Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

Some diagnostic methods have been used for a very long time. Congo red stain saw the light of day in 1883, and quickly became important in many fields of use. Nowadays we recognize the importance of Congo red in diagnose of amyloid diseases. However, the technique and experience needed throughout the process from a suspected case to the diagnose is of greate importance. When diagnostic difficulties appeared in a few patient cases at the local hospital in Gävle, Sweden, a solution was needed. A delayed diagnose could have a potential devastating outcome seen in the perspective of the patient. Therefore it is crucial to have both sensitive and specific diagnostic methods that are optimized against the sought pathogenesis. This study aspired to find the solution to the difficulties in diagnostic work, brought to light by a pathology doctor at the hospital. Several different methodical procedures are used throughout the process, and were evaluated with focus lying on the thickness of the tissue, the staining method and the microscopes used in diagnostics. Different thickness of the tissue was cut and stained. The results demonstrated the importance of proper techniques and methods in preparing the tissue, and the tools to analyse it with. The thickness of tissue and the lightsource in the microscope played a cruicial role in diagnostics. Additionally it showed the importance to continue to raise the quality of work and make progress in the diagnostic and scientific field, possibly by finding new applications for old methods.

Spectral interferometry, also known as spectral-domain white light or low coherence interferometry, has seen numerous applications in sensing and metrology of physical parameters. It can provide phase or optical path information of interest in single shot measurements with exquisite sensitivity and large dynamic range. As fast spectrometer became more available in 21st century, spectral interferometric techniques start to dominate over time-domain interferometry, thanks to its speed and sensitivity advantage. In this work, a dual-modality phase/birefringence imaging system is proposed to offer a quantitative approach to characterize phase, polarization and spectroscopy properties on a variety of samples. An interferometric spectral multiplexing method is firstly introduced by generating polarization mixing with specially aligned polarizer and birefringence crystal. The retardation and orientation of sample birefringence can then be measured simultaneously from a single interference spectrum. Furthermore, with the addition of a Nomarski prism, the same setup can be used for quantitative differential interference contrast (DIC) imaging. The highly integrated system demonstrates its capability for noninvasive, label-free, highly sensitive birefringence, DIC and phase imaging on anisotropic materials and biological specimens, where multiple intrinsic contrasts are desired. Besides using different intrinsic contrast regime to quantitatively measure different biological samples, spectral multiplexing interferometry technique also finds an exquisite match in imaging single anisotropic nanoparticles, even its size is well below diffraction limit. Quantitative birefringence spectroscopy measurement over gold nanorod particles on glass substrate demonstrates that the proposed system can simultaneously determine the polarizability-induced birefringence orientation, as well as the scattering intensity and the phase differences between major/minor axes of single nanoparticles. With the anisotropic nanoparticles' spectroscopic polarizability defined prior to the measurement with calculation or simulation, the system can be further used to reveal size, aspect ratio and orientation information of the detected anisotropic nanoparticle. Alongside developing optical anisotropy imaging systems, the other part of this research describes our effort of investigating the sensitivity limit for general spectral interferometry based systems. A complete, realistic multi-parameter interference model is thus proposed, while corrupted by a combination of shot noise, dark noise and readout noise. With these multiple noise sources in the detected spectrum following different statistical behaviors, Cramer-Rao Bounds is derived for multiple unknown parameters, including optical pathlength, system-specific initial phase, spectrum intensity as well as fringe visibility. The significance of the work is to establish criteria to evaluate whether an interferometry-based optical measurement system has been optimized to its hardware best potential. An algorithm based on maximum likelihood estimation is also developed to achieve absolute optical pathlength demodulation with high sensitivity. In particular, it achieves Cramer-Rao bound and offers noise resistance that can potentially suppress the demodulation jump occurrence. By simulations and experimental validations, the proposed algorithm demonstrates its capability of achieving the Cramer-Rao bound over a large dynamic range of optical pathlengths, initial phases and signal-to-noise ratios. / PHD / Optical imaging is unique for its ability to use light to provide both structural and functional information from microscopic to macroscopic scales. As for microscopy, how to create contrast for better visualization of detected objects is one of the most important topic. In this work, we are aiming at developing a noninvasive, label-free and quantitative imaging technique based on multiple intrinsic contrast regimes, such as intensity, phase and birefringence. Spectral multiplexing interferometry method is firstly introduced by generating spectral interference with polarization mixing. Multiple parameters can thus be demodulated from single-shot interference spectrum. With Jones Matrix analysis, the retardation and orientation of sample birefringence can be measured simultaneously. A dual-modality phase/birefringence imaging system is proposed to offer a quantitative approach to characterize phase, polarization and spectroscopy properties on a variety of samples. The high integrated system can not only deliver label-free, highly sensitive birefringence, DIC and phase imaging of anisotropic materials and biological specimens, but also reveal size, aspect ratio and orientation information of anisotropic nanoparticles of which the size is well below diffraction limit. Alongside developing optical imaging systems based on spectral interferometry, the other part of this research describes our effort of investigating the sensitivity limit for general spectral interferometry based systems. The significance of the work is using Cramer-Rao Bounds to establish criteria to evaluate whether an optical measurement system has been optimized to its hardware best potential. An algorithm based on maximum likelihood estimation is also developed to achieve absolute optical pathlength demodulation with high sensitivity. In particular, it achieves Cramer-Rao bound and offers noise resistance that can potentially suppress the demodulation jump occurrence.

Orientadores: Raquel Fernanda Gerlach, Frederico Barbosa de Sousa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-17T01:33:00Z (GMT). No. of bitstreams: 1 Porto_IsabelMaria_D.pdf: 20861055 bytes, checksum: 0299cefd6df5fb84fe2f7a034ca14273 (MD5) Previous issue date: 2010 / Resumo: O esmalte é o tecido mais mineralizado do corpo. Esta característica o torna um tecido altamente conservado no período post mortem. Por isso ele é citado como o melhor tecido para se estudar matéria orgânica em fósseis no campo da antropologia, paleontologia e arqueologia. É possível também a determinação do sexo na ciência forense. Porém a dificuldade está em se extrair o conteúdo orgânico destes dentes, já que o esmalte maduro contém menos de 1% de proteínas. Dois estudos da presente tese descrevem um método muito eficiente em extrair proteínas do esmalte. Esta mineralização do esmalte dentário pode ser prejudicada por doenças, como a fluorose dentária por exemplo. Em nosso terceiro estudo, analisamos o perfil dos aminoácidos para saber se o flúor interfere na clivagem das amelogeninas. Neste estudo, a clivagem da amelogenina não é afetada pelo flúor em dentes humanos fluoróticos erupcionados e inclusos. O quarto estudo traz como hipótese que a distribuição espacial dos conteúdos bioquímicos nas lesões fluoróticas do esmalte se assemelha aquela observada na última onda de mineralização durante o estágio de maturação. Observou-se neste estudo que há um aumento do conteúdo orgânico e uma hipomineralização na superfície no esmalte fluorótico, tanto nos terceiros molares erupcionados quanto inclusos. Estes dados sugerem que a lesões fluoróticas em esmalte humano refletem a composição do esmalte no período tardio de maturação da amelogênese / Abstract: The dental enamel is the most highly mineralized tissue in the body. Due to its characteristics, the enamel is highly inert to changes brought about by time and the environment, being a very important source of information for palaeo-, palaeanthropo-, and anthropologists. The enamel proteins are also used for sex identification in forensic science. But protein recovery from the enamel it is a challenging task. Here, we present two studies that describe procedures very effective in providing enamel samples that are adequate for protein analysis. This mineralization could be impaired by diseases like dental fluorosis, for example. In the third study, we analyzed the amino acid profile in the erupted and nonerupted fluorotic human teeth compared to control ones. In this study the cleavage of amelogenin was not affected by fluoride in the fluorotic teeth. In the fourth study we hypothesized that the composition of fluorotic lesions may resemble the enamel found during last wave of enamel mineralization. We found an increase in the organic content and a superficial hypomineralization of the fluorotic enamel, in both erupted and non-erupted human third molars. These data suggest that in both non-erupted and erupted human teeth the fluorotic lesions resembles the late maturation stage enamel, which is not mineralized until maturation is completed / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

Introdução: Apesar de controverso, questiona-se um papel deletério da endometriose nos resultados de procedimentos de reprodução assistida, o que pode estar relacionado ao comprometimento da qualidade oocitária. Para que o oócito maduro esteja preparado para a fertilização, é necessário que o fuso meiótico mantenha a sua integridade e funcionabilidade. Objetivos: Comparar a presença e localização do fuso meiótico e o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis sem e com endometriose. Comparar os resultados de Injeção Intracitoplasmática de espermatozóides (ICSI) entre os oócitos em telófase I e metáfase II, e entre aqueles com e sem fuso celular visível, nos grupos analisados. Metodologia: Estudo prospectivo e controlado com pacientes inférteis, submetidas à estimulação ovariana para realização de ICSI, selecionadas consecutivamente e divididas em dois grupos: Controle (fator tubário e/ou masculino) e Endometriose (subdividido em endometriose mínima e leve I/II versus moderada e severa III/IV). Os oócitos com extrusão do primeiro CP foram avaliados pela microscopia de polarização imediatamente antes da realização da ICSI e caracterizados quanto à presença/localização do fuso celular em relação ao primeiro CP e ao estágio de maturação nuclear (telófase I ou metáfase II). Foram analisados as taxas de fertilização, clivagem, número de embriões de boa qualidade no segundo (D2) e terceiro (D3) dia de desenvolvimento oriundos dos oócitos em telófase I versus metáfase II, e metáfase II com fuso visível versus sem fuso visível, nos grupos controle, endometriose, endometriose I/II e endometriose III/IV. Resultados: Foram analisados 441 oócitos, sendo 254 do grupo controle e 187 do grupo endometriose (115 do grupo endometriose I/II e 72 do grupo endometriose III/IV). Não observamos diferença significativa entre a percentagem de oócitos em metáfase II com fuso celular visível e não visível (88,6%, 91,3%, 88,2%, respectivamente, nos grupos controle, endometriose I/II e endometriose III/IV) e entre a percentagem de oócitos com fuso celular nas diferentes localizações nos grupos avaliados. Entre os oócitos aparentemente maduros, observamos um aumento significativo de oócitos em telófase I no grupo endometriose III/IV (5,6%) quando comparado ao grupo endometriose I/II (0%). Observamos uma tendência a menores taxas de fertilização dos oócitos injetados em telófase I quando comparados aos em metáfase II, nos grupos controle (p=0,08), endometriose (p=0,05) e endometriose III/IV (p=0,09). Comparando-se os oócitos com e sem fuso celular visível, não observamos diferença significativa nos resultados de ICSI entre os grupos analisados. Conclusão: Não observamos diferença significativa entre os grupos analisados quanto à visualização e localização do fuso celular em oócitos maturados in vivo com o primeiro CP visível. Todavia, observamos um aumento significativo de oócitos em telófase I nas portadoras de endometriose moderada e severa, sugerindo um retardo ou comprometimento na conclusão da meiose I. Considerando que os oócitos injetados em telófase I apresentam piores taxas de fertilização do que os injetados em metáfase II, este achado poderia justificar o comprometimento dos resultados de reprodução assistida em mulheres inférteis com endometriose moderada e severa, além de ser utilizado com ferramenta prognóstica pós-ICSI. / Introduction: Although it has been a controversial issue for decades, a deleterious role of endometriosis on assisted reproductive techniques (ART) outcomes is questioned, which may be related to oocyte quality. For a mature oocyte be prepared for fertilization is necessary that the meiotic spindle keeps its integrity and its function. Objectives: To compare the presence and localization of the meiotic spindle and the oocyte nuclear maturation with the visible first polar body of infertile patients with and without endometriosis. To compare ICSI outcomes between oocytes on telophase I and metaphase II, and the ones with and without visible meiotic spindle, on those two groups. Methodology: A prospective and controlled study with infertile patients who underwent ovarian stimulation for purposes of ICSI, selected consecutively and divided into two groups: control (tubal and/or male factor) and endometriosis (subdivided in minimum and mild stage I/II versus moderate and severe stage III/IV). The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged using a polarization microscopy immediately before ICSI and characterized according to the presence and localization of meiotic spindle and its relation to the first polar body and the nuclear maturation stage (telophase I and metaphase II). We have analyzed the fertilization rates, clivage, number of good quality embryos on the second (D2) and third (D3) day of development from oocytes on telophase I versus the ones on metaphase II, and metaphase II visible spindle versus the non-visible ones, on the control groups, endometriosis, endometriosis stage I/II and endometriosis stage III/IV. Results: A total of 441 oocytes were analyzed, 254 oocytes form the control group and 187 from the endometriosis one (115 from endometriosis stage I/II and 72 from endometriosis stage III/IV). No significant differences between the percentage of metaphase II with visible and non-visible meiotic spindle were found (88,6%, 91,3%, and 88,2%, in the control, endometriosis I/II and endometriosis III/IV groups, respectively). Among the apparently matured oocytes, we have observed a significant increase of oocytes on telophase I on the endometriosis III/IV group (5,6%) when compared with the endometriosis I/II group (0%). We have observed a tendency to fewer fertilization rates from the injected oocytes on telophase I when compared with the ones on metaphase II, on the control group (p=0,08), endometriosis (p=0,05) and endometriosis III/IV group (p=0,09). When we compared oocytes with and without visible meiotic spindle, we found no significant difference on ICSI outcomes among the studied groups. Conclusions: We have found no significant difference among the studied groups regarding the visualization and localization of the meiotic spindle from in vivo matured oocytes with a visible first polar body. However, we have observed a significant increase on the number of oocytes on telophase I from patients with moderate and severe endometriosis, suggesting a delay or an impairment in the completion of meiosis I. Since the injected oocytes on telophase I present a worse fertilization rates than the ones injected on metaphase II, this finding could explain the impairment on the outcomes of ART in infertile women with moderate and severe endometriosis, besides it could be used as a prognosis tool after ICSI procedures.

Introdução: Apesar de controverso, questiona-se um papel deletério da endometriose nos resultados de procedimentos de reprodução assistida, o que pode estar relacionado ao comprometimento da qualidade oocitária. Para que o oócito maduro esteja preparado para a fertilização, é necessário que o fuso meiótico mantenha a sua integridade e funcionabilidade. Objetivos: Comparar a presença e localização do fuso meiótico e o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis sem e com endometriose. Comparar os resultados de Injeção Intracitoplasmática de espermatozóides (ICSI) entre os oócitos em telófase I e metáfase II, e entre aqueles com e sem fuso celular visível, nos grupos analisados. Metodologia: Estudo prospectivo e controlado com pacientes inférteis, submetidas à estimulação ovariana para realização de ICSI, selecionadas consecutivamente e divididas em dois grupos: Controle (fator tubário e/ou masculino) e Endometriose (subdividido em endometriose mínima e leve I/II versus moderada e severa III/IV). Os oócitos com extrusão do primeiro CP foram avaliados pela microscopia de polarização imediatamente antes da realização da ICSI e caracterizados quanto à presença/localização do fuso celular em relação ao primeiro CP e ao estágio de maturação nuclear (telófase I ou metáfase II). Foram analisados as taxas de fertilização, clivagem, número de embriões de boa qualidade no segundo (D2) e terceiro (D3) dia de desenvolvimento oriundos dos oócitos em telófase I versus metáfase II, e metáfase II com fuso visível versus sem fuso visível, nos grupos controle, endometriose, endometriose I/II e endometriose III/IV. Resultados: Foram analisados 441 oócitos, sendo 254 do grupo controle e 187 do grupo endometriose (115 do grupo endometriose I/II e 72 do grupo endometriose III/IV). Não observamos diferença significativa entre a percentagem de oócitos em metáfase II com fuso celular visível e não visível (88,6%, 91,3%, 88,2%, respectivamente, nos grupos controle, endometriose I/II e endometriose III/IV) e entre a percentagem de oócitos com fuso celular nas diferentes localizações nos grupos avaliados. Entre os oócitos aparentemente maduros, observamos um aumento significativo de oócitos em telófase I no grupo endometriose III/IV (5,6%) quando comparado ao grupo endometriose I/II (0%). Observamos uma tendência a menores taxas de fertilização dos oócitos injetados em telófase I quando comparados aos em metáfase II, nos grupos controle (p=0,08), endometriose (p=0,05) e endometriose III/IV (p=0,09). Comparando-se os oócitos com e sem fuso celular visível, não observamos diferença significativa nos resultados de ICSI entre os grupos analisados. Conclusão: Não observamos diferença significativa entre os grupos analisados quanto à visualização e localização do fuso celular em oócitos maturados in vivo com o primeiro CP visível. Todavia, observamos um aumento significativo de oócitos em telófase I nas portadoras de endometriose moderada e severa, sugerindo um retardo ou comprometimento na conclusão da meiose I. Considerando que os oócitos injetados em telófase I apresentam piores taxas de fertilização do que os injetados em metáfase II, este achado poderia justificar o comprometimento dos resultados de reprodução assistida em mulheres inférteis com endometriose moderada e severa, além de ser utilizado com ferramenta prognóstica pós-ICSI. / Introduction: Although it has been a controversial issue for decades, a deleterious role of endometriosis on assisted reproductive techniques (ART) outcomes is questioned, which may be related to oocyte quality. For a mature oocyte be prepared for fertilization is necessary that the meiotic spindle keeps its integrity and its function. Objectives: To compare the presence and localization of the meiotic spindle and the oocyte nuclear maturation with the visible first polar body of infertile patients with and without endometriosis. To compare ICSI outcomes between oocytes on telophase I and metaphase II, and the ones with and without visible meiotic spindle, on those two groups. Methodology: A prospective and controlled study with infertile patients who underwent ovarian stimulation for purposes of ICSI, selected consecutively and divided into two groups: control (tubal and/or male factor) and endometriosis (subdivided in minimum and mild stage I/II versus moderate and severe stage III/IV). The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged using a polarization microscopy immediately before ICSI and characterized according to the presence and localization of meiotic spindle and its relation to the first polar body and the nuclear maturation stage (telophase I and metaphase II). We have analyzed the fertilization rates, clivage, number of good quality embryos on the second (D2) and third (D3) day of development from oocytes on telophase I versus the ones on metaphase II, and metaphase II visible spindle versus the non-visible ones, on the control groups, endometriosis, endometriosis stage I/II and endometriosis stage III/IV. Results: A total of 441 oocytes were analyzed, 254 oocytes form the control group and 187 from the endometriosis one (115 from endometriosis stage I/II and 72 from endometriosis stage III/IV). No significant differences between the percentage of metaphase II with visible and non-visible meiotic spindle were found (88,6%, 91,3%, and 88,2%, in the control, endometriosis I/II and endometriosis III/IV groups, respectively). Among the apparently matured oocytes, we have observed a significant increase of oocytes on telophase I on the endometriosis III/IV group (5,6%) when compared with the endometriosis I/II group (0%). We have observed a tendency to fewer fertilization rates from the injected oocytes on telophase I when compared with the ones on metaphase II, on the control group (p=0,08), endometriosis (p=0,05) and endometriosis III/IV group (p=0,09). When we compared oocytes with and without visible meiotic spindle, we found no significant difference on ICSI outcomes among the studied groups. Conclusions: We have found no significant difference among the studied groups regarding the visualization and localization of the meiotic spindle from in vivo matured oocytes with a visible first polar body. However, we have observed a significant increase on the number of oocytes on telophase I from patients with moderate and severe endometriosis, suggesting a delay or an impairment in the completion of meiosis I. Since the injected oocytes on telophase I present a worse fertilization rates than the ones injected on metaphase II, this finding could explain the impairment on the outcomes of ART in infertile women with moderate and severe endometriosis, besides it could be used as a prognosis tool after ICSI procedures.

Orientador: Milton Carlos Kuga / Resumo: O objetivo do estudo foi avaliar o efeito de agentes remineralizantes à base de fosfato ou silicato de cálcio em formar precipitados e obstruir túbulos dentinários e seus efeitos sobre o colágeno da dentina erodida. Assim mesmo, avaliar seu efeito na resistência de união, formação de camada hibrida e penetrabilidade dentinária do sistema adesivo autocondicionante Clearfil SE Bond. Material e métodos: Trezentos espécimes foram obtidos do segmento cervical de incisivos bovinos e previamente submetidos à erosão dentinária. Os espécimes foram distribuídos em 5 protocolos (n=30): NP, Desensibilize NanoP; RD, MI Paste Plus RecaldentTM; NR, Regenerate NR-5TM; KF, Desensibilize KF 2% e CO, sem tratamento. Cada um dos protocolos foi aplicado no total de 4 sessões, com intervalos de 7 dias. Duzentos espécimes (n=20) foram diariamente submetidos ao desafio ácido, por imersão em suco de laranja, por 5 minutos e, posteriormente mantidos em saliva artificial. Na sequencia, cem espécimes foram analisados em microscopia eletrônica de varredura para avaliar a formação de precipitados sobre a superfície dentinária (500x) e obter o número de túbulos dentinários abertos (2,000x), e em microscopia EDX para obter resultados qualitativos sobre a composição do precitado, e avaliados por meio de microscopia de luz polarizada, com coloração picrosirius red, para qualificar a morfologia do colágeno da dentina após a conclusão do tratamento. Nos duzentos espécimes restantes (n=20), cem sem desafio ácido... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Die rheumatoide Arthritis ist eine chronisch-entzündliche Bindegewebserkrankung mit symmetrischem Befall der Gelenke. Die genaue Ätiologie ist bisher unbekannt. Aktivierte synoviale Fibroblasten sollen durch gesteigerte Adhäsion und Produktion von proinflammatorischen Zytokinen und Matrix-lysierenden Proteasen maßgeblich an der Gelenkdestruktion beteiligt sein. Ziel dieser Arbeit war es, ein neues in-vivo-Knorpeldestruktions-Modell zu etablieren, in welchem unter immunkompetenten Bedingungen, die Invasion und Destruktion von Gelenkknorpel durch die Fibroblasten-Zelllinie LS48 über einen längeren Zeitraum simuliert werden kann. Die am Institut für klinische Immunologie der Medizinischen Fakultät der Universität Leipzig etablierte Zelllinie LS48 wurde in die ipsilateralen Kniegelenke von BALB/c-Mäusen injiziert. Die dadurch induzierte Gewebsdestruktion wurde über zehn Wochen in zweiwöchigem Abstand histopathologisch beurteilt und klassifiziert. Als vergleichende Fibroblasten-Zelllinie wurden nicht-invasive NIH/3T3-Zellen eingesetzt. An Hand der Score-Parameter Zellinvasion, Pannusformation und Knorpeldestruktion wurde eine mäßige bis schwer-wiegende Gewebsdestruktion durch die LS48-Zellen bereits ab der zweiten Untersuchungswoche lichtmikroskopisch nachgewiesen, ohne dass dabei pathologische Effekte in den kontralateralen Kniegelenken aufgetreten sind. Polarisationsmikroskopisch wurden für den Parameter Knorpeldestruktion vergleichbare Ergebnisse erzielt. Damit wurde gezeigt, dass das Modell BALB/c LS48 ein erfolgversprechendes Instrument darstellt, das zur Testung neuer therapeutischer Strategien gegen die Gelenkdestruktion verwendet werden kann. Inwieweit die Auseinandersetzung der LS48-Zellen mit dem spezifischen Immunsystem der BALB/c-Maus Auswirkungen auf den Verlauf der Gewebsdestruktion hat, sollte in weiterführenden Experimenten untersucht werden.