Incluir como ag?ncias financiadoras: Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES), Financiadora de Estudos e Projetos (FINEP) e Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG). / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-02-07T21:39:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_henrique_fernandes_ottoni.pdf: 3524322 bytes, checksum: c7675d522b34cfb2455e18dbca1699da (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-03-09T19:17:22Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_henrique_fernandes_ottoni.pdf: 3524322 bytes, checksum: c7675d522b34cfb2455e18dbca1699da (MD5) / Made available in DSpace on 2018-03-09T19:17:22Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_henrique_fernandes_ottoni.pdf: 3524322 bytes, checksum: c7675d522b34cfb2455e18dbca1699da (MD5) Previous issue date: 2017 / Conselho Nacional de Pesquisas (CNPq) / A presen?a de fluoresc?ncia interferente em uma dada amostra ? considerada como um importante problema em m?todos fluorim?tricos, devido ? poss?vel sobreposi??o espectral entre ela e a emiss?o fluorescente de sondas. Por isso, ? importante conhecer se h? fluoresc?ncia interferente em uma dada amostra e subst?ncias de interesse nas condi??es de trabalho pretendidas. No presente estudo, foi avaliada a presen?a de fluoresc?ncia interferente em linf?citos humanos ap?s o tratamento com extratos de tr?s diferentes plantas medicinais, sendo estas, objeto de estudo do nosso grupo de pesquisas. Os extratos s?o: o extrato etan?lico das partes a?reas de Ageratum fastigiatum, extrato etan?lico das partes a?reas de Eriosema campestre e o extrato etan?lico do caule de Pseudobrickellia brasiliensis. Foi coletado o sangue de tr?s volunt?rios para a separa??o das c?lulas mononucleares de sangue perif?rico, para a confec??o de culturas in vitro em meio RPMI devidamente suplementado. Foram feitas uma cultura controle n?o-tratada (CON), culturas tratadas com cada extrato em tr?s concentra??es diferentes, al?m de uma cultura de c?lulas tratadas com dimetilsulf?xido (DMSO), solvente usado na solubiliza??o dos extratos vegetais. As culturas celulares foram incubadas por 24 horas a 37 ?C e 5% de CO2. Ap?s esse per?odo, as c?lulas foram lavadas e avaliadas por citometria de fluxo ou por microscopia confocal. A presen?a de fluoresc?ncia interferente foi determinada com base em histogramas de intensidade de fluoresc?ncia feitos para oito intervalos de comprimento de onda distintos, tendo sido feitas an?lises quali e quantitativas dos dados. A fluoresc?ncia dos extratos vegetais e DMSO foram avaliadas isoladamente por fluorimetria, usando-se as mesmas concentra??es utilizadas para as culturas celulares. Atrav?s da citometria de fluxo, foi identificado que o tratamento de linf?citos com qualquer um dos tr?s extratos de plantas levou ao aparecimento de fluoresc?ncia interferente detect?vel em v?rias faixas de comprimento de onda. Culturas tratadas com DMSO n?o apresentaram fluoresc?ncia interferente. Pela fluorimetria foi visto que os extratos n?o emitem fluoresc?ncia, o que sugere que a fluoresc?ncia interferente foi induzida nas c?lulas ap?s intera??es entre elas e os extratos. Este estudo levanta precau??es que visam avaliar a poss?vel presen?a de fluoresc?ncia interferente em condi??es de trabalho, possibilitando evitar esse vi?s e aumentar a confiabilidade dos resultados. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The presence of interfering fluorescence in a given sample is considered as an important problem on fluorometric methods due to the possible spectral overlap between it and the fluorescent emission of probes. Therefore, it is important to know if there is interfering fluorescence in a given sample, as well as, in substances of interest, in the desired working conditions. In the present study, it was evaluated the presence of interfering fluorescence in human lymphocytes after the treatment with extracts from three different medicinal plants, being such plants the object of study of our research group. The extracts are: the ethanolic extract from the aerial parts of Ageratum fastigiatum, ethanolic extract from the aerial parts of Eriosema campestre and the ethanolic extract from the stem of Pseudobrickellia brasiliensis. Blood was collected from three volunteers to get the peripheral blood mononuclear cells, for in vitro culture preparation in properly supplemented RPMI medium. It was made an untreated control culture (CON), cultures treated with each extract at three different concentrations, and a culture of cells treated with dimethylsulfoxide (DMSO), the solvent used in the plant extracts solubilization. Cell cultures were incubated for 24 hours at 37?C and 5% CO2. After that, cells were washed and evaluated by flow cytometry or by confocal microscopy. The presence of interfering fluorescence was determined based on making fluorescence intensity histograms at eight wavelength ranges for each cell culture. Qualitative and quantitative data analyzes were performed with those graphs. Plant extracts and DMSO fluorescences were evaluated by fluorometry, using the same concentrations used for the cell cultures. In flow cytometry results, it was identified that the treatment of lymphocytes with any of the three plant extracts led to the appearance of interfering fluorescence detectable in several wavelength ranges. Cultures treated with DMSO showed no interfering fluorescence. By fluorometry it was seen that the extracts are not fluorescent, which suggests that the interfering fluorescence was induced in the cells by interactions between them and the extracts. This study raises precautions to evaluate the possible presence of interfering fluorescence in working conditions, making possible to avoid this bias and increase the results reliability.

Autofluorescence retinal images are obtained with a confocal laser scanning ophthalmoscope, and used for the diagnostic of glaucoma. Glaucoma causes a gradual death of nerve cells and can cause blindness. Retina autofluorescence is caused by pigment lipofuscin, which causes cell damage. The aim of this work was to study methods suitable for segmentation of autofluorescence zones and method for tracking objects in an image. In this project was implemented algorithm of autofluorescence zone detection using method of region growing, designed and realized method for tracking autofluorescence regions.

The aim of this work has been the development and optimization of methods for early diagnosis of lung cancer, their utility and integration into daily practice. Firstly, we developed a device for measurement of endobronchial temperature (thermobronchoscopy) and found significant difference in endoluminal temperature above tumors and infiltrated lymph nodes compared to healthy regions. We further designed an appliance for near infrared spectroscopy of the bronchial mucosa and identified spectroscopic features useful for localization of solitary pulmonary nodule. The use of the appliance improved yield of endobronchial biopsy compared to endobronchial ultrasound. In the next part of the study, we describe further techniques for early diagnosis of lung cancer including endobronchial ultrasound, optical coherence tomography, confocal fluorescence microendoscopy, reflectance spectroscopy, autofluorescence bronchoscopy, fluorescence bronchoscopy, and narrow band imaging with concise introduction of our experience gained in several pilot projects. Next, we showed the utility of measurement of acetic acid in exhaled air as a promising biomarker for non-invasive identification of patients with symptomatic acid gastroesophageal reflux. Lastly, we demonstrated significant difference in radiation dose in HRCT...

The aim of this work has been the development and optimization of methods for early diagnosis of lung cancer, their utility and integration into daily practice. Firstly, we developed a device for measurement of endobronchial temperature (thermobronchoscopy) and found significant difference in endoluminal temperature above tumors and infiltrated lymph nodes compared to healthy regions. We further designed an appliance for near infrared spectroscopy of the bronchial mucosa and identified spectroscopic features useful for localization of solitary pulmonary nodule. The use of the appliance improved yield of endobronchial biopsy compared to endobronchial ultrasound. In the next part of the study, we describe further techniques for early diagnosis of lung cancer including endobronchial ultrasound, optical coherence tomography, confocal fluorescence microendoscopy, reflectance spectroscopy, autofluorescence bronchoscopy, fluorescence bronchoscopy, and narrow band imaging with concise introduction of our experience gained in several pilot projects. Next, we showed the utility of measurement of acetic acid in exhaled air as a promising biomarker for non-invasive identification of patients with symptomatic acid gastroesophageal reflux. Lastly, we demonstrated significant difference in radiation dose in HRCT...

The optical touch pointer (OTP), a fluorescence spectroscopy based system, assists brain surgeons during guided brain tumor resection in patients with glioblastoma multiforme (GBM). After recording and analyzing the autofluorescence spectrum of the tissue, it is possible to distinguish malignant from healthy brain tissue. A challenge during the intraoperative measurements is the interference of blood. If it gets in contact with the laser pointer, the blood blocks the light transmission to and from the tissue. The purposes of the project were to study and categorize patterns of blood interference and to present possible solutions to avoid signal blocking by blood. To measure fluorescence and reflection two devices were used respectively, the OTP which has a spectrometer and a blue laser, and the diffused reflection spectroscopy system (DRS) which has a spectrometer and a white light source. Both operate independently from each other and are connected to a fiber optical probe. A similar scenario to the one in the operation theater was simulated in the lab. Fluorescence and diffuse reflection measurements with and without blood were realized on skin and on two different plastic fluorescent standards. The results were analyzed with the aid of MatLAB, and compared with data collected in the hospital during brain tumor resection. The highest autofluorescence of brain tissue and skin is reached at approximately 506 nm. Although skin and both plastic standards have different optical properties regarding color or rather fluorescence, all of them presented very similar curves when blood on them blocked partially or completely the light transmission. A blood layer of more than 0.1 mm thickness blocks the blue laser light. Blood absorption happens at 541 and 577 nm due to oxy-hemoglobin (HbO2) in both liquid and dried blood. When the fluorescence spectrum is available but weak, the reflection spectrum contains two dips (traces of HbO2 at 541 and 577 nm). In brain there were cases in which light absorption occurred additionally at other wavelengths than the absorption peaks of deoxyhemoglobin (Hb) and HbO2. Blood interference during the OP can be prevented if the probe rests in a saline solution after every measurement. In this way the fresh blood sticking on the probe dissolves in the solution. For dried or coagulated blood, additional manual cleansing is needed.

No description available.

The aromatic amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies. These investigations extract information on protein conformations and interactions from the emitted fluorescence's intensity, wavelength, and/or polarization. Like most fluorescent organic compounds, these amino acids also undergo transitions into dark meta-stable states, including triplet and photo-radical states. These transitions are notably sensitive to the surrounding environment, offering an additional set of parameters that reflect the protein's interactions, folding states, and immediate surroundings. Transient State (TRAST) monitoring has been developed to quantify fluorophore transition dynamics by recording the average fluorescence intensity in response to a modulated excitation. In this work, we performed TRAST experiments to investigate tyrosine autofluorescence and used it to detect conformational changes in calmodulin, a calcium-binding protein containing two tyrosine residues. A photophysical model for tyrosine was established, and it was revealed how tyrosine's dark state transitions changed with excitation intensity, solvent pH, and redox conditions. The TRAST experiments demonstrated that tyrosine's dark state transitions could serve as valuable information sources for label-free analyses of protein conformations and interactions. / De aromatiska aminosyrorna tryptofan, tyrosin och fenylalanin har använts i stor utsträckning för olika inmärkningsfria proteinstudier. Dessa undersökningar extraherar information om proteinkonformationer och interaktioner från den emitterade fluorescens intensiteten, dess våglängd och/eller polarisering. Liksom de flesta fluorescerande organiska föreningar genomgår dessa aminosyror också övergångar till mörka metastabila tillstånd, inklusive triplett- och fotoradikaltillstånd. Dessa övergångar är särskilt känsliga för den omgivande miljön, och erbjuder en extra uppsättning parametrar som återspeglar proteinets interaktioner, vikningstillstånd och omedelbara omgivningar. Transient State (TRAST) monitorering har utvecklats för att kvantifiera fluoroforövergångsdynamik genom att registrera den genomsnittliga fluorescensintensiteten som svar på en modulerad excitation. I detta arbete utförde vi TRAST-experiment för att undersöka tyrosinautofluorescens och använde den för att detektera konformationsförändringar i calmodulin, ett kalciumbindande protein som innehåller två tyrosiner. En fotofysikalisk modell för tyrosin etablerades, och hur tyrosins mörka tillståndsövergångar förändrades med excitationsintensitet, lösningsmedels pH och redoxförhållanden kunde faststållas. TRAST- experimenten visade att tyrosins mörka tillståndsövergångar kan fungera som värdefulla informationskällor för inmärkningsfria analyser av proteinkonformationer och interaktioner.

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

L'objectif de ce travail de recherche est le développement, la mise au point et la validation d'une méthode de spectroscopie multi-modalités en diffusion élastique et autofluorescence pour caractériser des tissus biologiques in vitro et in vivo. Ces travaux s'organisent en quatre axes.<br />La première partie des travaux présente l'instrumentation : développement, réalisation et caractérisation expérimentale d'un système de spectrométrie bimodale multi-points fibrée permettant l'acquisition de spectres in vivo (distances variables, acquisition rapide).<br />La deuxième partie porte sur la modélisation des propriétés optiques du tissu : développement et validation expérimentale sur fantômes d'un algorithme de simulation de propagation de photons en milieux turbides et multi-fluorescents.<br />La troisième partie propose une étude expérimentale conduite ex vivo sur des anneaux artériels frais et cryoconservés. Elle confirme la complémentarité des mesures spectroscopiques en diffusion élastique et autofluorescence et valide la méthode de spectroscopie multi-modalités et l'algorithme de simulation de propagation de photons. Les résultats originaux obtenus montrent une corrélation entre propriétés rhéologiques et optiques.<br />La quatrième partie développe une seconde étude expérimentale in vivo sur un modèle pré-clinique tumoral de vessie. Elle met en évidence une différence significative en réflectance diffuse et/ou en autofluorescence et/ou en fluorescence intrinsèque entre tissus sains, inflammatoires et tumoraux, sur la base de longueurs d'onde particulières. Les résultats de la classification non supervisée réalisée montrent que la combinaison de différentes approches spectroscopiques augmente la fiabilité du diagnostic.

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