynamic driving force for the electron transfer reaction, by lowering the energetic gap (LUMO-LUMO) between the isoalloxazine ring and the modified adenine. In order to characterize the excited state dynamics of the mutant chromophore, the transient absorption measurements were made of each free flavin in solution. These measurements indicate the pathway of electron transfer must be mediated via superexchange rather than a hopping mechanism. This important result shows that the role of adenine in photolyase is to facilitate a superexchange electron transfer mechanism, and a modified flavin can act as a reporter under these experimental conditions. By exploiting Corynebacterium ammoniagenes FAD synthetase adenylation promiscuity, we have enzymatically-synthesized and purified a novel dually fluorescent flavin cofactor. This new flavin adenine dinucleotide (FAD) analogue, flavin 2-aminopurine (2Ap) dinucleotide (F2ApD), can be selectively excited through the 2Ap moiety at 310 nm, a wavelength at which flavins have intrinsically low extinction. The dinucleotide 2Ap emits at 370 nm with high efficiency. This emission has excellent overlap with the absorption spectra of both oxidized and reduced hydroquinone flavin (FlOX and FlHQ respectively), which emit at ~525 and ~505 nm respectively. We have characterized the optical properties of this dually fluorescent flavin, iFAD. Steady state fluorescence excitation and emission spectra were obtained and contrasted with the other flavins. Temperature- and solvent-dependent emission spectra suggest that F2ApD stacking interactions are significantly different compared to FAD and etheno-FAD (FAD). The optical absorption spectra of these dinucleotides were compared with FMN to explore electronic interactions between the flavin and nucleobase moieties. To probe the evolution of the different excited state populations, femtosecond transient absorption measurements were made on the iFADs, revealing that F2ApD exhibited unique transient spectra as compared to either FAD or FAD. The significance of these results to flavins, flavoprotein function, and bioimaging are discussed. The reconstituted -FAD in E.coli photolyase was catalytically active and actually repaired more efficiently than the FAD-reconstituted photolyase. To validate that an enzymatically synthesized iFAD could be reconstituted into a flavoprotein, this work shows a DNA repair assay using F2ApD that was reconstituted into E. coli photolyase, generating the reconstituted analogue, ApPL. Activity assays were compared between FAD-PL and ApPL. This comparison further elucidates the importance of the driving force on the electron transfer reaction in PL. A comparison of fluorescence spectroscopies between the reconstituted PLs highlights their applicability as biosensors and/or mechanistic reporters. / Chemistry

SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector. / Master of Science in Life Sciences