Deoxyribonucleic acid (DNA) forms the basis of all known living organisms. Despite the essential role played by DNA, its dynamic system and functional behaviour are still not completely understood. The work presented in this thesis aims to explore the structural dynamics of DNA systems, using fluorescence-based approaches, and to attempt to develop a technique for the measurement of fluorescence decays of biological molecules on the ultrafast (femtosecond) timescale. Absorption of UV radiation by DNA is known to lead to mutations and damage to DNA structure and functionality. For the majority of absorbed photons, the excitation energy dissipates harmlessly as heat, but in some instances this energy transfers to regions of DNA that are more susceptible to damage. 2-Aminopurine (2AP), a fluorescent analogue of the native DNA base adenine, can be incorporated into DNA with minimal perturbation to the DNA structure, and can be used to investigate inter-base electronic energy transfer. By selectively exciting the native DNA base in 2AP-containing dinucleotides and utilising 2AP fluorescence as an energy acceptor, the mechanism of electronic energy transfer has been investigated. Analysis of the resulting fluorescence lifetimes of 2AP has revealed that energy transfer preferentially excites conformations in which the bases are highly stacked, and the fluorescence of 2AP is highly quenched. This has led to a re-evaluation of energy transfer efficiencies between the natural bases and 2AP, and has shown that transfer efficiencies cannot be determined correctly from steady-state fluorescence measurements. To investigate the influence of base dynamics on the quenching of 2AP fluorescence in DNA, time-resolved fluorescence measurements were carried out on 2AP-containing systems in frozen solution at 77 K. These studies included dinucleotides, single–strand oligonucleotides and their corresponding duplexes. In all cases, comparison of the fluorescence decay parameters measured at room temperature with those measured at 77 K showed that elimination of base dynamics prevented rapid quenching, on the 10s of ps timescale or faster, although quenching on the 100s of ps timescale persisted for 2AP in single strands and duplexes. The multi-exponential fluorescence decay of 2AP in DNA and its high sensitivity to local environment is commonly exploited to investigate DNA-enzyme interactions. Transposases are enzymes involved in the movement of sections of DNA (transposons) within the genome. The Mos1 transposase catalyses the movement of a transposon via a cut-and-paste mechanism involving several intermediate complexes. Understanding the complex mechanism by which the transposase can remove and insert a section of DNA would allow these enzymes to be used as biomolecular tools. The structure of the intermediate Mos1 strand-transfer complex (STC) has been investigated by incorporating 2AP into several regions of the transposon and analysing the fluorescence decay. The involvement of a base-flipping-like mechanism has been identified in the mechanism of strand transfer for the Mos1 transposon. The time-resolved fluorescence measurements performed in this thesis are limited to time resolution of ~20 ps and longer using TSCPC. However, an abundance of photophysical events in DNA occur on the femtosecond timescale. Development of a methodology utilising fluorescence gating techniques (such as sum-frequency generation or diffraction from a transient grating) have been attempted, in order to construct an experimental system that enables the broadband detection of ultrafast fluorescence decays. Despite the lack of immediate success in recording the fluorescence decay from a sample, due to technical issues and time-constraints, initial characterisation of the set-up was performed and the prospect of broadband detection was demonstrated. Overall, this thesis gives insight into some of the dynamic processes taking place in DNA and presents work performed to develop a system that would allow the extension of these studies to processes occurring on the fs timescale.

abstract: In this work, atomic force microscopy (AFM) and time resolved confocal fluorescence microscopy are combined to create a microscopy technique which allows for nanometer resolution topographic and fluorescence imaging. This technique can be applied to any sample which can be immobilized on a surface and which can be observed by fluorescence microscopy. Biological problems include small molecular systems, such as membrane receptor clusters, where very high optical resolutions need to be achieved. In materials science, fluorescent nanoparticles or other optically active nanostructures can be investigated using this technique. In the past decades, multiple techniques have been developed that yield high resolution optical images. Multiple far-field techniques have overcome the diffraction limit and allow fluorescence imaging with resolutions of few tens of nanometers. On the other hand, near-field microscopy, that makes use of optically active structures much smaller than the diffraction limit can give resolutions around ten nanometers with the possibility to collect topographic information from flat samples. The technique presented in this work reaches resolutions in the nanometer range along with topographic information from the sample. DNA origami with fluorophores attached to it was used to show this high resolution. The fluorophores with 21 nm distance could be resolved and their position on the origami determined within 10 nm. Not only did this work reach a new record in optical resolution in near-field microscopy (5 nm resolution in air and in water), it also gave an insight into the physics that happens between a fluorescent molecule and a dielectric nanostructure, which the AFM tip is. The experiments with silicon tips made a detailed comparison with models possible on the single molecule level, highly resolved in space and time. On the other hand, using silicon nitride and quartz as tip materials showed that effects beyond the established models play a role when the molecule is directly under the AFM tip, where quenching of up to 5 times more efficient than predicted by the model was found. / Dissertation/Thesis / Ph.D. Physics 2012

Orientador : Pedro Felício Estrada Bernabé / Resumo: As lesões cariosas de superfícies proximais se destacam por sua grande incidência, tanto pelo fato da região propiciar o acúmulo de biofilme bacteriano, quanto pelas dificuldades de higienização e de diagnóstico precoce. O objetivo deste estudo foi avaliar a performance da inspeção visual + sondagem (IV+S), laser de fluorescência (L), radiografia interproximal (RXI), separação dental + inspeção visual + sondagem (SD+IV+S) e separação dental + laser de fluorescência (SD+L) no diagnóstico de lesões cariosas proximais sem e com cavitação. Foram examinadas, por 2 profissionais, 167 superfícies de 30 pacientes de faixa etária entre 4 e 12 anos de idade, utilizando-se os 5 tipos de exames. Como método de validação para lesões cariosas com cavitação foi utilizada a dupla impressão das áreas interproximais com silicona de condensação. Em relação às lesões cariosas sem cavitação, os valores médios de sensibilidade encontrados para os exames de IV+S, L, RXI, SD+IV+S e SD+L foram de 44%, 33%, 49%, 100% e 48%, respectivamente. Em relação às lesões cariosas com cavitação, a sensibilidade foi de 15%, 40%, 59%, 59% e 41%, respectivamente. A correlação dos métodos de diagnóstico, em relação às lesões cariosas com cavitação, sugeriram uma maior precisão para a SD+IV+L. Os resultados sugeriram que a utilização da separação dental aumenta o desempenho no diagnóstico de lesões cariosas proximais, e que o método de impressão com silicona pode ser indicado como um método auxiliar definitivo de diagnóstico de lesões com cavitação. / Abstract: The approximals carious lesions stand out for its great incidence, so much for the fact of the area to propitiate the accumulation of bacterial plaque, as for the difficulties of toothbrushing and of precocious diagnosis. The objective of this study went evaluate the performance of the visual inspection + probing (IV+P), laser fluorescence (LF), bitewing radiographs (BW), dental separation + visual inspection + probing (SD+IV+P) and dental separation + laser fluorescence (SD+L) in the diagnosis of approximals carious lesions without and with cavitation. 167 surfaces of 30 patient of age group between 4 and 12 years were examined for 2 professional being used the 5 types of exams. As validation method for cavitated carious lesions were the double impression of the interproximals areas with condensation-cure silicone impression material. In relation to the carious lesions without cavitation, the medium values of sensibility found for the exams of IV+P, LF, BW, SD+IV+P and SD+LF were of 44%, 33%, 49%, 100% and 48%, respectively. In relation to the cavitated carious lesions, the sensibility was of 15%, 40%, 59%, 59% and 41%, respectively. The results suggested that the use of the dental separation increases the effectiveness in the diagnosis of approximal carious lesions, and the impression method can be indicated as a definitive auxiliary method of diganosis of lesions cavitated. / Doutor

>Magister Scientiae - MSc / The discovery of cell free foetal DNA (cffDNA) in 1997 allows for the combination of accuracy as well as non-invasiveness for prenatal diagnosis. This non-invasive genetic test requires only a maternal blood sample from which the cffDNA can be isolated and analysed. In this work cffDNA was isolated from a maternal blood sample using a micro-fluidic device which was fabricated using hot embossing and laser ablation techniques. The DNA sample was first pre-concentration by electrokinetic trapping (EKT) and then isotachophoresis (ITP). The concentrated sample was then separated by size using capillary electrophoresis (CE), all in a single device. All parameters and processes concerned with the micro-fluidic device were optimised sequentially. These parameters include both the chemical components as well as the physical processes which occur. The DNA used for the optimisation protocol was analysed using fluorescence spectroscopy, agarose gel electrophoresis as well as an Agilent Bioanalyser. The optimised protocol included a 9% acrylamide/pDMA matrix, 3 M N,N-dimethylurea as a denaturing agent, with tris based buffers for pre-concentration steps and 1X TBE (tris/borate/EDTA) buffer for capillary electrophoresis. The applied voltage of ITP was 300 V and CE was carried out at 180 V. The timing at which DNA was extracted from the device was kept at time = 60 s intervals. The optimised protocol was then used for real sample analysis and these samples were obtained from mothers pregnant with male foetuses. The DNA extracted from the micro-fluidic device was then analysed using real time PCR (RT-PCR) in order to distinguish which was maternal and which was foetal. This was carried out by amplification of male and general (present in male and female) genes respectively. RT-PCR results confirmed that only the male specific gene was amplified in initial samples exiting the device and it was thus successful in isolating cffDNA from a maternal plasma sample.

In this research, the use of gold nanostructures (AuNS) was explored to evaluate the interaction between molecules and the nanoparticle (NP) surface. In that way, three different projects were developed; one project using fluorescence and two projects using Raman spectroscopy as measuring technique. The fluorescence spectroscopy project used the fluorescence lifetime imaging microscope (FLIM) to evaluate the relative position of the molecules methylene blue (MB) and cucurbit[7]uril (CB) on the gold nanoparticle (AuNP) surface. Although the inclusion complex is favored in solution, it was found that MB forms an exclusion complex with CB, when CB is attached to the AuNP surface. The first project utilizing Raman spectroscopy, specifically surface enhanced Raman scattering (SERS), took advantage of a confined system (a reverse micelle) to evaluate the Raman signal of water molecules in close proximity to the AuNP surface. It was observed that the SERS water signal had a big shift to higher energies compared with the Raman signal of the bulk water; indicating the water molecules in the system are subjected to different bond-stretching energies. The second Raman project studied the modification of two different AuNS (specifically AuNP and gold nanorod -AuNR) with thiols. Different thiols were used to evaluate the kinetics of the modification of the AuNS surface, also the different AuNS presented different ligands on their surface. In general, and considering the difference in the bonding strength of the ligands present on the AuNS surface (by synthesis) and the size of the thiol, at least 2 h are required to modify the complete AuNS surface.

In freshwater fish, such as the rainbow trout (Oncorhynchus mykiss), higher ion concentrations in the body fluids relative to the dilute surrounding environment lead to diffusive ion loss that is countered by active ion uptake. Active ion uptake is achieved via specialised cells in the gill epithelium known as ionocytes, with the species studied to date exhibiting multiple ionocyte subtypes with specific complements of ion transport proteins. To better understand the functions and responses of each ionocyte subtype, methods are needed to isolate specific ionocyte subtypes. This thesis developed a method to use fluorescence-activated cell sorting (FACS) to isolate the peanut lectin agglutinin-positive (PNA+) ionocyte subtype of the trout gill, which is posited to be a base-secreting cell that takes up Cl- ions. A suspension of gill cells dissociated using ethylenediaminetetraacetic acid (EDTA) was labelled with biotinylated PNA that was detected using streptavidin conjugated to a fluorophore, and subjected to FACS to yield a population of PNA+ ionocytes of high viability and purity. To validate the utility of the approach, it was used in a proof-of-principle experiment to evaluate transcript abundance of cytosolic carbonic anhydrase (CAc) in PNA+ ionocytes in trout that were subjected to metabolic alkalosis. This experiment revealed that the relative transcript abundance of CAc was significantly elevated in PNA+ ionocytes of alkalotic trout relative to that of control trout (P = 0.001; N = 7), a response that is consistent with the expected role of PNA+ ionocytes in compensation for systemic alkalosis.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-01-04T17:34:19Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcio_marques_silva.pdf: 2786122 bytes, checksum: 4da1fad39fb206cdf4baf5459f0e0cb3 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-01-06T11:52:58Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcio_marques_silva.pdf: 2786122 bytes, checksum: 4da1fad39fb206cdf4baf5459f0e0cb3 (MD5) / Made available in DSpace on 2017-01-06T11:52:58Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcio_marques_silva.pdf: 2786122 bytes, checksum: 4da1fad39fb206cdf4baf5459f0e0cb3 (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento do Pessoal de N?vel Superior (CAPES) / O clomazone ? um herbicida inibidor da s?ntese de carotenoides. Esse herbicida ? facilmente solubilizado e volatilizado e por consequ?ncia, pode causar danos ao ambiente. Em vista do problema, objetivou-se com esta pesquisa: avaliar a sensibilidade de esp?cies forrageiras e daninhas a res?duos atmosf?ricos do clomazone e determinar a campo o efeito do res?duo atmosf?rico do clomazone sobre a fisiologia de plantas forrageiras e daninhas. Foram realizados dois experimentos. O primeiro foi conduzido em c?maras experimentais de 500 dm? em ambiente monitorado, delineado inteiramente casualizado com 5 repeti??es. Os tratamentos foram arranjados em esquema fatorial 6x5, sendo, seis esp?cies vegetais: triticale, milho, sorgo, braquiar?o, beldroega e campim braquiaria e o segundo cinco doses de clomazone 0, 90, 180, 270 e 360 g ha-1 (equivalentes ?s concentra??es atmosf?ricas de 0,0; 0,05; 0,10; 0,15 e 0,20 mg L-1, considerado o volume). As esp?cies ficaram expostas ao herbicida no interior das c?maras por per?odo de 96 horas em atmosfera controlada. Ap?s esse intervalo, as c?maras foram abertas, procedendo-se ? primeira avalia??o, repetida aos 7 e 14 dias ap?s a abertura. Avaliou-se a intoxica??o e o teor de clorofila. Com exce??o do milho, todas as esp?cies testadas mostraram-se sens?veis ?s concentra??es residuais de clomazone na atmosfera, podendo ser utilizadas no monitoramento da qualidade do ar. O segundo experimento foi conduzido a campo. Delineado em blocos causalizados com quatro repeti??es, em esquema fatorial 6x4, sendo seis esp?cies vegetais [quatro plantas forrageiras: lab lab, sorgo, braqui?r?o e java, e duas plantas daninhas: beldroega e sida] e quatro solu??es de aplica??o do clomazone (0, 360, 720 e 1.080 g ha-1, equivalentes a 0; 0,05; 0,10 e 0,20 mg L-1, considerado o volume). As plantas forrageiras e daninhas ficaram expostas ao clomazone, em tuneis cobertos por filme de polietileno de baixa densidade (150 ?m) de volume de 12m?, por per?odo de 72 horas. Ap?s esse tempo, os t?neis foram abertos, procedendo-se ?s seguintes avalia??es: intoxica??o das plantas, fluoresc?ncia inicial, fluoresc?ncia m?xima, a raz?o entre a fluoresc?ncia vari?vel e fluoresc?ncia m?xima, quenching fotoqu?mico e quenching n?o-fotoqu?mico, taxa de transporte de el?trons e do teor de clorofila. Mesmo em concentra??es que n?o promovem efeito visual, o clomazone ? capaz de causar danos significativos na atividade fotossint?tica das esp?cies. As vari?veis fisiol?gicas, clorofila total, rendimento qu?ntico m?ximo do PSII e fluoresc?ncia inicial da clorofila podem ser utilizadas de forma eficiente no monitoramento de res?duos do clomazone na atmosfera. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Produ??o Vegetal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The herbicide Clomazone is an inhibitor of carotenoids synthesis. This herbicide is easily solubilized and volatilized, and by consequence, can cause damage to environment. Due to this problem, the objective of this study was evalue the sensibility of forage and weeds species to atmospheric wastes of Clomazone and to determinate in field the effect of atmosferic waste of Clomazone in phisiology of forage and weeds plants. Two experiments were performed. The first was conducted in 500dm? experimental chambers in a monitored environment, completely randomized design design with five repetitions. The treatments was arranged in 6x5factorial scheme, being six vegetable species: triticale, corn, sorghum, braquiar?o, beldroega e capim braquiara and the second five doses of Clomazone 0, 90, 180, 270 e 360 g ha-1 (equivalent to atmospheric concentrations of 0,0; 0,05; 0,10; 0,15 e 0,20 mg L-1, volume considered). The species were exposed to the herbicide inside the chambers for 96 hours in controled atmosphere. After this interval, the chambers were open, proceeding the first evaluation, repeated at 7 and 14 days after the opening. Was rated the intoxication and the chlorophyll contente. Excepting the corn, all tested species proved to be sensitive to Clomazone residual concentration in atmosphere, and can be used in ais quality monitoring. The second experiment was conducted in field. An experiment was conducted in a randomized block design with four repetitions, in 6x4 factorial scheme, being six vegetable species [ four forage plants :lab lab, sorghum, braquiar?o and java, and two weed plants: beldroega and sida] and four Clomazone application solutions (0, 360, 720 e 1.080 g ha-1, equivalentes a 0; 0,05; 0,10 e 0,20 mg L-1, volume considered). The forage and weeds plants were exposed to Clomazone, in tunnels covered by polyethylene film of low density (150 ?m), volume of 12m?, for 72 hours. After this time, the tunnels were open, proceeding this following evaluations: plants intoxication, initial fluorescence, maximum fluorescence, ratio of the variable fluorescence and maximum fluorescence, photochemical quenching and non-photochemical quenching, el?ctron transport rate and chlorophyll contente. Even in concentrations that don?t promote visual effect, the Clomazone is able to cause significative damage in photosynthetic activity of species. The phisiologic variables, total chlorophyll, PSII maximum quantum yield and chlorophill initial fluorescence can be used efficiently in monitoring of Clomazone wastes in atmosphere.

The literary part of the work approaches the plant material, cucumbers (Cucumis sativus L.), the issue of greenhouse production and growing in hydroponics. A large part is devoted to describing the process of photosynthesis and the different factors affecting the impact and progress of the process. The practical part is devoted to monitoring and measuring the physiological characteristics of the photosynthetic apparatus using LCpro +: the intensity of photosynthesis, transpiration levels, water efficiency, stomatal conductance with cucumbers. Outside the parameters of photosynthesis is also evaluated chlorophyll content using the device CCM200plus Chlorophyll Content Meter and the ability of plants to use sunlight, thus fluorescence. Flourescence was measured with a fluorometer OS30p + Chlorophyll. Based on the evaluation, it was found that all measured characteristics were influenced by physiological and health status and degree of leaf damage.

Fluorescence single-molecule methods represent mighty tools for researchers in the field of structural and molecular biology. These methods are bringing in many advantages when compared to the statistical data processing of multi-molecular species. We can directly compare true statistical distributions and their kinetics. Here belongs fluorescence correlation spectroscopy, FRET and burst variance analysis. As the research advances, new methods are being developed which at the very beginning do not have proper analytical relations for data interpretation and which experimental limits we can't tell. That is the moment when the computer simulations can be used to our advantage. They can help us to specify the right direction of the future research, which is a great money-saver, while opening new perspectives and insights into the explored matter. Correct interpretation of the simulations results is key for the consequent defining of new theoretical models.

Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2016-09-05T18:41:37Z No. of bitstreams: 1 Dissertação_Humbervania Reis Gonçalves da Silva_2012.pdf: 1948968 bytes, checksum: 1e2bef735b027e934bccadc6da99dff4 (MD5) / Approved for entry into archive by Vanessa Reis (vanessa.jamile@ufba.br) on 2016-09-06T11:31:39Z (GMT) No. of bitstreams: 1 Dissertação_Humbervania Reis Gonçalves da Silva_2012.pdf: 1948968 bytes, checksum: 1e2bef735b027e934bccadc6da99dff4 (MD5) / Made available in DSpace on 2016-09-06T11:31:39Z (GMT). No. of bitstreams: 1 Dissertação_Humbervania Reis Gonçalves da Silva_2012.pdf: 1948968 bytes, checksum: 1e2bef735b027e934bccadc6da99dff4 (MD5) / CAPES / A monitoração da qualidade dos combustíveis é importante, não apenas em decorrência do desempenho dos veículos, mas também devido ao impacto ambiental das emissões de poluentes. Combustíveis adulterados resultam em aquecimento e aceleração do motor, além de aumentarem o consumo de combustível, a emissão de material particulado e de gases de exaustão. No Brasil, na década de 90 houve várias experiências de produção e uso de biodiesel a partir de diversas matérias-primas de origem vegetal ou animal, assim como de óleo residual. No entanto, apenas com a Portaria Nº 310 de 27/12/2001, a ANP estabeleceu as especificações para comercialização de óleo diesel e sua mistura com 2% de biodiesel e definiu obrigações dos agentes econômicos sobre o controle de qualidade do biocombustível. O trabalho visa simplificar os testes utilizados para caracterizar as misturas de biodiesel e diesel, de B00 (0%v/v de biodiesel no diesel) até B100 (100%v/v de biodiesel puro). Foi utilizada a técnica de espectrofluorimetria total 3D em combinação com Análise do Componente Principal (PCA) para identificar adulteração de bicombustível, pela mudança dos valores de concentrações de biodiesel em diesel. Os espectros de emissão fluorescente das amostras foram realizadas em um espectrofluorímetro, onde a emissão foi detectada de 230 a 800 nm em intervalos de 0,5 nm enquanto as moléculas presentes na amostra sofriam excitação em comprimentos de onda fixos na faixa de 200 a 775 nm. Outros espectros de emissão fluorescente das amostras foram obtidos excitando-se as amostras com LED de 365 nm e capturando-se a emissão na faixa de 385 a 1000 nm com incremento de 1 nm. Foram preparadas 76 amostras, todas foram analisadas através da Espectroscopia de Fluorescência Total associada à PCA e foi possível identificar adulterações de diesel em decorrência do excesso de biodiesel, ultrapassando os valores permitidos por lei. / Monitoring of fuel quality is important, not only because of the performance of vehicles, but due to the environmental impact of emissions of pollutants. Adulterated fuels result in heating and acceleration of the motor, besides increasing the fuel consumption, emissions and particulate matter exhaust gas . In Brazil, in the 90s there were several experiences of production and use of biodiesel from various raw materials of vegetable or animal origin, as well as residual oil. However, only with Ordinance No. 310, 27/12/2001, ANP established specifications for commercialization of diesel and its mixture with 2% biodiesel and defined obligations of economic agents about the quality control of the biofuel. The work aims to simplify the tests used to characterize mixtures of biodiesel and diesel, B00 (0% v / v biodiesel in C) to B100 (100% v / v of pure biodiesel). We used the technique of spectrofluorimetry full 3D in combination with PCA to identify adulteration of biofuel, by changing the values of concentrations of biodiesel in diesel. The fluorescence emission spectra of the samples were performed in a spectrofluorometer where the emission was detected at 230-800 nm in steps of 0.5 nm while the sample suffered excitation in wavelengths fixed at 200-775 nm. Other fluorescent emission spectra of the samples were obtained by exciting the sample with LED of 365 nm and capturing the emission in the range 385 to 1000 nm with an increment of 1 nm. Have been prepared 76 samples, all were analyzed by Fluorescence Spectroscopy Total associated with PCA and were able to identify adulteration of diesel fuel due to the excess biodiesel, exceeding the values allowed by law.