Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 198-212.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

A cinética enzimática dos derivados de quinizarina com cadeias homólogas por lipases imobilizadas foi investigada por espectroscopia de fluorescência. Este estudo foi realizado em nível macroscópico e microscópico. Para o estudo macroscópico, foi utilizada a lipase suportada CALB (Novozyme® 435) e para o estudo microscópico a lipase Rhizopus niveus imobilizada em nanopartículas de sílica. Os derivados de quinizarina são espécies que não apresentam fluorescência, porém, quando são hidrolisados, tornam-se fluorescentes (quinizarina). Com um modelo cinético considerando um mecanismo de dois processos sequenciais do tipo Michaelis-Menten, foi possível fazer uma descrição adequada da evolução temporal da formação da quinizarina. O tempo médio de reação da hidrólise enzimática, em nível macroscópico, foi determinado para os derivados diacetato, dibutirato, dihexanoato e dioctanoato de quinizarina nos solventes hexano, ciclo-hexano e decalina saturados com água. No estudo microscópico, a lipase de Rhizopus niveus foi incorporada em nanopartículas de sílica de 200nm. A hidrólise enzimática foi monitorada por imagens e pela flutuação da intensidade de fluorescência com o tempo, por meio da microscopia de fluorescência confocal. Os resultados mostraram que, após a adição do substrato (derivados da quinizarina), começam a aparecer regiões fluorescentes devido ao trabalho enzimático (formação da quinizarina). As imagens de microscopia de fluorescência confocal não mostraram uma nítida diferença entre os substratos avaliados. Entretanto, o estudo da flutuação da intensidade de fluorescência mostrou que há uma diferença entre os substratos e que é possível estimar constantes de tempo de relaxação da atividade enzimática. Além disso, a atividade da lipase depende da forma em que a mesma está distribuída nas nanopartículas (ligada ou adsorvida) e também do tamanho da cadeia de alquílica que compões os derivados. O decaimento de fluorescência da quinizarina produzida pela hidrólise dos derivados pela lipase foi adquirido por microscopia de fluorescência confocal usando excitação de 2-fótons. / The kinetics of enzymatic hydrolysis of quinizarin diester by supported lipase dispersed beads in organic solvents was investigated by fluorescence spectroscopy. This study was performed on macroscopic and microscopic levels. For the macroscopic study was used CALB immobilized lipase (Novozyme ® 435) on acrylate beads, and for microscopic study Rhizopus niveus lipase immobilized on silica nanoparticles. The quinizarin derivatives (substrates) are non-fluorescent species, and only the end product quinizarin has fluorescence. A kinetic model considering two sequential Michaelis-Menten mechanisms provides a suitable description of the time evolution of the quinizarin formation monitored by emission spectroscopy and photon counting measurements. The average reaction time of the enzymatic hydrolysis was determined for quinizarin diacetate, dibutirate, dihexanoate and dioctanoate in hexane, cyclohexane and decaline water saturated solvents. In the microscopic study, the Rhizopus niveus lipase was dispersed into and bound silica mesoporous 200nm particles. In both systems, dispersed silica nanoparticles and a small fraction of aggregates are found in thin film. The enzyme activity was monitored by images and fluctuations of fluorescence intensity over time using confocal fluorescence microscopy. The results showed that after addition of substrate fluorescent spots due to enzyme activity start to appear. Confocal fluorescence images showed no clear difference among substrates. However, the study of fluorescence intensity fluctuations showed that enzyme activity depends on the type of substrate and enzyme support. In addition, the lipase activity depends on the form in which it is distributed in the nanoparticles (bound or entrapped) and the size of the alkyl diester derivatives. The fluorescence decay of quinizarin produced by lipase hydrolysis of diester was measured by confocal fluorescence microscopy using 2-photon pulse excitation.

O citoesqueleto é a estrutura celular mais importante em células eucariotas e é responsável por manter a forma da célula e as junções celulares, auxiliando nos movimentos celulares. Esta é composta de filamentos de Actina, Microtúbulos e filamentos intermediários. Recentemente, a análise de duas dessas estruturas tornaram-se importantes, pois é possível obter micrografias usando microscópios de alta resolução, que contém microscopia de fluorescência, em combinação com métodos complexos de aplicação de substâncias de contraste para rotulagem e posterior análises visuais. A combinação dessas técnicas, entretanto, limita-se a ser descritiva e subjetiva. Neste trabalho, são avaliadas cinco técnicas de análise de imagens, as quais são: Bag of Visual Words (BoVW), Local Binary Local (LBP), Textons baseados em Discrete Fourier Transform (TDFT), Textons baseados em Gabor Filter Banks (TGFB) e Textons baseados em Complex Networks (TCN) sobre o conjunto de dados 2D Hela e FDIG Olympus. Experimentos extensivos foram conduzidos em ambos os conjuntos de dados, e seus resultados podem servir de base para futuras pesquisas como análises do citoesqueleto em imagens de microscopia fluorescente. Neste trabalho, é apresentada uma comparação quantitativa e qualitativa dos métodos acima mencionados para entender o comportamento desses métodos e propriedades dos microfilamentos de actina (MA) e Microtúbulos (MT) em ambos os conjuntos de dados. Os resultados obtidos evidenciam que é possível classificar o conjunto de dados da FDIG Olympus com uma precisão de até 90:07% e 98:94% para 2D Hela, além de obter 86:05% e 96:84%, respectivamente, de precisão, usando teoria de redes complexas. / The cytoskeleton is the most important cellular structure in eukaryotic cells and is responsible for maintaining the shape of the cell and cellular junctions, aiding in cell movements. This is composed of filaments of Actin, Microtubules and intermediate filaments. Recently, the analysis of two of these structures has become important because it is possible to obtain micrographs using microscopes of high resolution and fluorescence technology, in combination with complex methods of application of substances of contrast for labeling and later visual analysis. The use of these techniques, however, is limited to being descriptive and subjective. In this work, we evaluate some of the most popular image analysis techniques such as Bag of Visual Words (BoVW), Local Binary Pattern (LBP), Textons based on Discrete Fourier Transform(TDFT) , Gabor Filter banks (TGFB), and approaches based on Complex Networks theory (TCN) over the famous dataset 2D Hela and FDIG Olympus. Extensive experiments were conducted on both datasets in which their results can serve as a baseline for future research with cytoskeleton classification in microscopy fluorescence images. In this work, we present the quantitative and qualitative comparison of above mentioned methods for better understand the behavior of these methods and the properties of Actin microfilaments (MA) and Microtubules (MT) on both datasets. The results showed that it is possible to classify the FDIG Olympus data set with accuracy of up to 90:07% and 98:94% for 2D Hela, in addition to reaching 86:05% and 96:84% respectively, using complex network theory.

The influenza A virus genome consists of eight single-stranded segments of negativesense viral RNA (vRNA) with highly conserved, partially complementary termini. These termini associate in a double-stranded RNA structure, known as a panhandle, which is bound by the viral RNA-dependent RNA polymerase and can serve as a promoter in both viral transcription and replication. In part A of this thesis, I use a combination of classical biochemistry techniques and fluorescence techniques (both at the ensemble and single-molecule level) for a quantitative investigation of the interaction between purified influenza A RNA polymerase and the individual 5' and 3' conserved termini of the vRNA segments, as well as the double-stranded vRNA promoter. Furthermore, I report the first direct, real-time observation of the promoter changing its structure when bound by the polymerase and show that the structure assumed agrees best with the corkscrew model. In part B of this thesis, I use fluorescence to detect RNA: I design and test a singlemolecule biosensor aimed at probing the presence of influenza A RNA in a sample, on the one hand, and I use click-chemistry to fluorescently label very shorty RNAs (3-25nt) that have been generated in an in vitro transcription reaction, on the other. The biosensing assay I propose can be further developed for diagnostic purposed, while click-chemistry labelling of short RNAs can be optimised and extended such that it becomes a reliable alternative to the use of radiolabels.

O controle na formação do biofilme em implantes e próteses ortopédicas continua sendo um dos grandes desafios acerca da infeção relacionada aos dispositivos na área da saúde. O objetivo desta pesquisa foi investigar biomateriais com aplicabilidade na ortopedia, visando os avanços e enfrentamentos dos desafios na área da infectologia. Uma revisão integrativa foi realizada a respeito da formação de biofilme em biomateriais de próteses de quadril com a finalidade de contribuir com as medidas de prevenção e controle aos agravos infecciosos. Além disso, a formação in vitro do biofilme em função dos biomateriais (titânio e titânio revestido com biovidro F18), microrganismos (Staphylococcus epidermidis e Candida albicans) e tempos de incubação (2, 4 e 8 horas) foi avaliada por microscopia de fluorescência. A revisão integrativa foi realizada no portal PubMed da National Library of Medicine, bem como nas bases Cochrane, Embase, Web of Science, CINAHL e LILACS com a inclusão de estudos primários sobre a temática, publicados online até novembro de 2017, em português, inglês e espanhol. Na fase experimental / laboratorial, biofilmes de S. epidermidis (ATCC 12228) e C. albicans (ATCC 90028) foram formados em corpos de prova de titânio e titânio revestido com biovidro F18 após 2, 4 e 8 horas de incubação a 37?C sob agitação orbital. As áreas das imagens dos corpos de prova, em porcentagem, recobertas com biofilme (células vivas) foram avaliadas por microscopia de fluorescência. Os dados coletados foram submetidos à análise estatística empregando-se os testes de normalidade Shapiro Wilk, U de Mann-Whitney e t de Student por meio do software IBM SPSS Statistics (versão 25) e nível de significância ?=5%. Na revisão integrativa, os resultados demonstraram que dos 16 estudos primários, 81,25% eram pesquisas experimentais in vitro e que novos biomateriais foram desenvolvidos para prevenir a formação de biofilme. Com relação à fase experimental / laboratorial, houve menor formação de biofilme por S. epidermidis e C. albicans (p<0,001) no titânio revestido com biovidro F18 do que no titânio, após 8 horas de incubação. Entretanto, houve maior formação de biofilme por S. epidermidis e C. albicans após 8 horas do que em 2 horas de incubação, tanto no titânio quanto no titânio revestido com biovidro F18 (p<0,05). Em suma, a revista da literatura mencionou o desenvolvimento de biomateriais novos para prevenir a formação de biofilme. Na fase laboratorial / experimental, o titânio revestido com biovidro F18 apresentou atividade antibiofilme em comparação com o titânio, e os tempos de incubação de 2 para 8 horas aumentaram a formação de biofilme em ambos os biomateriais. Ainda, pesquisas futuras acerca do biovidro F18 fundamentadas nos aspectos físicoquímicos, bioquímicos e microbiológicos são importantes para a elucidação dos mecanismos de ação relacionados ao controle dos biofilmes / The control of biofilm formation on implants and orthopedic prostheses still is one of the major challenges concerning infection related to devices in the health field. The objective of this research was to investigate biomaterials with applicability in orthopedics, aiming for advances and facing challenges in the infectology area. An integrative review was performed regarding biofilm formation on hip prosthesis biomaterials in order to contribute to the preventive and infection control measures. Moreover, the in vitro biofilm formation according to biomaterials (titanium and titanium coated with F18 bioglass), microorganisms (Staphylococcus epidermidis and Candida albicans) and incubation times (2, 4 and 8 hours) was evaluated by fluorescence microscopy. The integrative review was performed on PubMed portal from National Library of Medicine as well as on Cochrane, Embase, Web of Science, CINAHL and LILACS databases with the inclusion of primary studies about the topic, published online up until November 2017, in Portuguese, English and Spanish. In the experimental / laboratory step, S. epidermidis (ATCC 12228) and C. albicans (ATCC 90028) biofilms were formed on proof bodies of titanium and titanium coated with F18 bioglass after 2, 4 and 8 hours of incubation at 37?C under orbital shaking. The image areas of proof bodies, in percentage, coated with biofilm (living cells) were evaluated by fluorescence microscopy. The data collected were submitted to statistical analysis using normality tests Shapiro Wilk, U from Mann-Whitney and t from Student through IBM SPSS Statistics (version 25) software and significance level ?=5%. In the integrative review, the results showed that among 16 primary studies, 81.25% were in vitro experimental studies and that new biomaterials were developed to prevent biofilm formation. Regarding experimental / laboratory step, there was less biofilm formation by S. epidermidis and C. albicans (p<0.001) on titanium coated with F18 bioglass than on titanium, after 8 hours of incubation. However, there was more biofilm formation by S. epidermidis and C. albicans after 8 hours than in 2 hours of incubation, both on titanium and on titanium coated with F18 bioglass (p<0.05). In sum, the literature review mentioned the development of new biomaterials to prevent biofilm formation. In laboratory / experimental step, titanium coated with F18 bioglass presented antibiofilm activity in comparison with titanium, and the incubation times of 2 to 8 hours increased biofilm formation on both materials. Besides, future studies about F18 bioglass based on physicochemical, biochemical and microbiological aspects are important for the elucidation of action mechanisms related to biofilms control

Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.

For the proper function of the Fluorescence olographic microscope, it is necessary to adjust all the optical components of the microscope. Furthermore, the precise adjustment is the very critical condition for proper imaging of the Coherence-controlled holographic microscope. Therefore, it is necessary to create a sight collimator for these microscopes for their adjustment. The fluorescence holographic microscope is based on an interference and holographic principles, whose history is mentioned in the theoretical part of the thesis. The existing state of the art of laser sight collimators and their use in practice is also mentioned. The optical and mechanical design of the laser sight collimator and its realization are described in the next part of the thesis. The software for detecting the black sight cross was created for the use of the laser sight collimator in practice. The software is necessary to evaluate the correctness of the alignment of the adjusted microscope. The descriptions of the adjustment procedures for the laser sight collimator and for the Fluorescence holographic microscope are mentioned in the last part of the thesis. These procedures are necessary for proper manipulation and use with the proposed laser sight collimator.

The Coherence Controlled Holographic Microscope (CCHM) was developed at BUT Brno for a quantitative phase imaging of living cells. Nowadays it ocurres that its imaging properties are enhanced by the use of additional modules. In the present the microscope is equipped with the epifluorescence module, which allows observation of fluorescently marked living cells. This thesis is going to follow up on the development of this module and is going to extend its options by confocal imaging. The disadvantage of current multi-channel confocal microscopes is a mechanical rotation of the Nipkow discs, which causes undesired mechanical vibrations. That is why in this thesis it is replaced by Digital Micromirror Device. With its use was developed optical system of the whole confocal model, whose correct funcion was simulated in optical CAD. The experimentally verified prototype serves to test the imaging properties. On this basis is designed an application idea of the fluorescence confocal module, which will be possible to connect to the CCHM microscope.