O propósito deste trabalho foi avaliar a interação de matriz óssea bovina inorgânica, (Gen-ox®) como material de enxerto e implantes de titânio em mandíbula de coelhos. A amostra constituiu-se de 32 coelhos da raça Botucatu de aproximadamente 4kg. Os incisivos inferiores destes animais foram extraídos e foi realizada um defeito de aproximadamente 4mm de diâmetro. Um dos lados foi mantido apenas com coágulo (grupo controle) e o outro lado (grupo experimental) recebeu o enxerto do material proposto. Após 60 dias foram instalados implantes bilateralmente na região do defeito. Os animais foram eutanaziados imediatamente após a instalação dos implantes e após 1, 2 e 6 meses (n=6) para o grupo microscópico a após 2 e 6 meses para o grupo do teste biomecânico (n=4). A análise constituiu de medidas da distância da crista óssea alveolar à plataforma do implante, da densidade radiográfica em valores de pixel ma região do alvéolo, de teste biomecânico de torque de remoção e microscópico através de medidas de contato osso-implante (BIC) (em µm e %) e área de osso (em µm2 e %). Os resultados demonstraram que não houve diferenças significantes nem durante o tempo e nem entre os grupos na analise de distancia entre crista óssea alveolar e plataforma do implante. Na medida de densidade óssea, não houve variação significante ao longo do tempo, mas houve diferença estatisticamente significante entre os grupos, sendo maior no grupo experimental. Para o teste biomecânico também não houve variação significante do torque de remoção ao longo do tempo e nem houve diferença significante entre os grupos. Na análise de contato osso implante (BIC) não houve variação significante ao longo do tempo, mas houve diferença estatisticamente significante entre os grupos, sendo maior no grupo experimental e houve interação entre tempos e grupos. E com relação a medida de área de osso não houve diferenças significantes nem durante o tempo e nem entre os grupos controle e experimental nos períodos analisados. Os resultados sugerem que o material foi bem aceito na região e teve comportamento semelhante ao coágulo em contato com a superfície do implante. / The aim of this study was to evaluate the interaction of an inorganic bovine bone matrix (Gen-ox®) as bone graft and titanium implants in rabbits mandible. Thirty-two Botucatu rabbits weighting about 4kg were used on this experiment. The mandible incisives were extracted and a defect of 4 mm of diameter was performed. One socket and defect was filled with experimental material. The opposite site was left to heal naturally and served as control. After 60 days, the implants were installed on the defect region. The animals were killed immediately after the implant installation and after 1, 2 and 6 months. Vertical bone height, bone density, biomechanical test, bone implant contact (BIC) and bone area near to implant threads were evaluated. The results showed that there was no significant differences on the distance between the alveolar bone crest to the implant platform (vertical bone height) along the time, neither between groups. On the bone density (pixel value) there was no significant difference along the time, but there was significant difference between groups, being the experimental group higher. For the biomechanical test there was no significant difference along the time, neither between groups. There was no significant differences on the bone implant contact (BIC) measures along the time, but there was significant difference between groups, being the experimental groups higher and there was interaction between time and groups. On bone area evaluation, there was no significant difference along the time, neither between the groups during the analyzed time. The graft behavior observed by the authors was similar to that of the control group, suggesting high acceptance of the material as graft option associated to titanium implants.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein α (C/EBPα) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBPα. Additionally, the original clone found to interact with C/EBPα consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBPα mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBPα, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBPα-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBPα-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBPα. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBPα was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBPα, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBPα.

It is my hope that this thesis may serve as a guide for future students wishing to build a microscope from scratch. The design and construction of a scanning, confocal fluorescence microscope equipped with shaped microwave excitation is detailed. The use of the microscope is demonstrated by coherently manipulating single Nitrogen-Vacancy centres in diamond. Further the instrument is used to investigate a dual Halbach array magnet system.

Micro-Electro-Mechanical Systems (MEMS) micromirrors have been developed for more than two decades along with the development of MEMS technology. They have been used into many application fields: optical switches, digital light projector (DLP), adoptive optics (AO), high definition (HD) display, barcode reader, endoscopic optical coherence tomography (OCT) and confocal microscope, and so on. Especially, MEMS mirrors applied into endoscopic OCT and confocal microscope are the intensive research field. Various actuation mechanisms, such as electrostatic, electromagnetic, electro bimorph thermal, electrowetting, piezoelectric (PZT) and hybrid actuators, are adopted by different types of micromirrors. Among these actuators, the electrostatic is easily understood and simple to realize, therefore, it is broadly adopted by a large number of micromirrors. This thesis reports the design, fabrication, and characterization of a 2-D Silicon-on-insulation (SOI) MEMS micromirror with sidewall (SW) electrodes for endoscopic OCT or confocal microscope imaging. The biaxial MEMS mirror with SW electrodes is actuated by electrostatic actuators. The dimension of mirror plate is 1000micron×1000micron, with a thickness of a 35micron. The analytical modeling of SW electrodes, fabrication process, and performance characteristics are described. In comparison to traditional electrostatic actuators, parallel-plate and comb-drive, SW electrodes combined with bottom electrodes achieve a large tilt angle under a low drive voltage that the comb-drive does and possess fairly simple fabrication process same as that of the parallel-plate. A new fabrication process based on SOI wafer, hybrid bulk/surface micromachined technology, and a high-aspect-ratio shadow mask is presented. Moreover, the fabrication process is successfully extended to fabricate 2×2 and 4×4 micromirror arrays. Finally, a biaxial MEMS mirror with SW electrodes was used into Confocal MACROscope for imaging. Studied optical requirements in terms of two optical configurations and frequency optimization of the micromirror, the biaxial MEMS mirror replaces the galvo-scanner and improves the MACROscope. Meanwhile, a new Micromirror-based Laser Scanning Microscope system is presented and allows 2D images to be acquired and displayed.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein α (C/EBPα) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBPα. Additionally, the original clone found to interact with C/EBPα consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBPα mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBPα, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBPα-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBPα-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBPα. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBPα was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBPα, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBPα.

Optical beam induced current (OBIC) mapping has found wide-spread applications in characterizing semiconductor devices and integrated circuitry. In this study, we have used a two-photon scanning microscope to investigate InGaN light emitting diodes (LED). The defects induced by electrostatic discharge (ESD) can be clearly identified by DC-OBIC images. Additionally, we have combined an E-O modulator and a high frequency phase sensitive lock-in amplifier to conduct time-resolved study on the dynamical properties of the LEDs. The defects also exhibit different delay time when compared with the normal parts.

Micro-Electro-Mechanical Systems (MEMS) micromirrors have been developed for more than two decades along with the development of MEMS technology. They have been used into many application fields: optical switches, digital light projector (DLP), adoptive optics (AO), high definition (HD) display, barcode reader, endoscopic optical coherence tomography (OCT) and confocal microscope, and so on. Especially, MEMS mirrors applied into endoscopic OCT and confocal microscope are the intensive research field. Various actuation mechanisms, such as electrostatic, electromagnetic, electro bimorph thermal, electrowetting, piezoelectric (PZT) and hybrid actuators, are adopted by different types of micromirrors. Among these actuators, the electrostatic is easily understood and simple to realize, therefore, it is broadly adopted by a large number of micromirrors. This thesis reports the design, fabrication, and characterization of a 2-D Silicon-on-insulation (SOI) MEMS micromirror with sidewall (SW) electrodes for endoscopic OCT or confocal microscope imaging. The biaxial MEMS mirror with SW electrodes is actuated by electrostatic actuators. The dimension of mirror plate is 1000micron×1000micron, with a thickness of a 35micron. The analytical modeling of SW electrodes, fabrication process, and performance characteristics are described. In comparison to traditional electrostatic actuators, parallel-plate and comb-drive, SW electrodes combined with bottom electrodes achieve a large tilt angle under a low drive voltage that the comb-drive does and possess fairly simple fabrication process same as that of the parallel-plate. A new fabrication process based on SOI wafer, hybrid bulk/surface micromachined technology, and a high-aspect-ratio shadow mask is presented. Moreover, the fabrication process is successfully extended to fabricate 2×2 and 4×4 micromirror arrays. Finally, a biaxial MEMS mirror with SW electrodes was used into Confocal MACROscope for imaging. Studied optical requirements in terms of two optical configurations and frequency optimization of the micromirror, the biaxial MEMS mirror replaces the galvo-scanner and improves the MACROscope. Meanwhile, a new Micromirror-based Laser Scanning Microscope system is presented and allows 2D images to be acquired and displayed.

It is my hope that this thesis may serve as a guide for future students wishing to build a microscope from scratch. The design and construction of a scanning, confocal fluorescence microscope equipped with shaped microwave excitation is detailed. The use of the microscope is demonstrated by coherently manipulating single Nitrogen-Vacancy centres in diamond. Further the instrument is used to investigate a dual Halbach array magnet system.

O propósito deste trabalho foi avaliar a interação de matriz óssea bovina inorgânica, (Gen-ox®) como material de enxerto e implantes de titânio em mandíbula de coelhos. A amostra constituiu-se de 32 coelhos da raça Botucatu de aproximadamente 4kg. Os incisivos inferiores destes animais foram extraídos e foi realizada um defeito de aproximadamente 4mm de diâmetro. Um dos lados foi mantido apenas com coágulo (grupo controle) e o outro lado (grupo experimental) recebeu o enxerto do material proposto. Após 60 dias foram instalados implantes bilateralmente na região do defeito. Os animais foram eutanaziados imediatamente após a instalação dos implantes e após 1, 2 e 6 meses (n=6) para o grupo microscópico a após 2 e 6 meses para o grupo do teste biomecânico (n=4). A análise constituiu de medidas da distância da crista óssea alveolar à plataforma do implante, da densidade radiográfica em valores de pixel ma região do alvéolo, de teste biomecânico de torque de remoção e microscópico através de medidas de contato osso-implante (BIC) (em µm e %) e área de osso (em µm2 e %). Os resultados demonstraram que não houve diferenças significantes nem durante o tempo e nem entre os grupos na analise de distancia entre crista óssea alveolar e plataforma do implante. Na medida de densidade óssea, não houve variação significante ao longo do tempo, mas houve diferença estatisticamente significante entre os grupos, sendo maior no grupo experimental. Para o teste biomecânico também não houve variação significante do torque de remoção ao longo do tempo e nem houve diferença significante entre os grupos. Na análise de contato osso implante (BIC) não houve variação significante ao longo do tempo, mas houve diferença estatisticamente significante entre os grupos, sendo maior no grupo experimental e houve interação entre tempos e grupos. E com relação a medida de área de osso não houve diferenças significantes nem durante o tempo e nem entre os grupos controle e experimental nos períodos analisados. Os resultados sugerem que o material foi bem aceito na região e teve comportamento semelhante ao coágulo em contato com a superfície do implante. / The aim of this study was to evaluate the interaction of an inorganic bovine bone matrix (Gen-ox®) as bone graft and titanium implants in rabbits mandible. Thirty-two Botucatu rabbits weighting about 4kg were used on this experiment. The mandible incisives were extracted and a defect of 4 mm of diameter was performed. One socket and defect was filled with experimental material. The opposite site was left to heal naturally and served as control. After 60 days, the implants were installed on the defect region. The animals were killed immediately after the implant installation and after 1, 2 and 6 months. Vertical bone height, bone density, biomechanical test, bone implant contact (BIC) and bone area near to implant threads were evaluated. The results showed that there was no significant differences on the distance between the alveolar bone crest to the implant platform (vertical bone height) along the time, neither between groups. On the bone density (pixel value) there was no significant difference along the time, but there was significant difference between groups, being the experimental group higher. For the biomechanical test there was no significant difference along the time, neither between groups. There was no significant differences on the bone implant contact (BIC) measures along the time, but there was significant difference between groups, being the experimental groups higher and there was interaction between time and groups. On bone area evaluation, there was no significant difference along the time, neither between the groups during the analyzed time. The graft behavior observed by the authors was similar to that of the control group, suggesting high acceptance of the material as graft option associated to titanium implants.

abstract: The cell is a dense environment composes of proteins, nucleic acids, as well as other small molecules, which are constantly bombarding each other and interacting. These interactions and the diffusive motions are driven by internal thermal fluctuations. Upon collision, molecules can interact and form complexes. It is of interest to learn kinetic parameters such as reaction rates of one molecule converting to different species or two molecules colliding and form a new species as well as to learn diffusion coefficients. Several experimental measurements can probe diffusion coefficients at the single-molecule and bulk level. The target of this thesis is on single-molecule methods, which can assess diffusion coefficients at the individual molecular level. For instance, super resolution methods like stochastic optical reconstruction microscopy (STORM) and photo activated localization microscopy (PALM), have a high spatial resolution with the cost of lower temporal resolution. Also, there is a different group of methods, such as MINFLUX, multi-detector tracking, which can track a single molecule with high spatio-temporal resolution. The problem with these methods is that they are only applicable to very diluted samples since they need to ensure existence of a single molecule in the region of interest (ROI). In this thesis, the goal is to have the best of both worlds by achieving high spatio-temporal resolutions without being limited to a few molecules. To do so, one needs to refocus on fluorescence correlation spectroscopy (FCS) as a method that applies to both in vivo and in vitro systems with a high temporal resolution and relies on multiple molecules traversing a confocal volume for an extended period of time. The difficulty here is that the interpretation of the signal leads to different estimates for the kinetic parameters such as diffusion coefficients based on a different number of molecules we consider in the model. It is for this reason that the focus of this thesis is now on using Bayesian nonparametrics (BNPs) as a way to solve this model selection problem and extract kinetic parameters such as diffusion coefficients at the single-molecule level from a few photons, and thus with the highest temporal resolution as possible. / Dissertation/Thesis / Source code related to chapter 3 / Source code related to chapter 4 / Doctoral Dissertation Physics 2020