Biological systems present many challenges to researchers attempting to study them using spectroscopy. Low specificity, low sensitivity, and broad and overlapping lineshapes limit the amount of information that can be obtained in experiments. Two-dimensional fluorescence spectroscopy (2D FS) is a highly sensitive and information-rich spectroscopic technique that was developed to study the conformations and excited state dynamics of systems exhibiting exciton coupling. In this dissertation, I describe a variety of extensions of 2D FS that further increase its utility for the study of biological systems. I describe experiments on a dimer of zinc tetraphenylporphyrin embedded in a membrane, in which the signals from two conformational subpopulations were separated in order to study the thermodynamics of their interconversion. I present proof-of-principle experiments on nucleic acids that utilize fluorescence resonance energy transfer to separate signals from different subpopulations. I also describe experiments in which 2D FS was performed using ultraviolet excitation to determine the conformation of a dinucleotide of a fluorescent analogue of the nucleic acid base adenine. I discuss experiments on porphyrin dimers in which 2D FS was used as a probe of excited state dynamics. Finally, I present model calculations for a proposed variation of 2D FS in which entangled photons would be used as the excitation source. These calculations suggest that this approach has the potential to yield significantly narrower spectral lineshapes than conventional 2D FS. These experiments and calculations yield new insight into the systems investigated and establish a `toolbox' of variations of 2D FS that can be used to gain as much information as possible from experiments on challenging systems such as protein-DNA complexes. This dissertation contains previously published and unpublished co-authored material.

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Made available in DSpace on 2014-06-11T19:33:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-04-28Bitstream added on 2014-06-13T19:23:00Z : No. of bitstreams: 1 rodrigues_ja_dr_arafo.pdf: 687578 bytes, checksum: c18d341ec7fb82f28c518c4dc3ec4fac (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo do presente estudo foi avaliar in vitro o desempenho dos métodos baseados na medição da fluorescência induzida pela luz para detecção de lesões de cárie oclusal em dentes permanentes. Para tanto, foram realizadas três pesquisas: (1) O objetivo dessa pesquisa foi avaliar a influência da subtração do valor de fluorescência zero no desempenho de dois aparelhos que induzem fluorescência na detecção de cárie. Desse modo, foram utilizados 119 molares permanentes, nos quais três áreas da superfície vestibular (cúspide, central e cervical) das porções mesial e distal foram selecionadas. Além disso, uma lesão de cárie oclusal por dente foi escolhida como “sítio teste”. Dois examinadores mensuraram tanto as áreas nas superfícies vestibulares quanto as lesões oclusais utilizando o DIAGNOdent 2095 (DD, KaVo, Alemanha) e o DIAGNOdent 2190 (DDpen). Foi observada influência da subtração do valor de fluorescência zero no desempenho do DD, com diminuição da sensibilidade. Pode-se concluir que as mensurações com o DD podem ser realizadas sem a subtração do valor de fluorescência zero. No entanto, para o DDpen esse procedimento não pode ser eliminado. (2) Foram selecionados 119 dentes com o objetivo de comparar o desempenho na detecção de lesões de cárie oclusal de três aparelhos que induzem fluorescência, exame radiográfico convencional e sistema ICDAS II. Uma lesão de cárie por dente foi escolhida como “sítio teste”. Dois examinadores realizaram duas medidas independentes utilizando o DD, DDpen, câmera VistaProof (VP, Dürr Dental, Alemanha), além de exame radiográfico convencional e exame visual através do sistema ICDAS II. Conclui-se que cada aparelho variou seu desempenho de acordo com os valores de sensibilidade e especificidade. / The aim of this in vitro study was to asses the performance of light-induced fluorescence as auxiliary to conventional methods is detecting occlusal caries lesions in permanent teeth. For this reason, three studies were carried out: (1) The aim of this study was to evaluate the influence of zero value subtraction on the performance of two laser fluorescence devices for detecting occlusal caries. 119 permanent molars were selected. Three areas (cuspal, middle and cervical) of both mesial and distal portions and one occlusal site were assessed by two examiners using the DIAGNOdent 2095 (LF, KaVo, Germany) and DIAGNOdent 2190 (LFpen) devices. It was observed an influence of the zero value subtraction in the LF performance: the sensitivity decreased. However, because of the trend, it could be concluded that the LF readings could be performed without the zero value subtraction. Despite of that, it does not imply in wrong results clinically. For the LF pen, the zero value subtraction influences the performance though and should however not be eliminated. (2) This in vitro study compared the performance of fluorescence-based methods, radiographic examination, and ICDAS II system on occlusal surfaces. 119 molars were independently assessed twice by two experienced dentists using the laser fluorescence (LF and LFpen) and fluorescence camera (FC, Dürr Dental, Germany) devices, ICDAS II system and bitewing radiographs (BW). It can be concluded that the performance of each method changes according to the sensitivity and specificity.

The advent and establishment of systems biology has cemented the idea that real understanding of biological systems requires quantitative models, that can be integrated to provide a complete description of the cell and its complexities. At the same time, synthetic biology attempts to leverage such quantitative models to efficiently engineer novel, predictable behaviour in biological systems. Together, these advances indicate that the future understanding and application of biology will require the ability to create, parameterise and discriminate between quantitative models of cellular processes in a rigorous and statistically sound manner. In this thesis we take the regulation of GAL1 expression in Saccharomyces cerevisiae as a test case and look at all aspects of this process: from reporter selection to data acquisition and statistical analysis. In chapter B we will discuss optimal fluorescent reporter selection and construction for investigating transcriptional dynamics, as well as procedures for quantifying and correcting the various sources of error in our microscope system. In chapter 3 we will describe software developed to analyse fluorescent microscopy images and convert them to gene expression data. A number of iterations of the software are tested against manually curated data sets, and the measurement error produced by its imperfections is quantified and discussed. In chapter 4 a method, based on fluctuations in photobleaching, is developed for quantifying both measurement error and the relationship between protein concentration and measured fluorescence. The method is refined and its efficacy discussed. In the last section I make a preliminary application of these methods to investigating the regulatory effect of the GAL10-lncRNA. Interesting phenomena are observed and further investigated using two new strains: genetic variants expressing a fluorescent reporter from the GAL1 promoter, one harbouring a wild type GAL1 promoter and one in which the binding site for the Gal10 noncoding RNA has been removed. The methods developed throughout the thesis are applied and the data analysed. A heterogeneous response, distinguishable between the two strains, is observed and related to cell-to-cell variations in growth rate.

Atualmente os maiores gastos da citricultura concentram-se na sanidade do pomar. Os custos fitossanitários, juntamente com fertilizantes, ultrapassam 60% do custo da produção. O controle de doenças como a Morte Súbita dos Citros (MSC), Declínio e o Greening, também conhecida como Huanglongbing, são feitos de forma visuais os que conferem ao método um elevado grau de subjetividade e imprecisão. O presente trabalho teve por finalidade desenvolver uma metodologia rápida e segura de diagnóstico para as doenças acima citadas utilizando espectroscopia de fluorescência e fluorescência induzida por laser (FIL). Foram utilizados neste estudo dois porta-enxertos, Cravo e Cleópatra, e quatro variedades de copa, Pêra, Natal, Valência e Hamilim. As doenças estudadas também foram classificadas em diferentes níveis de severidade. O índice 1 foi utilizado para baixa severidade da MSC, e 2 para o estágio mais avançado da doença. Para o Greening fez-se uma distinção entre folhas sintomáticas e assintomáticas. A doença Declínio dos Citros também fez parte deste estudo em função dos sintomas serem muito parecidos aos da fase inicial de MSC, sendo este um problema sério para as avaliações de contaminação no campo. Inicialmente, foram realizados estudos a respeito da emissão de fluorescência dos pigmentos fotossintéticos provenientes dos extratos de folhas doentes e sadias através de um fluorímetro de bancada. Para as análises foliares utilizou-se um sistema portátil de espectroscopia de fluorescência induzida por laser desenvolvido no LOLEIA. O estudo dos extratos revelaram que as maiores variações ocorrem nas concentrações de clorofila a e b e que os pigmentos acessórios como ? e ?- caretonos não fluorescem no comprimento de onda estudado. De uma maneira geral, é possível dizer que plantas doentes sofrem uma diminuição das clorofilas a e b. Os resultados das folhas foram analisados por um método estatístico de Análises de Componentes Principais (PCA) para facilitar a observação das alterações espectrais. Através da metodologia desenvolvida foi possível a identificação e a distinção das doenças com nível de acerto superior a 93%. Contudo, estes resultados contribuíram para o desenvolvimento de uma metodologia para diagnóstico das doenças de citros de uma forma rápida, precisa e economicamente viável. / Currently the largest portion of money applied to citriculture focus on health of the orchard. The phytosanitary costs, along with fertilizers, exceed 60% of the production cost. The control of diseases such as Sudden Death of Citrus (SDC), Decline and Greening, also known as Huanglongbing, is done by visual inspection, which confers to the method a high level of subjectivity and imprecision. This work was intended to develop a quick and safe methodology of diagnosis for the diseases mentioned above using fluorescence spectroscopy and laser-induced fluorescence (LIF). It was used in this study two rootstocks, Cravo and Cleopatra, and four varieties of crown: Pera, Natal, Valencia and Hamlin. The diseases studied were also classified into different levels of severity. The index 1 was used for low severity SDC, and 2 for a more advanced stage of this disease. For Greening has been made a distinction between symptomatic and asymptomatic leaves. The Decline of Citrus disease was also part of this study because its symptoms are very similar to those in the initial phase of SDC, and this is a serious problem for the evaluation of contamination in the field. Initially, studies have been made on the fluorescence of photosynthetic pigments from the extracts of ill and healthy leaves using a non-portable fluorimeter. For the leaf analyses we used a portable system of laser-induced fluorescence spectroscopy developed in LOLEIA. The study of extracts revealed that the largest cariations occur in the concentrations of chlorophyll a and b, and that acessory pigments like ? and ?-carotones do not fluoresce in the wavelength used. In general, it is possible to say that ill plants have a decrease of chlorophylls a e b. The leaf results were analyzed through the statistical method Principal Component Analysis (PCA) in order to facilitate the observation of spectral changes. Through the developed methodology it was possible to identify and distinguish the diseases with efficiency greater than 93%. However, these results contributed for the development of a methodology for diagnosis of citrus diseases in a quick, accurate and economically viable way.

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Le tricyanofurane est un groupement extrêmement électro-attracteur grâce à trois groupements nitriles conjugués. Cette caractéristique électronique nous a permis de synthétiser de nouvelles sondes fluorescentes intéressantes pour l’imagerie biologique : de petites molécules possédant un caractère dipolaire très marqué qui leur confère une fluorescence très décalée vers le rouge.Une première application de ce genre de molécules est la détection du pH intracellulaire à l’aide de groupement phénol conjugué au tricyanofurane. Grâce à une rétro-synthèse judicieuse, il a été possible de développer une famille complète possédant une très large gamme de pKa couvrant tous les pH intracellulaires possibles. En fonctionnalisant ces sondes, il est possible de cibler certains organites tels les mitochondries. En remplaçant le groupement hydroxyle par un ester pinacolboronique, ces sondes sont sensibles au peroxyde d’hydrogène. Ce type de sondes est appelé OFF/ON car la sonde est initialement non-fluorescente et la fluorescence est restaurée par l’action du peroxyde d’hydrogène. Une étude de la fluorescence à l’état solide a aussi été menée sur des molécules présentant une émission intense dans le rouge. Celles-ci ont été utilisées pour créer des sondes enzymatiques solubles en milieu physiologique qui libèrent un fluorophore insoluble après activité enzymatique. Ce fluorophore, après précipitation émet une fluorescence non présente avant activation. / Tricyanofuran is a strong electro-withdrawing group due to its three conjugated nitrile groups. This electronic characteristic was used to synthetize new fluorescent probes for biological imaging: small molecules owing a strong dipolar behavior that strongly shifts the fluorescence to the red. A first application of this kind of molecules is intracellular pH detection with a phenol moiety conjugated to the tricyanofuran. Thanks to a convenient retro-synthesis, a large family was developed displaying a large range of pKa spanning all the possible intracellular pHs. When these probes are functionalized, it is possible to target organelles such as mitochondria. When the hydroxyl group is replaced by a pinacolboronic ester, it is possible to detect hydrogen peroxide. This kind of probe is called turn-on probe because the probe is initially non-fluorescent and emission is restored by the reaction with hydrogen peroxide.Solid-state fluorescence was also studied on molecules presenting a strong and red fluorescence. This kind of molecules has then been used to design enzymatic probes soluble in physiological medium. After enzymatic cleavage, a non-soluble fluorophore is liberated and, after precipitation, leads to a strong emission.

Orientador: Teresa Dib Zambon Atvars / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T13:29:22Z (GMT). No. of bitstreams: 1 Cossiello_RafaelDiFalco_D.pdf: 19466365 bytes, checksum: 4613f44ad7300b66e793fa6de43b2c82 (MD5) Previous issue date: 2007 / Resumo: O copolímero SAA (poli(estireno-co-ácido acrílico-co-1-metacrilato de pirenilmetila)) foi sintetizado por via radicalar em agua e caracterizado por analises termicas (DSC, TGA), espectroscopicas (NMR C e H, UV-VIS, FTIR, fluorescência estacionária e resolvida no tempo) e elétricas (eletroluminescência). As blendas com poli[2-metóxi-5-(2¿-etilóxi)-p-fenilenovinileno] MEH-PPV foram estudadas desde 0,01; 0,05; 0,10; 0,50; 1,0; 10,0; 25; 50; 75 e 90 (% em massa) de MEH-PPV em SAA para ánalise da evolucao da morfologia por microscopia óptica, de fluorescência e eletrônica de varredura em espessuras de 70 mm (fratura criogênica) e 70 nm (espessura do dispositivo). Em baixas concentracões os domínios de MEH-PPV se apresentam em forma de núcleos dispersos e empacotados e conforme se aumenta a concentracao de MEH-PPV, observa-se que há a coalescência destes núcleos até a formação de uma fase interconectada de uma rede tridimensional. As medidas de difração à altos angulos mostram que há uma preferência de orientação do MEH-PPV ao longo do substrato e nao influencia a isotropia do SAA devido a baixa miscibilidade entre estes polímeros. As unidades de 1-pirenil metila foram utilizadas para avaliar a polaridade do microambiente e o mecanismo de transferencia de energia entre o doador (SAA) e o receptor (MEH-PPV). A dependência do tempo de fluorescência das unidades 1-pirenilmetila com a concentracao do MEH-PPV fornece subsídios para concluir que ocorre interpenetração de cadeias e, portanto uma baixíssima solubilidade entre os dois polímeros. O melhor desempenho de eletroluminescência foi do dispositivo preparado a partir da mistura 50:50 de MEH-PPV:SAA, cuja corrente elétrica foi aumentada em 2,5 vezes e a luminância em 4 vezes se comparado com o dispositivo de MEH-PPV puro. A morfologia de fase interconectada se mostrou importante para um melhor processo de transporte de cargas. O copolímero de SAA permitiu uma melhor injeção de cargas devido aos grupos carboxila presentes das unidades de ácido acrílico / Abstract: The copolymer SAA poly(styrene-co- acrylic acid -co-1-pyrenylmethyl methacrylate) was synthesized by radical emulsion in water and characterized by thermal analyses (DSC, TGA), spectroscopic (NMR 13C and 1:00, UV-VIS, FTIR, stationary and time-resolved fluorescence) and electric (electroluminescence). The blends with poly[2-methoxy-5(2'ethylhexyloxy)-p-phenylenevinylene] (MEH-PPV) were studied from 0. 01; 0. 05; 0.10; 0.50; 1.0; 10.0; 25; 50; 75 and 90 (% in mass) of MEH-PPV in SAA to analyze the morphology evolution by optical, epifluorescence and electronic microscopy. The films were prepared in two thicknesses: one around 70 mm to be analyzed by cryogenic fractures and 70 nm, to be comparable with the device thickness. In low concentrations the domains of MEH-PPV appears dispersed nuclei and accrete when the concentration of MEH-PPV is increased forming an interconnected phase. The diffraction measures (WAXD) show a preference orientation of MEH-PPV along the substrate plane and it do not influences the SAA due the low miscibility between each other. The increase of the MEH-PPV concentration showed a redshift on electroluminescence spectrum due the inner filter and auto-absorption and re-emission effects. The units of 1-pyrenylmethyl were used to evaluate the polarity of the microenvironment and the mechanism of energy transfer of among the donor (SAA) and the acceptor (MEH-PPV). The timedependence of fluorescence with the increase of MEH-PPV concentration show that occurs an chain interpenetration following the fluorescence non-radiative energy transfer model. The best electroluminescent device was prepared using the mixture 50:50 of MEH-PPV:SAA. The interconnected phases are important for a better process of charge transport. The copolymer SAA allowed for a dilution effect avoiding the aggregation process and facilitating the charge injection processes. This technology of polymeric blends gave good results the increase of the luminance intensity and reduction of the voltage operation / Doutorado / Físico-Química / Doutor em Ciências

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