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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.

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HER2 breast cancer is an aggressive disease that occurs in 20 - 30% of the breast cancer population. Treatment for HER2 breast cancer includes use of an anti-HER2 monoclonal antibody, trastuzumab. Testing for HER2 is of critical importance due to the adverse side effects and substantial costs associated with this anti-HER2 treatment. Currently, two kinds of tests, Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC), are FDA approved for determination of HER2 status in breast cancers. Clinical and non clinical factors that affect the choice HER2 test and the use of anti-HER2 therapy in breast cancer were analyzed using a data set containing information from six outpatient oncology clinics in the United States. The analysis showed that geographic location, cancer stage, and diagnosis date (pre- or post-publication of testing guidelines) have significant effects on choice of test. With regard to trastuzumab prescription, geographic location and HER2 status have significant effects on the prescription of trastuzumab. In addition, there was a non-significant trend for certain Medicare patients not to receive trastuzumab therapy. These findings indicate that disparities are present in breast cancer care based on geography and cancer stage, and highlight the importance of testing guidelines. The cost effectiveness of FISH vs. IHC was determined, by considering the financial and health-related costs associated with testing and subsequent treatment as well as the accuracy of each test. The results show that FISH is the optimal choice for HER2 testing and is more cost-effective than IHC.

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