Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis.

Dans la première partie de cette thèse, nous étudions la cinétique de la complexation d'un double brin d'ADN par la protéine NCp7. Pour ce faire, nous étudions l'évolution des propriétés mécaniques de l'ADN au fur et à mesure de sa complexation, en étirant la complexe ADN/NCp7 à l'aide d'un montage de piégeage optique. Nous avons observé que la longueur de persistance du complexe diminue au fur et à mesure de la complexation. En utilisant un modèle statistique décrivant l'évolution de la flexibilité de l'ADN complexé par NCp7. Notre principal résultat est que la fraction//phi de paires de bases ayant réagi n'est pas une fonction linéaire du temps aux faibles //phi. Nous interprétons nos résultats en supposant que l'adsorption de NCp7 sur l'ADN est fortement coopérative. Dans deuxième chapitre, nous décrivons la dynamique de particules sondes dans une suspension vitreuse colloïdale de Laponite. La Laponite est une particule colloïdale discoïdale de 25nm de diamètre et de 0.92 nm d'épaisseur. Nous utilisons une expérience de microscopie en onde évanescente, et suivons le mouvement de particules fluorescentes de latex. Nous imageons ensuite ces particules. Nous montrons que, pour un mouvement possédant une seule échelle de temps caractéristique, elle est simplement une fonction linéaire du temps. Nous obtenons que, quelle que soit leur taille, le mouvement des particules sondes peut être décrit par une succession de deux modes dynamiques, où le mode le plus rapide correspond à la diffusion des particules dans un fluide viscoélastique. / In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.

Este trabalho teve por objetivo desenvolver e caracterizar um sensor de força utilizando uma fibra óptica modificada pelo processo conhecido como Fiber tapering. A fibra quando modificada deixa exposto o campo evanescente, o que a torna sensível a influências externas, e a luz guiada na fibra pode vir a sofrer reflexão interna total frustrada ao entrar em contato com materiais. Ao envolver a região modificada por um material elastomérico, a área de contato e consequentemente a atenuação torna-se uma função da intensidade da força aplicada, possibilitando então relacionar a força a atenuação da luz. Baseando-se nesse efeito, foi criado um sensor de dimensões reduzidas, de rápida resposta, linear, altamente sensível e de boa repetibilidade. Foi criado também um circuito eletrônico utilizando amplificadores operacionais para a aquisição e processamento do sinal proveniente da fibra e selecionado um sensor comercial comum para a realização de experimentos e comparações. Ambos os sensores foram posicionados sobre uma balança de precisão e submetidos a diversos esforços obtendo-se dados sobre a resposta estática. Em seguida utilizando um shaker eletrodinâmico foram medidos os tempos de resposta a uma entrada degrau, e realizando esforços repetitivos foram analisados os desvios das medidas lidas pelos sensores. / The aim of this research was to develop and characterize a force sensor using a modified optical fiber by a process known as Fiber tapering. The modified fiber leaves the evanescent field exposed and prone to external influences and the guided light may suffer frustration of total internal reflection upon contact with materials. When covering the modified fiber section with an elastomeric material, the contact area and therefore the attenuation becomes a function of the applied pressure, making it possible to relate force to attenuation in light intensity. Based on this effect, a small sensor was created, having a quick response time, with high linearity, high sensitivity and good repeatability. Along with the sensor, an electronic circuit using operational amplifiers was designed for acquisition and processing of the signal obtained from the optical fiber. In addition, in order to perform experiments and comparisons, a standard force sensor was chosen. Both sensors were placed over a precision weighing scale and had different intensities of force applied on them, and after that, data regarding static measurements was gathered. The response time was obtained using an electrodynamic shaker and applying a step input. Furthermore, data was gathered about the deviations on the measurements by performing a repetitive set of compressions.

Neste trabalho apresentamos uma nova técnica para a medida da rotação da polarização da luz por uma substância que possui atividade óptica. O sistema utiliza um LED, dois polarizadores, um prisma de vidro semicilíndrico, uma cubeta, uma CCD e um computador para análise de dados. Luz proveniente do LED passa pelo primeiro polarizador, cujo eixo de transmissão se encontra a 45°, e incide no prisma pelo lado semicilíndrico, ocorrendo reflexão na sua base, num ângulo próximo do ângulo crítico. Devido ao tamanho finito do feixe e o formato curvo da superfície do prisma, vários ângulos de incidência são observados na base da lente semicilíndrica. A luz refletida passa então pela cubeta e depois por um analisador, cujo eixo se encontra paralelo ao primeiro polarizador, e então o sinal é captado pela CCD. De forma alternativa, a cubeta pode ser posicionada após o primeiro polarizador, antes do prisma. Quando a cubeta é preenchida com água, observa-se na CCD uma interferência destrutiva exatamente no ângulo crítico caracterizado por um mínimo de intensidade nesse ângulo. Se uma substância opticamente ativa é utilizada para preencher a cubeta, a posição desse mínimo é alterada dependendo do ângulo de rotação da polarização imposto pela substância. Uma calibração é necessária e pode ser feita utilizando-se soluções de concentração conhecida de sacarose ou frutose, por exemplo. O aparato obtido foi utilizado para medir a rotação causada por uma amostra normal (0,26 g/ml) de soluções de sacarose e frutose e apresenta uma precisão de 0,04°. Equivalentemente, a precisão em concentração é de 0,001 g/ml ou aproximadamente 0,1% (m/m). Isso corresponde a uma precisão que é uma ordem de grandeza acima dos aparelhos comerciais e técnicas mais comuns utilizadas atualmente. Em contrapartida, o custo da montagem experimental é duas ordens de grandeza menor que os mesmos aparelhos comerciais. A produção de uma gama de ângulos de incidência devido à focalização na superfície cilíndrica do prisma substitui a necessidade de se produzir rotação no eixo de polarização do analisador após a passagem da luz pela amostra opticamente ativa, como ocorre em alguns aparelhos comercializados. Este dispositivo, por ser de baixo custo, compacto e de fácil manuseio, é de grande importância porque pode ser utilizado na indústria sucroalcooleira para a medida da quantidade de sacarose em cana e também na indústria farmacêutica para a identificação de substâncias opticamente ativas dextrogiras ou levógiras. / In this thesis we present a novel technique for measuring light polarization rotation caused by an optically active substance. The system is composed by a LED, two polarizers, a semi cylindrical glass prism, a cuvette, a linear CCD camera and a computer for data analysis. Light from the LED passes through the first polarizer, whose transmission axis is set at 45°. After that, the linear polarized light enters the prism by the semi cylindrical face, occurring reflection in the flat face at critical angle approximately. Several incidence angles are accessed due to the beams finite size and the shape of the semi cylindrical lens. The reflected light passes through the cuvette and then through the analyzer, whose transmission axis is set parallel to the first polarizer. Finally, the light is detected by the CCD. When the cuvette is filled up with water, a destructive interference at the critical angle is observed, characterized by a narrow valley centered at this angle. If the cuvette is filled up with an optically active substance, the center of this valley is shifted depending on the substance, its concentration and the optical path travelled by the light in the substance. A calibration is needed and is performed using a set of solutions of known concentrations. Our apparatus was used to measure the angle rotation caused by a normal solution (0,26 g/ml) of sucrose and fructose solutions and has a precision of 0,04°. It corresponds to a precision that is one order of magnitude above most used commercial apparatus and developed techniques. In contrast, the cost of our experimental setup is two orders of magnitude less than the same commercial devices. The production of a range of angles of incidence due to focusing on the cylindrical surface of the prism replaces the need of producing a rotation of the analyzer polarization axis after the light passes through the optically active sample, as it happens in some commercial devices. Since this device has a low cost, is compact and easy to handle, it may be of great importance for applications in the pharmaceutical industry to identify enantiomers, and in the sugar industry for measuring sugar content in sugar cane juice.

Les matériaux artificiellement structurés que sont les cristaux photoniques sont couramment utilisés pour leurs propriétés dispersives. Leur constante diélectrique varie périodiquement à l'échelle de la longueur d'onde selon deux ou trois directions avec un contraste d'indice suffisamment élevé. La relation de dispersion ω = ω(k) qui résulte de cette variation périodique a la forme d'une structure de bande à l'intérieur de laquelle il existe des bandes interdites photoniques où la propagation du champ électromagnétique est interdite. En dehors de ces bandes, i.e. dans les bandes photoniques, se trouvent les propriétés de dispersion des cristaux photoniques.Le but de ce travail de thèse est de concevoir, de fabriquer et de caractériser des dispositifs à cristal photonique à gradient. Ces dispositifs ont été conçus de façon à s'appliquer dans les domaines allant des micro-ondes à l'optique. Nous avons conçu des dispositifs à partir de cristaux photoniques dont les propriétés dispersives les rendent analogues à des milieux linéaires, homogènes et isotropes (LHI). À la maille élémentaire de ces cristaux photoniques LHI, nous avons appliqué un gradient pour réaliser des lentilles à gradient 1D. Des résultats importants concernant la conception, la fabrication et la caractérisation expérimentale d'une lentille plate à gradient d'indice fonctionnant dans la bande X des micro-ondes sont reportés. Celle lentille focalise une onde plane incidente et collimate l'onde émise par une source ponctuelle situés dans son plan focal. Si cette lentille constitue en soi un démonstrateur et valide la démarche mise en œuvre pour la concevoir, ses applications potentielles concernent particulièrement les antennes. Nous réalisons également plusieurs lentilles à gradient 2D dont des lentilles de Lüneburg et Half Maxwell Fisheye; leurs applications aux antennes sont importantes. Nous nous intéressons aussi à la réalisation de lentilles optiques à gradient d'indice dites « SELFOC® ». Dans le but de confirmer les propriétés dispersives remarquables qui ont été mises en évidence, nous avons poursuivi dans ce sens en revisitant une expérience classique qui met en évidence l'existence des ondes évanescentes : celle du « double prisme à angle droit ». Nous mettons également en évidence le phénomène de « réflexion totale frustrée » ainsi que le décalage, découvert par Goos et Hänchen, que subit l'onde réfléchie sur le dioptre. Ce sont ces deux points — réflexion totale frustrée et effet Goos-Hänchen — que nous vérifions dans le cas de cristaux photoniques LHI. / Artificially structured materials that are photonic crystals are commonly used for their dispersive properties. Their dielectric constant varies periodically across the wavelength in two or three directions with a sufficiently high index contrast. The resulting dispersion relation ω = ω(k) of the periodic variation has the form of a band structure within which there are photonic bandgaps in which the propagation of the electromagnetic field is prohibited. Outside of these bands, i.e. in the photonic band, there are the dispersion properties of the photonic crystals.The aim of this thesis is to design, fabricate and characterize graded photonic crystal devices. These devices were designed to be applied in areas ranging from microwaves to optics. We designed devices from photonic crystals with dispersive properties which make them similar to linear, homogeneous and isotropic media (LHI). In the unit cell of the LHI photonic crystal, we applied a gradient to achieve 1D graded lenses. Important results regarding the design, manufacturing and experimental characterization of a flat lens GRIN operating in X-band microwaves are deferred. This lens focuses an incident plane wave and collimates the wave emitted by a point source located in its focal plane. If this lens is itself a demonstrator and validates the approach implemented for the design, its potential applications particularly concern antennas. We also carry several 2D graded lenses including Lüneburg and Half Maxwell Fisheye lenses; their applications to the antennas are important. We are also interested in making optical graded index lenses called "SELFOC®".In order to confirm the remarkable dispersive properties that have been identified, we continued in that direction by revisiting a classic experiment that highlights the existence of evanescent waves: the "double right angle prism". We also highlight the phenomenon of "frustrated total internal reflection" and the shift discovered by Goos and Hänchen suffered by the reflected wave on the interface. It's these two points – frustrated total internal reflection and Goos-Hänchen effect - that we check in the case of LHI photonic crystals.

To effectively design systems of microchannels it is necessary for scientists and engineers to understand thermal transport characteristics of microchannels. To experimentally determine the convective heat transfer coefficient of microchannels it is necessary to measure both the bulk and surface temperature fields. This investigation aims to develop a technique, named Total Internal Reflection Fluorescent Micro-Thermometry (TIR-FMT), to measure the temperature of water within several hundred nanometers of a wall--effectively, the surface temperature of the wall. In TIR-FMT, an evanescent-wave is generated in the water near the wall. The intensity of this evanescent-wave decays exponentially with distance from the wall. A fluorophore if illuminated by the evanescent-wave can absorb a photon. Excited fluorophores subsequently emit red-shifted photons, which are called fluorescence. The probability of a fluorescent emission is temperature-dependent. Therefore, by monitoring the intensity of the fluorescence a correlation can be made to the temperature of the region of illumination. Using the TIR-FMT technique the temperature dependence of the fluorescence intensity from buffered fluorescein (pH=9.2) was determined to be 1.35%/C. TIR-FMT can be used to measure the temperature of a fluorophore solution within 600 nm of a wall across a temperature range of 12.5-55C. The average uncertainties (95% confidence) of the temperature measured was determined to be 2.3C and 1.5C for a single 1.5x1.5 and #956;m pixel and the entire 715x950 and #956;m viewfield. By spatial averaging, average uncertainties of 2.0C and 1.8C were attained with spatial resolutions of 16x16 and 100x100 and #956;m, respectively.

In this dissertation, we describe a novel method that we call Diffusing Colloidal Probe Microscopy (DCPM), which integrates Total Internal Reflection Microscopy (TIRM) and Video Microscopy (VM) methods to monitor three dimensional trajectories in colloidal ensembles levitated above macroscopic surfaces. TIRM and VM are well established optical microscopy techniques for measuring normal and lateral colloidal excursions near macroscopic planar surfaces. The interactions between particle-particle and particle-substrate in colloidal interfacial systems are interpreted by statistical analyses from distributions of colloidal particles; dynamic properties of colloidal assembly are also determined from particle trajectories. Our studies show that DCPM is able to detect many particle-surface interactions simultaneously and provides an ensemble average measurement of particle-surface interactions on a homogeneous surface to allow direct comparison of distributed and average properties. A benefit of ensemble averaging of many particles is the diminished need for time averaging, which can produce orders of magnitude faster measurement times at higher interfacial particle concentrations. The statistical analyses (Ornstein- Zernike and three dimensional Monte Carlo analyses) are used to obtain particle-particle interactions from lateral distribution functions and to understand the role of nonuniformities in interfacial colloidal systems. An inconsistent finding is the observation of an anomalous long range particle-particle attraction and recovery of the expected DLVO particle-wall interactions for all concentrations examined. The possible influence of charge heterogeneity and particle size polydispersity on measured distribution functions is discussed in regard to inconsistent particle-wall and particle-particle potentials. In the final part of this research, the ability of DCPM is demonstrated to map potential energy landscapes on patterned surfaces by monitoring interactions between diffusing colloidal probes with Au pattern features. Absolute separation is obtained from theoretical fits to measured potential energy profiles and direct measurement by sticking silica colloids to Au surfaces via electrophoretic deposition. Initial results indicate that, as colloidal probe and pattern feature dimensions become comparable, measured potential energy profiles suffer some distortion due to the increased probability of probes interacting with surfaces at the edges of adjacent pattern features. Measurements of lateral diffusion via analysis of mean square displacements also indicated lateral diffusion coefficients in excellent agreement with rigorous theoretical predictions.

In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.