The basis of this investigation is to characterize growth property differences in normal versus virally transformed cells. Using a temperature-sensitive mutant of Rous sarcoma virus, the cells' transformation state is regulated by the growth temperature; at 33°C the cells are transformed, while at 39°C the cells have normal characteristics. The morphology of NRK cells is elongated and fibroblastic; when transformed the cells are rounded. Normal cells grow to a monolayer and stop, while transformed cells grow to saturation densities greater than just a monolayer amount. Transformed cells can form foci when grown in mixture with normal cells. Normal cells must be in contact with the culture vessel in order to grow, but transformed cells lack anchorage dependence for growth.

LA31-NRK and B77-NRK are established cell lines that were normal rat kidney cells transformed with temperature-sensitive mutant (LA31) and wild-type Bratislava 77 (B77) of Rous sarcoma virus. It is recognized that many transformation-induced changes differentiate between normal and transformed cells. Morphology and four parameters of transformed cells such as saturation density, anchorage independence, plasminogen activator, and colony stimulating factor were used as indicators to observe the effect of human alpha interferon on the growth of NRK, LA31-NRK and B77-NRK. The results show that interferon could neither reverse the transformed cells to normal fashion nor change their behaviors or cause release of protease.

Tissue engineered scaffolds were constructed to mimic the native extracellular matrix (ECM) and promote cell migration of keratinocytes and fibroblasts. Electrospinning technology was used to fabricate these nano-scale matrices that consist of varying compositions and fiber diameters. The purpose of this study was to examine how average fiber diameter and scaffold composition regulate cell migration. Odyssey infrared scanning evaluated this on a macroscopic level, whereas confocal microscopy focused on a more microscopic approach. The expression of proteases released into the culture media was also examined. The results from this study suggest that fiber diameter increases as a function of electrospinning starting concentration. Altering the composition by adding a basement membrane-like material, Matrigel, does not statistically affect the average fiber diameter. Fibroblast migration is greater on collagen scaffolds than gelatin scaffolds based on surface area measurements. Confocal images illustrate a distinct cell polarity and various cell morphologies of fibroblasts on electrospun collagen scaffolds. Cell-matrix interactions are more prominent on intermediate to large scale fibers. However, cell-cell contacts are more prevalent at the smallest fiber diameters, suggesting that this scaffold acts like or as a two-dimensional surface. The expression of matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, by fibroblasts during in vivo cell migration assays, suggests that the greatest amount of matrix remodeling is at the two extremes of fiber diameters.

A fermentação alcoólica é susceptível à presença de contaminantes e controlar este problema é um desafio para a indústria produtora de etanol, sejam os contaminantes bacterianos ou leveduras selvagens. As leveduras selvagens ocorrem naturalmente no ambiente e nos substratos utilizados na fermentação e são mais difíceis de controlar do que contaminações por bactérias; podem causar excesso de produção de espuma, levando a perda do teor alcoólico e perda de eficiência fermentativa. Características morfológicas particulares de leveduras Saccharomyces cerevisiae podem exacerbar estes traços indesejáveis para a fermentação. Métodos de isolamento e identificação de leveduras selvagens podem consistir de testes de resistência à temperatura, avaliação por microscopia e a utilização de meios diferenciais sintéticos, que podem ser utilizados no isolamento de leveduras selvagens dos mais diversos substratos e identificação de características morfofisiológicas. O emprego de técnicas moleculares como PCR (Polymerase Chain Reaction) e DNA-fingerprinting também são empregadas, sendo capazes de detectar contaminações em pequenas quantidades, além de permitir a diferenciação a nível intra-específico. Foram avaliados 14 isolados de levedura proveniente de ambiente industrial, utilizando as leveduras CAT-1 e PE-2 como linhagens de referência, sendo avaliadas as características morfofisiológicas através de plaqueamento em meio diferenciais como WLN, BiGGY e Nagai, em conjunto com a análise microscópica das células. Os isolados foram também avaliados através de um teste de resistência de temperatura, testes de capacidade fermentativa e crescimento em microplacas em diversos meios. Por fim, foi extraído o DNA das amostras e este foi avaliado através de reação de PCR e sequenciamento a nível intra-específico. Os resultados mostraram grande variação de padrões morfológicos, presença de células deficientes respiratórias petite e crescimento consistente em temperaturas elevadas, característica esta geralmente presente em leveduras selvagens. Quanto à capacidade fermentativa, 12 dos 14 isolados apresentaram esta característica nos meios utilizados. No crescimento em microplaca, os isolados apresentaram comportamentos distintos em diferentes meios, quando comparados com as linhagens referência. Por fim, a análise molecular das amostras apresentou diferentes padrões de banda entre si e em comparação com as linhagens referência, e o sequenciamento a nível intra-específico mostrou que todos os isolados pertencem a espécie Saccharomyces cerevisiae. / Alcoholic fermentation is susceptible to the presence of contaminants and controlling this problem is a challenge for the ethanol industry, whether it\'s bacterial contaminants or wild yeasts. Wild yeast naturally occur in the environment and on substrates used in fermentation and are more difficult to control than contamination by bacteria. They may cause excessive foaming, leading to loss of the alcoholic yield and decrease of fermentative efficiency. Particular morphological traits of Saccharomyces cerevisiae yeasts may exacerbate these undesirable traits for fermentation. Methods of isolation and identification of wild yeast may consist of temperature resistance tests, evaluation by microscopy and also the use of synthetic differential media, which can be used to isolate wild yeasts from the most diverse substrates and to identify their morphophysiological characteristics. The use of molecular techniques such as PCR (Polymerase Chain Reaction) and DNA-fingerprinting are also employed, being able to detect contaminations in small quantities, besides allowing intra-specific differentiation. A total of 14 yeast isolates from an industrial environment were evaluated, using yeasts CAT-1 and PE-2 as reference strains. Morphophysiological characteristics were evaluated by plating in differential media such as WLN, BiGGY and Nagai, alongside with microscopic analysis of the yeast cells. The isolates were also evaluated according a temperature resistance test, fermentation capacity tests and a microplate growth assay in several media. Finally, the DNA was extracted from the samples and was evaluated through PCR reaction and sequencing at intra-specific level. The results showed a great variation of morphological patterns, presence of petite respiratory deficient cells and consistent growth at high temperatures, a characteristic that is generally present in wild yeasts. Regarding the fermentative capacity, 12 of the 14 isolates showed this trait in the media used. In the microplate growth assay, the isolates presented different behaviors in different media when compared with the reference strains. Finally, the molecular analysis of the samples presented different band patterns among themselves and in comparison with the reference lines, and the intra-specific sequencing showed that all the isolates belong to the species Saccharomyces cerevisiae.

A proposta desse projeto de pesquisa foi analisar e caracterizar células-tronco encontradas na polpa dentária e papila apical de um mesmo doador tratadas sob as mesmas condições de cultivo utilizando abordagens que permitem analises de componentes intracelulares que nunca antes haviam sido analisados nessas células. Para tanto, populações enriquecidas pela expressão CD146, STRO-1 e CD90 foram isoladas de terceiros molares inclusos indicados para exodontia, totalizando 16 pacientes e 16 dentes. Como controles negativos, foram utilizadas as células negativas para esses marcadores. Células positivas e negativas para cada marcador foram comparadas entre si e com os resultados dos outros marcadores. Para cada um dos marcadores e respectivos controles foram realizadas analises de cinética celular, ensaios morfológicos e ensaios subcelulares utilizando microscopia de luz sincrotron. Os dados obtidos foram submetidos à teste ANOVA e teste de Tukey (a=0,05) quando pertinente e/ou a análises descritivas. As células isoladas da polpa dentária e papila apical se comportaram diferentemente uma das outras. Nos ensaios de cinética celular, as células enriquecidas (positivas) apresentaram crescimento mais lento quanto comparados com as células não enriquecidas (negativas), independente do marcador em questão. Nos ensaios morfológicos, células CD 90+ da polpa dentária exibiram a menor área e menor perímetro quando comparadas com as CD 146+ e STRO-1+. A presença de compostos iônicos vistos por luz sincrotron mostraram maior fração de massa nas células positivas da polpa dentária. Dentre os elementos traços estatisticamente mais prevalentes foram o fósforo, cobre, zinco, potássio, estrôncio, cálcio e cloro, sendo este último presente na polpa e papila nos 3 marcadores estudados. Concluímos que tanto da polpa dentária quanto na papila apical de dentes humanos, há presença de células tronco multipotentes expressados pelos três marcadores e que apesar de serem obtidos do mesmo dente e doador e cultivados de forma iguais ela tem comportamentos diferentes. As alterações bioquímicas celulares estudadas pelos elementos traços em células separadas por diferentes marcadores foi o primeiro passo para possibilitar os conhecimentos mecanicistas vitais celulares que não são observadas em microscopia padrão. Porém, novos estudos como permitir a visualização da localização espacial de biomarcadores espectrais caracterizados, pode ajudar a consolidar os resultados aqui encontrados. Assim, a análise e classificação desse método de estudo pode ser refinado em pesquisas futuras, incluindo no uso de outros tipos de tecidos dentais para caracterizar as células tronco dentais. / The purpose of this research project was to analyze and characterize stem cells found in dental pulp and apical papilla from the same donor treated under the same culture conditions using approaches that allow analysis of intracellular components that had never before been analyzed in these cells. Therefore, populations enriched for CD146 expression, STRO-1 and CD90 were isolated from third molars indicated for extraction, totaling 16 patients and 16 teeth. As negative controls, cells negative for these markers were used. Positive and negative cells for each marker were compared and the results of other markers. For each of the markers and their controls were carried out analysis of cell kinetics, morphological tests and subcellular assays using synchrotron light microscopy. The data were submitted to ANOVA and Tukey\'s test (a = 0.05) where relevant and / or descriptive analyzes. Cells isolated from the apical papilla and dental pulp behaved differently from each other. In cell kinetics assays, enriched cells (positive) showed slower growth as compared with non-enriched cells (negative), regardless of the marker in question. In morphological studies, CD 90+ cells in the dental pulp exhibited a smaller area and lower perimeter compared to CD 146+ and STRO-1 +. The presence of ionic compounds seen by synchrotron light showed higher mass fraction of positive cells in the dental pulp. Among the most prevalent statistical trace elements are phosphorous, copper, zinc, potassium, strontium, calcium and chlorine, the latter being present in the pulp, and the papilla 3 markers studied. We conclude that both the dental pulp as the apical papilla of human teeth, there is presence of multipotent stem cells expressed the three markers and that although they are obtained from the same tooth and donor and grown in the same way it has different behaviors. The biochemical cellular changes studied by trace elements in separate cells with different markers was the first step to allow mechanistic cellular vital knowledge that is not observed in standard microscopy. However, new studies as to visualize the spatial location characterized spectral biomarkers can help consolidate the present results. Thus, the analysis and classification of the study method can be refined in future research including the use of other types of dental tissues to characterize the dental stem cell.

A proposta desse projeto de pesquisa foi analisar e caracterizar células-tronco encontradas na polpa dentária e papila apical de um mesmo doador tratadas sob as mesmas condições de cultivo utilizando abordagens que permitem analises de componentes intracelulares que nunca antes haviam sido analisados nessas células. Para tanto, populações enriquecidas pela expressão CD146, STRO-1 e CD90 foram isoladas de terceiros molares inclusos indicados para exodontia, totalizando 16 pacientes e 16 dentes. Como controles negativos, foram utilizadas as células negativas para esses marcadores. Células positivas e negativas para cada marcador foram comparadas entre si e com os resultados dos outros marcadores. Para cada um dos marcadores e respectivos controles foram realizadas analises de cinética celular, ensaios morfológicos e ensaios subcelulares utilizando microscopia de luz sincrotron. Os dados obtidos foram submetidos à teste ANOVA e teste de Tukey (a=0,05) quando pertinente e/ou a análises descritivas. As células isoladas da polpa dentária e papila apical se comportaram diferentemente uma das outras. Nos ensaios de cinética celular, as células enriquecidas (positivas) apresentaram crescimento mais lento quanto comparados com as células não enriquecidas (negativas), independente do marcador em questão. Nos ensaios morfológicos, células CD 90+ da polpa dentária exibiram a menor área e menor perímetro quando comparadas com as CD 146+ e STRO-1+. A presença de compostos iônicos vistos por luz sincrotron mostraram maior fração de massa nas células positivas da polpa dentária. Dentre os elementos traços estatisticamente mais prevalentes foram o fósforo, cobre, zinco, potássio, estrôncio, cálcio e cloro, sendo este último presente na polpa e papila nos 3 marcadores estudados. Concluímos que tanto da polpa dentária quanto na papila apical de dentes humanos, há presença de células tronco multipotentes expressados pelos três marcadores e que apesar de serem obtidos do mesmo dente e doador e cultivados de forma iguais ela tem comportamentos diferentes. As alterações bioquímicas celulares estudadas pelos elementos traços em células separadas por diferentes marcadores foi o primeiro passo para possibilitar os conhecimentos mecanicistas vitais celulares que não são observadas em microscopia padrão. Porém, novos estudos como permitir a visualização da localização espacial de biomarcadores espectrais caracterizados, pode ajudar a consolidar os resultados aqui encontrados. Assim, a análise e classificação desse método de estudo pode ser refinado em pesquisas futuras, incluindo no uso de outros tipos de tecidos dentais para caracterizar as células tronco dentais. / The purpose of this research project was to analyze and characterize stem cells found in dental pulp and apical papilla from the same donor treated under the same culture conditions using approaches that allow analysis of intracellular components that had never before been analyzed in these cells. Therefore, populations enriched for CD146 expression, STRO-1 and CD90 were isolated from third molars indicated for extraction, totaling 16 patients and 16 teeth. As negative controls, cells negative for these markers were used. Positive and negative cells for each marker were compared and the results of other markers. For each of the markers and their controls were carried out analysis of cell kinetics, morphological tests and subcellular assays using synchrotron light microscopy. The data were submitted to ANOVA and Tukey\'s test (a = 0.05) where relevant and / or descriptive analyzes. Cells isolated from the apical papilla and dental pulp behaved differently from each other. In cell kinetics assays, enriched cells (positive) showed slower growth as compared with non-enriched cells (negative), regardless of the marker in question. In morphological studies, CD 90+ cells in the dental pulp exhibited a smaller area and lower perimeter compared to CD 146+ and STRO-1 +. The presence of ionic compounds seen by synchrotron light showed higher mass fraction of positive cells in the dental pulp. Among the most prevalent statistical trace elements are phosphorous, copper, zinc, potassium, strontium, calcium and chlorine, the latter being present in the pulp, and the papilla 3 markers studied. We conclude that both the dental pulp as the apical papilla of human teeth, there is presence of multipotent stem cells expressed the three markers and that although they are obtained from the same tooth and donor and grown in the same way it has different behaviors. The biochemical cellular changes studied by trace elements in separate cells with different markers was the first step to allow mechanistic cellular vital knowledge that is not observed in standard microscopy. However, new studies as to visualize the spatial location characterized spectral biomarkers can help consolidate the present results. Thus, the analysis and classification of the study method can be refined in future research including the use of other types of dental tissues to characterize the dental stem cell.

<p>One of the fields where image processing finds its application but that remains as anunexplored territory is the analysis of cell morphology. This master thesis proposes a systemto carry out this research and sets the necessary technical basis to make it feasible, rangingfrom the processing of time-lapse sequences using image segmentation to the representation,description and classification of cells in terms of morphology.</p><p>Due to the highly variability of cell morphological characteristics several segmentationmethods have been implemented to face each of the problems encountered: Edge-detection,region-growing and marked watershed were found to be successful processing algorithms.This variability inherent to cells and the fact that human eye has a natural disposition to solvesegmentation problems finally lead to the development of a user-friendly interactiveapplication, the <em>Time Lapse Sequence Processor</em> (TLSP). Although it was initially consideredas a mere interface to perform cell segmentation, TLSP concept has evolved into theconstruction of a complete multifunction tool to perform cell morphology analysis:segmentation, morphological data extraction, analysis and management, cell tracking andrecognition system, etc. In its last version, TLSP v0.2 Alpha contains several segmentationtools, improved user interface and, data extraction and management capabilities.</p><p>Finally, a wide set of recommendations and improvements have been discussed, pointing the path for future development in this area.</p>

One of the fields where image processing finds its application but that remains as anunexplored territory is the analysis of cell morphology. This master thesis proposes a systemto carry out this research and sets the necessary technical basis to make it feasible, rangingfrom the processing of time-lapse sequences using image segmentation to the representation,description and classification of cells in terms of morphology. Due to the highly variability of cell morphological characteristics several segmentationmethods have been implemented to face each of the problems encountered: Edge-detection,region-growing and marked watershed were found to be successful processing algorithms.This variability inherent to cells and the fact that human eye has a natural disposition to solvesegmentation problems finally lead to the development of a user-friendly interactiveapplication, the Time Lapse Sequence Processor (TLSP). Although it was initially consideredas a mere interface to perform cell segmentation, TLSP concept has evolved into theconstruction of a complete multifunction tool to perform cell morphology analysis:segmentation, morphological data extraction, analysis and management, cell tracking andrecognition system, etc. In its last version, TLSP v0.2 Alpha contains several segmentationtools, improved user interface and, data extraction and management capabilities. Finally, a wide set of recommendations and improvements have been discussed, pointing the path for future development in this area.

Tendons, essential tissues that connect muscles to bones, are susceptible to rupture/degeneration due to their continuous use for enabling movement. Often surgical intervention is required to repair the tendon; relieving the pain and fixing the limited mobility that occurs from the damage. Unfortunately, post-surgery immobilization techniques required to restore tendon properties frequently lead to scar formation and reduced tendon range of motion. Our ultimate goal is to create an optimal tendon prosthetic that can stabilize the damaged muscle-bone connection and then be remodeled by resident cells from the surrounding tissues over time to ensure long-term function. To achieve this, we must first understand how cells respond to and interact with candidate replacement materials. The most abundant extracellular matrix (ECM) protein found in the body, collagen, is chosen as the replacement material because it makes up the majority of tendon dry mass and it can be remodeled by cell-based homeostatic processes. Previous studies found that Di-catechol nordihydroguaiaretic acid (NDGA) cross-linked fibers have greater mechanical strength than native tendons; and for this reason this biomaterial could be used for tendon replacement. This work focuses on investigating the behavior of fibroblasts on NDGA cross-linked and uncross-linked collagen samples to determine if cross-linking disrupts the cell binding sites affecting cell spreading, attachment, and migration. The in-vitro platform was designed by plasma treating 25 mm diameter cover slips that were exposed to 3-aminopropyl-trimetoxysilane/toluene and glutaraldehyde/ethanol solutions. The collagen solution was then dispensed onto the glutaraldehyde-coated cover slip and incubated for fibrillar collagen matrix formation. The collagen matrices were submerged in NDGA cross-linking solution for 24 hours to ensure the surface was completely cross-linked. Collagen films were made by allowing the uncross-linked gels to dry overnight before and after NDGA treatment, resulting in a more compacted structure. A spinning disk device was employed to quantify the ability of cells to remain attached to the collagen samples when exposed to hydrodynamic forces. To avoid any cell-cell interaction and focus on cell-surface interactions, 50-100 cells/mm2 were seeded carefully on each sample. Temporal studies demonstrated that cell adhesion strength and spreading area reached steady-state by 4 hr. Adhesion and spreading studies along with migration experiments demonstrated that NDGA treatment affects cellular behavior on films, partially reducing adhesion strength, migration, and spreading area. However, on the cross-linked gels which are less dense, the only change in cell behavior observed was in migration speed. We hypothesize that these differences are due to the collapsing of the collagen films. This compaction suggests a less open organization and could be allowing the collagen fibers to form more inter-chain bonds as well as bonds with the small NDGA cross-linker; while NDGA treatment of the fully hydrated gels may rely more on NDGA polymerization to span the greater distance between collagen fibrils. From these results, we can determine that the chemical/physical masking of the adhesion sites by NDGA on collagen films affects cellular behavior more than the masking that occurs in the cross-linked gels. Although this study shows an effect in cell behavior on the cross-linked films, it also demonstrates that cells can adhere and migrate to this NDGA biomaterial supporting the idea that this biomaterial can be utilized for tendon replacement.

A fermentação alcoólica é susceptível à presença de contaminantes e controlar este problema é um desafio para a indústria produtora de etanol, sejam os contaminantes bacterianos ou leveduras selvagens. As leveduras selvagens ocorrem naturalmente no ambiente e nos substratos utilizados na fermentação e são mais difíceis de controlar do que contaminações por bactérias; podem causar excesso de produção de espuma, levando a perda do teor alcoólico e perda de eficiência fermentativa. Características morfológicas particulares de leveduras Saccharomyces cerevisiae podem exacerbar estes traços indesejáveis para a fermentação. Métodos de isolamento e identificação de leveduras selvagens podem consistir de testes de resistência à temperatura, avaliação por microscopia e a utilização de meios diferenciais sintéticos, que podem ser utilizados no isolamento de leveduras selvagens dos mais diversos substratos e identificação de características morfofisiológicas. O emprego de técnicas moleculares como PCR (Polymerase Chain Reaction) e DNA-fingerprinting também são empregadas, sendo capazes de detectar contaminações em pequenas quantidades, além de permitir a diferenciação a nível intra-específico. Foram avaliados 14 isolados de levedura proveniente de ambiente industrial, utilizando as leveduras CAT-1 e PE-2 como linhagens de referência, sendo avaliadas as características morfofisiológicas através de plaqueamento em meio diferenciais como WLN, BiGGY e Nagai, em conjunto com a análise microscópica das células. Os isolados foram também avaliados através de um teste de resistência de temperatura, testes de capacidade fermentativa e crescimento em microplacas em diversos meios. Por fim, foi extraído o DNA das amostras e este foi avaliado através de reação de PCR e sequenciamento a nível intra-específico. Os resultados mostraram grande variação de padrões morfológicos, presença de células deficientes respiratórias petite e crescimento consistente em temperaturas elevadas, característica esta geralmente presente em leveduras selvagens. Quanto à capacidade fermentativa, 12 dos 14 isolados apresentaram esta característica nos meios utilizados. No crescimento em microplaca, os isolados apresentaram comportamentos distintos em diferentes meios, quando comparados com as linhagens referência. Por fim, a análise molecular das amostras apresentou diferentes padrões de banda entre si e em comparação com as linhagens referência, e o sequenciamento a nível intra-específico mostrou que todos os isolados pertencem a espécie Saccharomyces cerevisiae. / Alcoholic fermentation is susceptible to the presence of contaminants and controlling this problem is a challenge for the ethanol industry, whether it\'s bacterial contaminants or wild yeasts. Wild yeast naturally occur in the environment and on substrates used in fermentation and are more difficult to control than contamination by bacteria. They may cause excessive foaming, leading to loss of the alcoholic yield and decrease of fermentative efficiency. Particular morphological traits of Saccharomyces cerevisiae yeasts may exacerbate these undesirable traits for fermentation. Methods of isolation and identification of wild yeast may consist of temperature resistance tests, evaluation by microscopy and also the use of synthetic differential media, which can be used to isolate wild yeasts from the most diverse substrates and to identify their morphophysiological characteristics. The use of molecular techniques such as PCR (Polymerase Chain Reaction) and DNA-fingerprinting are also employed, being able to detect contaminations in small quantities, besides allowing intra-specific differentiation. A total of 14 yeast isolates from an industrial environment were evaluated, using yeasts CAT-1 and PE-2 as reference strains. Morphophysiological characteristics were evaluated by plating in differential media such as WLN, BiGGY and Nagai, alongside with microscopic analysis of the yeast cells. The isolates were also evaluated according a temperature resistance test, fermentation capacity tests and a microplate growth assay in several media. Finally, the DNA was extracted from the samples and was evaluated through PCR reaction and sequencing at intra-specific level. The results showed a great variation of morphological patterns, presence of petite respiratory deficient cells and consistent growth at high temperatures, a characteristic that is generally present in wild yeasts. Regarding the fermentative capacity, 12 of the 14 isolates showed this trait in the media used. In the microplate growth assay, the isolates presented different behaviors in different media when compared with the reference strains. Finally, the molecular analysis of the samples presented different band patterns among themselves and in comparison with the reference lines, and the intra-specific sequencing showed that all the isolates belong to the species Saccharomyces cerevisiae.