Faithful replication of genomic DNA is crucial for the survival of a cell. In order to achieve high-level accuracy in copying its genome, all cells employ replicative DNA polymerases that have intrinsic high fidelity. When an error occurs on the template DNA strand, in the form of lesions caused by diverse chemicals, reactive oxygen species, or UV light, the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, cells harbor multiple specialized translesion DNA polymerases that are error-prone and therefore able to accommodate the lesions and continue DNA synthesis. As a result of their low fidelity, the translesion polymerases are associated with increased mutagenesis, drug resistance, and cancer. Therefore, the access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate. This Thesis presents the development of a co-localization single-molecule spectroscopy (CoSMoS) method to directly visualize the loading of the Escherichia coli replicative polymerase on DNA, as well as the exchange between the replicative polymerase and the translesion polymerases Pol II and Pol IV. In contrast to the toolbelt model for the exchange between the polymerases, this work shows that the translesion polymerases Pol II and Pol IV do not form a stable complex with the replicative polymerase Pol IIIα on the β-clamp. Furthermore, we find that the sequential activities of the replication proteins: clamp loader, clamp, and Pol IIIα, are highly organized while the exchange with the translesion polymerases is disordered. This exchange is not determined by lesion-recognition but instead a concentration-dependent competition between the replicative and translesion polymerases for the hydrophobic groove on the surface of the β-clamp. Hence, our results provide a unique insight into the temporal organization of events in DNA replication and translesion synthesis.

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.

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.

Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects. This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20633号 / 工博第4371号 / 新制||工||1679(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies promise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electrical, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to manipulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within microfluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique.

L'exocytose régulée, est un processus qui permet la communication entre les cellules à travers la sécrétion des hormones et des neurotransmetteurs. Dans les neurones et les cellules neuroendocrines, l'exocytose est strictement contrôlée par des signaux extracellulaires tels que le potentiel trans-membranaire et la fixation des ligands sur des récepteurs. Des progrès substantiels ont été effectués afin de comprendre le mécanisme moléculaire de l'exocytose. Les composants majeurs de la machinerie de sécrétion ont été dévoilés. Maintenant, la question qui émerge concerne le rôle de la plateforme de protéines qui semble avoir une action coordonnée entre chaque protéine. Dans le cas de la famille des annexines, qui est bien connue pour son action dans l'exocytose, leurs modes d'interactions séquentielles ou concertées avec d'autres protéines ainsi que leurs effets régulateurs sur l'exocytose ne sont pas encore bien établis. Des résultats précédents indiquent que l'Annexine A6 (AnxA6) affecte l'homéostasie du calcium et la sécrétion de la dopamine à partir des cellules PC12, utilisées comme un modèle cellulaire de neurosécrétion (Podszywalow Bartnicka et al., 2010). Afin de déterminer l'effet inhibiteur de l'AnxA6 sur l'exocytose de la dopamine, nous cherchons des partenaires moléculaires de l'AnxA6 dans les cellules PC12. Nous faisons l'hypothèse que l'AnxA6 interagit avec la PLD1, une enzyme active dans l'étape de la fusion des vésicules avec la membrane plasmique. En utilisant la microscopie confocale et la microscopie à onde évanescente, nous avons trouvé que l'isoforme 1 de l'AnxA6 et la PLD1 sont tous les deux recrutés sur la surface des vésicules au cours de la stimulation des cellules PC12. AnxA6 inhibait l'activité de la PLD comme indiqué par notre méthode d'analyse enzymatique au moyen de la spectroscopie infrarouge. En conclusion, nous proposons que l'AnxA6 n'est pas seulement impliquée dans la réorganisation des membranes par ses capacités à se lier avec des phospholipides négativement chargés et avec le cholestérol, mais elle influence également l'activité de la PLD1, changeant la composition lipidique des membranes / The regulated exocytosis is a key process allowing cell-cell communication through the release of hormone and neurotransmitters. In neurons and neuroendocrine cells, it is strictly controlled by extracellular signal such as transmembrane potential and ligand bindings to receptors. Substantial progress has been made to understand the molecular mechanism of exocytosis. Major components of secretory machinery have been brought to light. Now the emergent question concerns the role of scaffolding proteins that are thought to coordinate the action of each other. In the case of annexin family well known to be involved in exocytosis, their modes of –sequential or concerted- interactions with other proteins, and their regulatory effects on exocytosis are not very well established. Previous findings indicated that Annexin A6 (AnxA6) affected calcium homeostasis and dopamine secretion from PC12 cells, used as cellular model of neurosecretion (Podszywalow-Bartnicka et al., 2010). To determine the inhibitory effect of AnxA6 on exocytosis of dopamine, we were looking for molecular partners of AnxA6 in PC12 cells. We hypothesized that AnxA6 interacts with phospholipase D1 (PLD1), an enzyme involved in the fusion step. By using confocal microscopy and total internal reflection fluorescence microscopy, we found that isoform 1 of AnxA6 and Phospholipase D1 are both recruited on the surface of vesicles upon stimulation of PC12 cells. AnxA6 inhibited phospholipase D activity as revealed by our enzymatic assay based on infrared spectroscopy. To conclude, we propose that AnxA6 is not only implicated in membrane organization by its capacity to bind to negative charged phospholipids and to cholesterol, but AnxA6 is also affecting PLD1 activity, changing membrane lipids composition