Secretory processes underpin the emergence of cellular diversity in complex multicellular organisms. However, our understanding of the basic mechanisms controlling the different secretory and endosomal compartments involved remains surprisingly incomplete. During my DPhil I have studied a specialised epithelial cell type in the male Drosophila accessory glands, the secondary cell, which contains unusually large intracellular compartments that are accessible to detailed morphological study. I characterise the organisation, ultrastructure and molecular composition of this cell's secretory and endosomal compartments, and I employ specific Rab GTPases, conserved coordinators of membrane trafficking and identity, to define multiple compartmental subtypes. By developing super-resolution and time-lapse microscopy approaches in these cells, I show that numerous intraluminal vesicles (ILVs) are formed within Rab11-labelled secretory compartments and released into the accessory gland lumen as exosomes, the first clear demonstration in eukaryotic cells of exosome biogenesis within a non-late endosomal compartment. Biogenesis of these ILVs is dependent on evolutionarily conserved Endosomal Sorting Complexes Required for Transport (ESCRT) 0-III genes and involves loading of compartment-specific cargoes. Work by others, some in collaboration with me, has shown that these novel mechanisms are conserved in human cells. I show that dense-core granules, the structures employed to package proteins and other molecules destined for regulated secretion, form within large non-cored Rab6- positive compartments, in a process that seems to involve inputs from both the Golgi and recycling endosomal pathways. Further analysis has revealed roles for specific Rabs, for ILVs, and for the conserved fibrillar protein Mfas/TGFBI in different aspects of DCG formation. I also show that DCGs are not only secreted, but can also be degraded by fusion to acidic endosomal compartments. Remarkably, there is evidence that mammalian cells may employ all of these mechanisms and defects in these processes may be linked to diseases like cancer, diabetes and neurodegenerative disorders. Hence my work has established a new system to study complex secretory mechanisms, which can now be developed to model specific disease processes in the future. In summary, I have discovered several novel cell biological mechanisms controlling exosome biology, dense-core granule biogenesis, regulated secretion, and endolysosomal trafficking. Some of these already appear relevant to human health and disease, suggesting that the secondary cell system has considerable further potential for unravelling the fundamental processes underlying eukaryotic secretion in the future.

Fluorescence microscopy is an indispensable tool in the areas of cell biology, histology and material science as it enables non-invasive observation of specimen in their natural environment. The main advantage of fluorescence microscopy is that, it is non-invasive and capable of imaging with very high contrast and visibility. It is dynamic, sensitive and allows high selectivity. The specificity and sensitivity of antibody-conjugated probes and genetically-engineered fluorescent protein constructs allows the user to label multiple targets and the precise location of intracellular components. However, its spatial reso- lution is limited to one-quarter of the excitation wavelength (Abbe’s diffraction limit). The advent of new and sophisticated optics and availability of fluorophores has made fluorescence imaging a flourishing field. Several advanced techniques like TIRF, 4PI, STED, SIM, SPIM, PALM, fPALM, GSDIM and STORM, have enabled high resolution imaging by breaking the diffraction barrier and are a boon to medical and biological research. Invention of confocal and multi-photon microscopes have enabled observation of the specimen embedded at depth. All these advances in fluorescence microscopy have made it a much sought-after technique. The first chapter provides an overview of the fundamental concepts in fluorescence imag- ing. A brief history of emergence of the field is provided in this chapter along with the evolution of different super-resolution microscopes. An introduction to the concept of fluorophores, their broad classification and their characteristics is discussed in this chap- ter. A brief explanation of different fluorescence imaging techniques and some trending techniques are introduced. This chapter provides a thorough foundation for the research work presented in the thesis. Second chapter deals with different microscopy techniques that have changed the face of biophotonics and nanoscale imaging. The resolution of optical imaging systems are dictated by the inherent property of the system, known as impulse response or more popularly “point spread function”. A basic fluorescence imaging system is presented in this chapter and introduces the concept of point spread function and resolution. The introduction of confocal microscope and multi-photon microscope brought about improved optical sectioning. 4PI microscopy technique was invented to improve the axial resolution of the optical imaging system. Using this microscopy modality, an axial resolution of upto ≈ 100nm was made possible. The basic concepts of these techniques is provided in this chapter. The chapter concludes with a discussion on some of the optical engineering techniques that aid in improved lateral and axial resolution improvements and then we proceed to take on these engineering techniques in detail in the next chapter. Introduction of spatial masks at the back aperture of the objective lens results in gen- eration of a Bessel-like beam, which enhances our ability to see deeper inside a spec- imen with reduced aberrations and improved lateral resolution. Bessel beams have non-diffracting and self-reconstructing properties which reduces the scattering while ob- serving cells embedded deep in a thick tissue. By coupling this with the 4PI super- resolution microscopy technique, multiple excitation spots can be generated along the optical axis of the two opposing high-NA objective lenses. This technique is known as multiple excitation spot optical (MESO) microscopy technique. It provides a lateral resolution improvement upto 150nm. A detailed description of the technique and a thorough analysis of the polarization properties is discussed in chapter 3. Chapters 4 and 5 bring the focus of the thesis to the main topic of research - multi- dimensional image reconstruction for fluorescence microscopy by employing the statis- tical techniques. We begin with an introduction to filtering techniques in Chapter 4 and concentrate on an edge-preserving denoising filter: Bilateral Filter for fluorescence microscopy images. Bilateral filter is a non-linear combination of two Gaussian filters, one based on proximity of two pixels and the other based on the intensity similarity of the two. These two sub-filters result in the edge-preserving capability of the filter. This technique is very popular in the field of image processing and we demonstrate the application of the technique for fluorescence microscopy images. The chapter presents a through description of the technique along with comparisons with Poisson noise mod- eling. Chapters 4 and 5 provide a detailed introduction to statistical iterative recon- struction algorithms like expectation maximization-maximum likelihood (EM-ML) and maximum a-posteriori (MAP) techniques. The main objective of an image reconstruc- tion algorithm is to recover an object from its noisy degraded images. Deconvolution methods are generally used to denoise and recover the true object. The choice of an appropriate prior function is the crux of the MAP algorithm. The remaining of chapter 5 provides an introduction to different potential functions. We show some results of the MAP algorithm in comparison with that of ML algorithm. In chapter 6, we continue the discussion on MAP reconstruction where two new potential functions are introduced and demonstrated. The first one is based on the application of Taylor series expansion on the image. The image field is considered to be analytic and hence Taylor series produces an accurate estimation of the field being reconstructed. The second half of the chapter introduces an interpolation function to approximate the value of a pixel in its neighborhood. Cubic B-splines are widely used as a basis function during interpolation and they are popular technique in computer vision and medical imaging techniques. These novel algorithms are tested on di_erent microscopy data like, confocal and 4PI. The results are shown at the _nal part of the chapter. Tagging cell organelles with uorescent probes enable their visualization and analysis non-invasively. In recent times, it is common to tag more than one organelle of interest and simultaneously observe their structures and functions. Multicolor uorescence imaging has become a key technique to study speci_c processes like pH sensing and cell metabolism with a nanoscale precision. However, this process is hindered by various problems like optical artifacts, noise, autouorescence, photobleaching and leakage of uorescence from one channel to the other. Chapter 7 deals with an image reconstruction technique to obtain noise-free and distortion-less data from multiple channels when imaging a multicolor sample. This technique is easily adaptable with the existing imaging systems and has potential application in biological imaging and biophysics where multiple probes are used to tag the features of interest. The fact that the lateral resolution of an optical system is better than the axial resolution is well known. Conventional microscopes focus on cells that are very close to the cover-slip or a few microns into the specimen. However, for cells that are embedded deep in a thick sample (ex: tissues), it is di_cult to visualize them using a conventional microscope. A number of factors like, scattering, optical aberrations, mismatch of refractive index between the objective lens and the mounting medium and noise, cause distortion of the images of samples at large depths. The system PSF gets distorted due to di_raction and its shape changes rapidly at large depths. The aim of chapter 8 is to introduce a technique to reduce distortion of images acquired at depth by employing image reconstruction techniques. The key to this methodology is the modeling of PSF at large depths. Maximum likelihood technique is then employed to reduce the streaking e_ects of the PSF and removes noise from raw images. This technique enables the visualization of cells embedded at a depth of 150_m. Several biological processes within the cell occur at a rate faster than the rate of acquisition and hence vital information is missed during imaging. The recorded images of these dynamic events are corrupted by motion blur, noise and other optical aberrations. Chapter 9 deals with two techniques that address temporal resolution improvement of the uorescence imaging system. The _rst technique focuses on accelerating the data acquisition process. This includes employing the concept of time-multiplexing to acquire sequential images from a dynamic sample using two cameras and generating multiple sheets of light using a di_raction grating, resulting in multi-plane illumination. The second technique involves the use of parallel processing units to enable real-time image reconstruction of the acquired data. A multi-node GPU and CUDA architecture effciently reduce the computation time of the reconstruction algorithms. Faster implementation of iterative image reconstruction techniques can aid in low-light imaging and dynamic monitoring of rapidly moving samples in real time. Employing rapid acquisition and rapid image reconstruction aids in real-time visualization of cells and have immense potential in the _eld of microbiology and bio-mechanics. Finally, we conclude the thesis with a brief section on the contribution of the thesis and the future scope the work presented. Thank you for using www.freepdfconvert.com service! 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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.

Resumo: A utilização da PDT para tratamento de diferentes tipos de infecções, tal como a candidose bucal, tem sido estudada. Entretanto, poucos são os dados científicos que relatam os possíveis efeitos tóxicos dessa terapia. Dessa forma, o objetivo deste estudo foi avaliar os efeitos da irradiação na mucosa bucal de ratos com LED azul (de 460 nm e potência de 200 mW/cm2) em presença do fotossensibilizador (FS) Photogem®, em duas diferentes concentrações (500 mg/L e 1000 mg/L). Para isso, foram utilizados 101 ratos (Rattus Norvegicus Albinus Holtzman) distribuídos em 6 grupos, de acordo com os seguintes tratamentos: Grupo 1 - controle; Grupo 2 - aplicação do FS (500 mg/L); Grupo 3 - aplicação do FS (500 mg/L) e irradiação com LED; Grupo 4 - aplicação do FS (1000 mg/L); Grupo 5 - aplicação do FS (1000 mg/L) e irradiação com LED; e Grupo 6 - irradiação com LED. O FS foi aplicado por 30 minutos (tempo de pré-incubação) e o tempo de irradiação da mucosa foi de 20 minutos (dose de 144 J/cm2). Decorridos os 4 períodos de avaliação propostos (0 dia, 1dia, 3 dias e 7 dias), os animais tiveram a mucosa palatina fotografada para análise macroscópica, sendo então imediatamente sacrificados para remoção cirúrgica do palato e posterior análise em microscopia de luz e de fluorescência. Um mapeamento térmico foi realizado a fim de avaliar a variação de temperatura ocorrida no tecido durante a irradiação com LED. Macroscopicamente, em todos os grupos experimentais e para todos os períodos de avaliação propostos na presente pesquisa, observou-se que a mucosa apresentava-se intacta, com aspecto de normalidade semelhante ao do Grupo 1 (controle). Microscopicamente, alterações teciduais, caracterizadas especialmente por discreta inflamação, puderam ser observadas na mucosa palatina de apenas 4 de um total de 80 animais submetidos a PDT... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of PDT has been investigated for the treatment of different types of infection, like oral candidosis. There are, however, few research-based data that report the possible toxic effects of this therapy. Therefore, this study evaluated the effects of irradiating the palatal mucosa of rats with blue LED (460 nm; 200 mW/cm²) in the presence of the photosensitizer Photogem® at two concentrations (500 and 1000 mg/L). Then, 101 rats (Rattus norvegicus albinus Holtzman) were randomly distributed in six groups, according to the treatment performed on the palatal mucosa: Group 1: control; Group 2: Photogem® (500 mg/L); Group 3: Photogem® (500 mg/L) + blue LED; Group 4 - Photogem® (1000 mg/L); Group 5: (1000 mg/L) + blue LED; and Group 6: blue LED. The exposure times to the photosensitizing agent and to the light source were 30 min (pre-incubation time) and 20 min (144 J/cm2 energy density), respectively. At 0, 1, 3 and 7 days posttreatment, the animals had their palatal mucosa photographed for macroscopic analysis and were immediately sacrificed. The palate was removed for further analysis by light and fluorescence microscopy. Thermal mapping was made to evaluate the temperature change occurred in the tissue during LED irradiation. In all experimental groups and periods, the macroscopic analysis revealed intact mucosa with normal aspect similar to that of Group 1 (control). Tissue alterations, characterized primarily by a mild inflammation, were observed microscopically on the mucosa of only 4 out of 80 animals subjected to PDT. Photosensitizer penetration into the treated mucosa was identified by the fluorescence emitted by Photogem® and was limited to the epithelial layer. The thermal mapping revealed a temperature increase from 35 to 41ºC during the 20-min irradiation. In conclusion, under the tested conditions, PDT using Photogem® at 500 and 1000 mg/L concentrations... (Complete abstract click electronic access below) / Orientador: Ana Cláudia Pavarina / Coorientador: Carlos Alberto de Souza / Banca: Eunice Teresinha Giampaolo / Banca: Cristina Kuraschi / Mestre

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.

Made available in DSpace on 2016-06-02T19:04:00Z (GMT). No. of bitstreams: 1 6814.pdf: 27157124 bytes, checksum: ccc98f69d1fc4cdc487ac2e9917edfc0 (MD5) Previous issue date: 2015-03-09 / Microelectrode arrays (MEA) are devices that allow chemical and electrical stimulation and recording of the extracellular electrical activity from entire neuronal cultures over long periods of time, such as several weeks. Some MEA models have transparent substrate, which enables the imaging of culture using optical microscopy. The images are taken from two channels: fluorescence light and transmitted light channels. In the first one, it is possible to visualize the neurons, while in the other one, it is possible to observe the microelectrodes. The objective of this work is to develop methods that enable performing quantitative analysis of the dissociated culture of rat dorsal root ganglion (DRG) neurons plated on MEA by means of the processing of the images, obtained from confocal fluorescence microscopy. We proposed and developed the following methods in order to achieve this objective: (A) A method to automatically identify the microelectrodes in the transmitted light channel using circular Hough Transform and error correction based on the Delaunay triangulation; (B) the registration of a number of images taken at different parts of the MEA in order to generate a unique and high-resolution representation of the whole culture; (C) the segmentation of the neuron in 2D images taken from the fluorescence channel, composed by the steps: preprocessing, thresholding, morphological filtering, neurons occlusion correction, watershed transform and object classification; (D) 2D quantitative analysis based on the identified microelectrodes and on the segmented neurons; (E) a method for generating 3D polygonal models of the neurons from the volumetric images, to be used for visualizing the culture on the MEA by different points of view and zoom levels; and (F) 3D quantitative analysis performed by the processing of the polygonal surfaces in conjunction with the information about the microelectrodes positioning. The results show that the methods are capable to identify the neurons and microelectrodes on the 2D images efficiently. In the 3D images, the preprocessing step which uses information from the 2D segmentation method, showed to be capable to generate correct polygonal models efficiently. Most of the studies involving the analysis of neuron cultures on MEAs consider only qualitative analysis or simple quantitative measures. However, the methods proposed in this thesis enables to obtain important measures related to the neuron culture, such as: the density and morphology of the neurons, and the spatial and topological distribution of the neurons and microelectrodes. The information about neuron morphology is important because they are related to the behavior of this kind of neuron. The spatial and topological distribution of neurons and microelectrodes are used for providing models of the interface between these elements, for supporting the analysis of the electrophysiological signal recorded by the microelectrodes, as well as in the computational simulations of the neuron culture behavior. / Matrizes de Microeletrodos (MEAs) são dispositivos que permitem estimular quimicamente ou eletricamente e registrar a atividade elétrica extracelular de culturas de neurônios durante um longo período de tempo, da ordem de várias semanas. Modelos de MEAs com o substrato transparente permitem imagear a cultura por meio de microscopia óptica. As imagens são obtidas em dois canais: um de luz de fluorescência e outro de luz de transmissão. O primeiro permite visualizar os neurônios, enquanto o segundo os microeletrodos. O objetivo deste trabalho é desenvolver métodos que permitam realizar análises quantitativas de culturas dissociadas de neurônios de gânglio da raiz dorsal (Dorsal Root Ganglion DRG) de ratos em MEAs por meio do processamento de imagens obtidas por microscopia confocal de fluorescência. Os seguintes métodos foram propostos e desenvolvidos para atingir este objetivo: (A) Identificação automática dos microeletrodos nas imagens do canal de luz de transmissão utilizando a transformada de Hough circular e correção de erros baseado na triangulação de Delaunay; (B) Registro de várias imagens tomadas de diferentes regiões da MEA para gerar uma única imagem em alta resolução que contemple a cultura toda; (C) Segmentação dos neurônios em imagens 2D obtidas a partir do canal de fluorescência, composto por etapas de pré-processamento, segmentação, filtragem morfológica, correção da oclusão de neurônios, transformada watershed e classificação de objetos; (D) Análise quantitativa 2D baseada nos microeletrodos identificados e nos neurônios segmentados; (E) Método para geração de modelos poligonais 3D dos neurônios a partir de imagens volumétricas, modelos os quais são utilizados para visualização da cultura na MEA por diferentes pontos de vista e níveis de zoom; e (F) Análise quantitativa 3D realizada por meio do processamento das superfícies poligonais juntamente com as informações sobre a posição dos microeletrodos. Os resultados mostram que os métodos são capazes de identificar com eficiência os neurônios e microeletrodos presentes nas imagens 2D. Nas imagens 3D, a etapa de pré-processamento utilizando informações resultantes do método de segmentação 2D se mostrou eficiente na geração dos modelos poligonais corretos. Enquanto a maioria das análises de imagens de culturas de neurônios em MEA consideram apenas análises quantitativas simples, os métodos aqui propostos permitem obter importantes medidas quantitativas relacionadas às culturas, tais como: a densidade e morfologia dos neurônios, assim como a distribuição espacial e topológica dos neurônios em relação aos microeletrodos. As informações sobre a morfologia são importantes, pois estão relacionadas com o comportamento desse tipo de neurônio. A distribuição espacial e topológica dos neurônios e microeletrodos permitem modelar a interface entre neurônios e microeletrodos e auxiliar nos estudos dos sinais eletrofisiológicos capturados pelos microeletrodos, assim como em simulações computacionais do comportamento dessas culturas.

Le principal objectif de cette thèse a consisté au développement d’une méthode de screenning pour la découverte de nouvelles réactions de ligations bioorthogonales ainsi que son application sur une bibliothèque développée pour cette étude. Par conséquent, un système de screening a été conçu en trois étapes consistant au départ en une analyse HPLC, puis une évaluation basée sur la fluorescence de haute résolution et finalement un test de microscopie confocal in cellulo. Puis, nous avons standardisé toutes les analyses avec les réactions CuAAC et SpAAC. En outre, nous avons synthétisés 18 réactifs d’intérêts et effectué un screening de 58 expériences de ligation avec une évaluation par méthode HPLC. Parmi les 9 réponses positives obtenues figure 6 réactions impliquant de nouveaux réactifs et les analyses LC‐MS ont pu tous les valider comme des réactions de cycloaddition directe à l’exception d’une réaction. Finalement, nous avons pu appliquer la méthode in cellulo développée, afin d’évaluer la pertinence des réactions de chélation CuAAC pour une application sur cellules. / The main goal of this thesis was the development of a screening method for the discovery of new bioorthogonal ligation reactions as well as its application on a self‐designed library. Therefore we designed a three step screening system consisting of a preliminary HPLC assay, a high resolution fluorescence based assay and a final in cellulo confocal microscopy assay.Subsequently we standardized all assays with the highly established CuAAC and SpAAC. Furthermore, we successfully synthesized 18 reagents of interest and screened 58 ligation experiments with the help of the HPLC setup. The 9 positive hits from this screening contained 6 reactions involving novel reagents and LCMS analysis was able to validate all but one as straight forward cycloaddition reaction. Finally we were able to apply the newly developed in cellulo assay to assess the suitability of chelating CuAAC for in cell application.

Orientadores: Jose Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-09T15:25:46Z (GMT). No. of bitstreams: 1 Soriano_DiogoCoutinho_M.pdf: 3276695 bytes, checksum: c47b4ce326955b65ff10f5c2891b25c2 (MD5) Previous issue date: 2007 / Resumo: No presente trabalho foi desenvolvido um instrumento capaz de quantificar de forma simultânea o encurtamento celular e a concentração citosólica de Ca2+ ([Ca2+]i), utilizando o indicador fluorescente fluo-3. A instrumentação foi aplicada para estudar o papel da ATPase de Ca2+ do RS (SERCA, principal transportador responsável pelo relaxamento celular e pela reposição do estoque de Ca2+ intracelular) na remoção de Ca2+ do citosol em miócitos cardíacos isolados de rato, o que exigiu o desenvolvimento de um protocolo experimental específico. O protocolo experimental desenvolvido consistiu em tornar inoperante o principal competidor da SERCA pelo Ca2+ citosólico, o trocador Na+/Ca2+ (NCX), pela perfusão da célula com solução sem Ca2+ e sem Na+. Nesta situação, considera-se que os demais transportadores de Ca2+ (ATPase de Ca2+ do sarcolema e uniporter mitocondrial de Ca2+) transportem o íon a uma taxa baixa demais para competir com a SERCA. A liberação do Ca2+ do RS foi induzida por pulsos rápidos de cafeína, e foram medidos a amplitude (?[Ca2+]i) e tempo para 50 % de queda (t1/2) dos transientes de [Ca2+]i na ausência e na presença de fármacos que afetam a taxa de captação de Ca2+ pela SERCA. Foram analisados os efeitos do agonista de receptores beta-adrenérgicos isoproterenol (ISO), que promove estimulação da SERCA, e de 2,5-di-(tert-butil) hydroquinona (tBQ), que atua como inibidor da enzima. ISO causou redução significativa do t1/2 de queda do [Ca2+]i (p< 0,01), sem alteração significativa de ?[Ca2+]i (p> 0,05). Já no caso do tBQ, foi observado um aumento significativo do t1/2 de queda do [Ca2+]i (p< 0,01) e redução da ?[Ca2+]i, (p< 0,05). Um modelo teórico da literatura foi adaptado para descrever matematicamente o modelo experimental proposto. As simulações com alterações nos parâmetros cinéticos da SERCA pelos fármacos (de acordo com dados da literatura) foram razoavelmente bem sucedidas em reproduzir os dados experimentais com relação ao tempo de remoção do Ca2+ do citosol. Portanto, foi apresentada aqui uma nova ferramenta experimental e teórica para estudar a captação de Ca2+ pela SERCA em miócitos cardíacos intactos / Abstract: The goal of this work was to develop an instrument for simultaneous measurement of cell shortening and cytosolic Ca2+ concentration ([Ca2+]i), by using the fluorescent Ca2+ indicator fluo-3. The instrument was applied to study the uptake of cytosolic Ca2+ by the sarcoplasmic reticulum (RS) Ca2+- ATPase (SERCA, the main transporter responsible for cell relaxation in intact isolated rat ventricular myocytes). For this purpose, the development of a specific experimental protocol was required. In this protocol, the main competitor of SERCA by cytosolic Ca2+, the Na+/Ca2+ exchanger (NCX), was disabled by removal of extracellular Ca2+ and Na+. In this situation, it may be assumed that the other Ca2+ transporters (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter) are too slow to compete with SERCA. SR Ca2+ release was induced by short, rapid caffeine pulses, and the amplitude (?[Ca2+]i) and time for 50% [Ca2+]i decline (t1/2) of the resulting [Ca2+]I transients were measured in the absence and in the presence of drugs that affect the rate of SR Ca2+ uptake by SERCA, namely the beta-adrenergic receptor agonist isoproterenol (ISO), which causes increase in SERCA activity, and 2,5-di-(tert-butyl) hydroquinone (tBQ), which inhibits the enzyme. ISO caused significant reduction of t1/2 (p< 0,01), without any significant change in ?[Ca2+]i (p> 0,05). In case of tBQ, significant increase of t1/2 (p< 0,01) and reduction of ?[Ca2+]I (p< 0,05) were observed. A theoretical model (Tang & Othmer, 1994) was adapted for mathematical description of the experimental model proposed. Simulation of the effects of the drugs, in which SERCA kinetic parameters were changed according to data obtained from literature, were reasonably successful at reproducing the cytosolic [Ca2+]i kinetics observed experimentally / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

A resposta do sistema nervoso para diversas lesões acarreta a ativação das células gliais entéricas. Este trabalho tem como objetivo analisar o efeito da isquemia e reperfusão intestinal (I/R-i) sobre as células gliais entéricas, neurônios e receptores P2X2 e P2X7. Foram analisados o íleo de ratos Controle, Sham e I/R-i com 0 hora, 24 horas e 14 dias de reperfusão. Foram realizadas dupla marcação dos receptores P2X2 e P2X7 com Hu e S100, densidade, área do perfil e marcação de proliferação celular. Os resultados mostraram dupla marcação de células gliais entéricas e neurônios com os receptores P2X2 e P2X7; a densidade apresentou um aumento de células gliais e diminuição de neurônios imunorreativos ao Hu. A área do perfil de células gliais entericas S100-IR apresentaram diminuição nos grupos I/R-i e foi detectada proliferação de células gliais entéricas nos grupos I/R-i 0 hora e 24 horas. Conclui-se que a isquemia levou a alterações diferenciadas nos receptores P2X2 e P2X7, células gliais entéricas e neurônios, que podem causar disfunções gastrointestinais. / The nervous system response to various injuries involves the activation of enteric glial cells. The aim of the work was to analyze the effect of ischemia and reperfusion (I/R-i) on enteric glial cells, neurons and receptors P2X2 and P2X7. We analyzed the ileum of Control, Sham and I/R-i with 0 hour, 24 hours and 14 days of reperfusion. Double staining were performed P2X2 and P2X7 receptors with Hu and S100, density, area profile and marking of cellular proliferation. The results show double staining of neurons and enteric glial cell with the P2X2 and P2X7; density increased by glial cells and decrease of neurons immunoreactive to Hu. The area profile of enteric glial cell S100-IR showed decreased in Groups I/R-I and enteric glial cell proliferation was observed in groups I/R-i 0 hours and 24 hours. It is concluded that ischemia has led to changes in differential P2X2 and P2X7 receptors, neurons and enteric glial cells, which can cause gastrointestinal dysfunction.