Intracellular ice formation (IIF), a major cause of cryoinjury in biological cells, is significantly more pronounced during freezing of tissue than during freezing of suspended cells. While extensive studies of IIF have been conducted for single cells in suspension, few have investigated IIF in tissue. Due to the increased complexity that arises from both cell-substrate and cell-cell interactions in tissue, knowledge of cryobiology of isolated cells cannot simply be extrapolated to tissue. Different theories have been hypothesized for the mechanisms of IIF in tissue, but none have been conclusively proven. Towards the goal of developing mathematical models to accurately predict the probability of IIF in tissues of one or more cell types, we have developed a novel high-speed video cryomicroscopy system capable of image acquisition at sampling rates up to 32,000 Hz. Specifically, the effects of cell adhesion to the substrate and cell-cell interactions were investigated with experimental (micropatterned endothelial cell constructs) and mathematical models (Monte Carlo simulations). We have reported the first direct observations of the IIF process recorded at unprecedented sub-millisecond and sub-micron resolution. For the majority of our experiments, IIF nucleation was determined to occur preferentially at the cell perimeter. This observation was not consistent with the commonly accepted hypotheses of ice nucleation in suspended cells and suggests that an alternative mechanism of IIF initiation is dominant in adherent cells. In addition, the kinetics of ice nucleation were shown to be influenced by time in culture, attached cell perimeter, fibronectin coating density, and degree of cell-cell contact. Moreover, an additional phenomenon, paracellular ice penetration was identified, and the frequency of formation was correlated with focal adhesion formation. The data and mathematical models presented in this thesis bring closer the goal of elucidating the primary mechanisms contributing to IIF in tissue; providing important contributions to both the fields of cryopreservation (minimizing IIF) and cryosurgery (maximizing IIF).

O Toxoplasma gondii é um parasito coccídio que se localiza intracelularmente em vários órgãos e tecidos de uma ampla gama de hospedeiros. O estudo da soroepidemiologia, deste parasito, na espécie felina é de grande relevância, pois o estreito convívio de seres humanos com esses animais pode acarretar na transmissão de algumas doenças como a toxoplasmose. Com o objetivo de contribuir com dados sobre a freqüência de anticorpos para Toxoplasma gondii em felinos domiciliados da cidade de Porto Alegre, os soros desses animais foram avaliados pelas técnicas de Hemaglutinação Indireta (HAI) e Reação de Imunofluorescência Indireta (RIFI). A freqüência de anticorpos de T. gondii para a amostragem em 245 soros felinos foi de 26,94% pela técnica de HAI e 37,96% pela técnica de RIFI. Dados epidemiológicos foram incluídos no trabalho, como gênero, raça, idade, acesso ou não à rua e tipo de alimentação. Estes parâmetros foram analisados estatisticamente para mensurar suas influências nos resultados obtidos com os testes. A percentagem de co-positividade e co-negatividade nas duas técnicas foi de 56% e 90%, respectivamente, e uma percentagem de concordância total de 77,5%, enquanto que o valor Kappa foi de 0.49. Este estudo mostra que os valores encontrados são relativamente altos, levando-nos a crer, que estes felinos, em algum momento de sua existência poderiam ser fonte de contaminação ambiental, como potenciais eliminadores de oocistos, principalmente aqueles que têm livre acesso à rua. / Toxoplasma gondii is an intracellular coccidian parasite that infects several organs and tissues in a large variety of hosts. Its seroepidemiology in feline species is of great value since the close relationship between human beings and cats may serve as a vector for the transmission of some diseases such as toxoplasmosis. The sera of 245 cats from Porto Alegre, southern Brazil, were submitted to indirect hemagglutination antibody (IHA) test and to indirect immunofluorescence (IIF) assay in order to determine the frequency of antibodies against Toxoplasma gondii. The IHA test showed that 26.94% of the cats had antibodies against Toxoplasma gondii compared to 37.96% in the IIF assay. Epidemiological data such as gender, race, age, access or not to the street and eating behavior were assessed. These parameters were statistically analyzed to measure the influence on test results. The co-positive and co-negative values amounted to 56 and 90% for the IHA test and IIF assay, respectively, yielding an overall agreement of 77.5% and a kappa coefficient of 0.49. The rates obtained by this study are relatively high, leading us to the assumption that these cats, mainly those with free access to the street, could be a source of environmental contamination, due to oocyst shedding, at some time over the course of their lifetime.

O Toxoplasma gondii é um parasito coccídio que se localiza intracelularmente em vários órgãos e tecidos de uma ampla gama de hospedeiros. O estudo da soroepidemiologia, deste parasito, na espécie felina é de grande relevância, pois o estreito convívio de seres humanos com esses animais pode acarretar na transmissão de algumas doenças como a toxoplasmose. Com o objetivo de contribuir com dados sobre a freqüência de anticorpos para Toxoplasma gondii em felinos domiciliados da cidade de Porto Alegre, os soros desses animais foram avaliados pelas técnicas de Hemaglutinação Indireta (HAI) e Reação de Imunofluorescência Indireta (RIFI). A freqüência de anticorpos de T. gondii para a amostragem em 245 soros felinos foi de 26,94% pela técnica de HAI e 37,96% pela técnica de RIFI. Dados epidemiológicos foram incluídos no trabalho, como gênero, raça, idade, acesso ou não à rua e tipo de alimentação. Estes parâmetros foram analisados estatisticamente para mensurar suas influências nos resultados obtidos com os testes. A percentagem de co-positividade e co-negatividade nas duas técnicas foi de 56% e 90%, respectivamente, e uma percentagem de concordância total de 77,5%, enquanto que o valor Kappa foi de 0.49. Este estudo mostra que os valores encontrados são relativamente altos, levando-nos a crer, que estes felinos, em algum momento de sua existência poderiam ser fonte de contaminação ambiental, como potenciais eliminadores de oocistos, principalmente aqueles que têm livre acesso à rua. / Toxoplasma gondii is an intracellular coccidian parasite that infects several organs and tissues in a large variety of hosts. Its seroepidemiology in feline species is of great value since the close relationship between human beings and cats may serve as a vector for the transmission of some diseases such as toxoplasmosis. The sera of 245 cats from Porto Alegre, southern Brazil, were submitted to indirect hemagglutination antibody (IHA) test and to indirect immunofluorescence (IIF) assay in order to determine the frequency of antibodies against Toxoplasma gondii. The IHA test showed that 26.94% of the cats had antibodies against Toxoplasma gondii compared to 37.96% in the IIF assay. Epidemiological data such as gender, race, age, access or not to the street and eating behavior were assessed. These parameters were statistically analyzed to measure the influence on test results. The co-positive and co-negative values amounted to 56 and 90% for the IHA test and IIF assay, respectively, yielding an overall agreement of 77.5% and a kappa coefficient of 0.49. The rates obtained by this study are relatively high, leading us to the assumption that these cats, mainly those with free access to the street, could be a source of environmental contamination, due to oocyst shedding, at some time over the course of their lifetime.

O Toxoplasma gondii é um parasito coccídio que se localiza intracelularmente em vários órgãos e tecidos de uma ampla gama de hospedeiros. O estudo da soroepidemiologia, deste parasito, na espécie felina é de grande relevância, pois o estreito convívio de seres humanos com esses animais pode acarretar na transmissão de algumas doenças como a toxoplasmose. Com o objetivo de contribuir com dados sobre a freqüência de anticorpos para Toxoplasma gondii em felinos domiciliados da cidade de Porto Alegre, os soros desses animais foram avaliados pelas técnicas de Hemaglutinação Indireta (HAI) e Reação de Imunofluorescência Indireta (RIFI). A freqüência de anticorpos de T. gondii para a amostragem em 245 soros felinos foi de 26,94% pela técnica de HAI e 37,96% pela técnica de RIFI. Dados epidemiológicos foram incluídos no trabalho, como gênero, raça, idade, acesso ou não à rua e tipo de alimentação. Estes parâmetros foram analisados estatisticamente para mensurar suas influências nos resultados obtidos com os testes. A percentagem de co-positividade e co-negatividade nas duas técnicas foi de 56% e 90%, respectivamente, e uma percentagem de concordância total de 77,5%, enquanto que o valor Kappa foi de 0.49. Este estudo mostra que os valores encontrados são relativamente altos, levando-nos a crer, que estes felinos, em algum momento de sua existência poderiam ser fonte de contaminação ambiental, como potenciais eliminadores de oocistos, principalmente aqueles que têm livre acesso à rua. / Toxoplasma gondii is an intracellular coccidian parasite that infects several organs and tissues in a large variety of hosts. Its seroepidemiology in feline species is of great value since the close relationship between human beings and cats may serve as a vector for the transmission of some diseases such as toxoplasmosis. The sera of 245 cats from Porto Alegre, southern Brazil, were submitted to indirect hemagglutination antibody (IHA) test and to indirect immunofluorescence (IIF) assay in order to determine the frequency of antibodies against Toxoplasma gondii. The IHA test showed that 26.94% of the cats had antibodies against Toxoplasma gondii compared to 37.96% in the IIF assay. Epidemiological data such as gender, race, age, access or not to the street and eating behavior were assessed. These parameters were statistically analyzed to measure the influence on test results. The co-positive and co-negative values amounted to 56 and 90% for the IHA test and IIF assay, respectively, yielding an overall agreement of 77.5% and a kappa coefficient of 0.49. The rates obtained by this study are relatively high, leading us to the assumption that these cats, mainly those with free access to the street, could be a source of environmental contamination, due to oocyst shedding, at some time over the course of their lifetime.

In an effort to minimize the harmful effects of intracellular ice formation (IIF) during cryopreservation of confluent tissues, computer simulations based on Monte Carlo methods were performed to predict the probability of IIF in confluent monolayers during various freezing procedures. To overcome the prohibitive computational costs of such simulations for large tissues, the well-known Johnson-Mehl-Avrami (JMA) model of crystallization kinetics was implemented as a continuum approximation of IIF in tissues. This model, which describes nucleation, growth, and impingement of crystals in a supercooled melt, is analogous to the process of intracellular ice formation and propagation in biological tissues. Based on the work of Weinberg and Kapral (1989), the JMA model was modified to account for finite-size effects, and was shown to predict accurately the results of freezing simulations in 1-D tissue constructs, for various propagation rates and tissue sizes. An initial analysis of IIF kinetics in 2-D tissues is also presented. The probability of IIF in 2-D liver tissue was measured experimentally during freezing of HepG2 cells cultured in monolayers, and compared to Monte Carlo simulations and predictions of the continuum model. The Avrami coefficient and exponent for IIF in HepG2 tissue were estimated to be k = 0.19 and n = 0.45.

The L5 signal of the Global Positioning System (GPS) is becoming available on an increasing number of Block IIF satellites. As the third civilian signal, L5 is superior in signal design to the L1 C/A and L2C civilian signals. This new signal has been marked healthy for use on selected satellites since 2010, yet the hardware capable of tracking the L5 signal is still in the early stages of development. This work investigates the characteristics of the new signal and the quality of data produced by L5-tracking receivers. Commonly used receiver models chosen for this study are the Leica GRX1200+GNSS, the Trimble NetR8, and the Javad Delta TRE-G3TH. The metrics used in this analysis to assess the quality of data produced by these receivers are signal strength, receiver phase noise, receiver code noise, and multipath. The data used in these analyses were obtained from the International GNSS Service for the days of the year 275 to 281 in 2011. Metrics averaged over the GPS week 1656 provide a good indication of the overall performance of the receivers. / text

Grâce à un grand nombre d’études biochimiques, génétiques et structurales effectuées dans les dernières années, des avancements considérables ont été réalisés et une nouvelle vision du processus par lequel la machinerie transcriptionnelle de l’ARN polymérase II (Pol II) décode l’information génétique a émergé. De nouveaux indices ont été apportés sur la diversité des mécanismes de régulation de la transcription, ainsi que sur le rôle des facteurs généraux de transcription (GTFs) dans cette diversification. Les travaux présentés dans cette thèse amènent de nouvelles connaissances sur le rôle des GTFs humains dans la régulation des différentes étapes de la transcription. Dans la première partie de la thèse, nous avons analysé la fonction de la Pol II et des GTFs humains, en examinant de façon systématique leur localisation génomique. Les patrons obtenus par immunoprécipitation de la chromatine (ChIP) des versions de GTFs portant une étiquette TAP (Tandem-Affinity Purification) indiquent de nouvelles fonctions in vivo pour certains composants de cette machinerie et pour des éléments structuraux de la Pol II. Nos résultats suggèrent que TFIIF et l’hétérodimère Rpb4–Rpb7 ont une fonction spécifique pendant l’étape d’élongation transcriptionnelle in vivo. De plus, notre étude amène une première image globale de la fonction des GTFs pendant la réaction transcriptionnelle dans des cellules mammifères vivantes. Deuxièmement, nous avons identifié une nouvelle fonction de TFIIS dans la régulation de CDK9, la sous-unité kinase du facteur P-TEFb (Positive Transcription Elongation Factor b). Nous avons identifié deux nouveaux partenaires d’interaction pour TFIIS, soit CDK9 et la E3 ubiquitine ligase UBR5. Nous montrons que UBR5 catalyse l’ubiquitination de CDK9 in vitro. De plus, la polyubiquitination de CDK9 dans des cellules humaines est dépendante de UBR5 et TFIIS. Nous montrons aussi que UBR5, CDK9 and TFIIS co-localisent le long du gène  fibrinogen (FBG) et que la surexpression de TFIIS augmente les niveaux d’occupation par CDK9 de régions spécifiques de ce gène, de façon dépendante de UBR5. Nous proposons que TFIIS a une nouvelle fonction dans la transition entre les étapes d’initiation et d’élongation transcriptionnelle, en régulant la stabilité des complexes CDK9-Pol II pendant les étapes précoces de la transcription. / Biochemical, genetic and structural studies made over the last years bring a new view on the RNA polymerase II (Pol II) machinery and the process by which it decodes the genetic information. They provided new insights into the diversity of the transcriptional regulation mechanisms, and on the role played by the general transcription factors (GTFs). The studies presented in this thesis provide new evidence on the role of human GTFs in the regulation of different stages of transcription. In the first part of the thesis, we investigated the function of the human Pol II and GTFs in living cells, by systematically analyzing their genomic location. The location profiles obtained by chromatin immunoprecipitation (ChIP) of TAP (tandem-affinity purification) tagged versions of these factors indicate new in vivo functions for several components of this machinery, and for structural elements of the Pol II. These results suggest that TFIIF and the heterodimer Rpb4–Rpb7 have a specific function during the elongation stage in vivo. Additionally, our study offers for the first time a general picture of GTFs function during the Pol II transcription reaction in live mammalian cells, and provides a framework to uncover new regulatory hubs. Secondly, we report on the identification of a new function of the factor TFIIS in the regulation of CDK9, the kinase subunit of the Positive Transcription Elongation Factor b (P-TEFb). We identify two interaction partners for TFIIS, namely CDK9 and the E3 ubiquitin ligase UBR5. We show that UBR5 catalyzes the ubiquitination of CDK9 in vitro. Moreover, the polyubiquitination of CDK9 in human cells is dependent upon both UBR5 and TFIIS, and does not signal its degradation. We also show that UBR5, CDK9 and TFIIS co-localize along specific regions of the  fibrinogen (FBG) gene, and that the overexpression of TFIIS increases the occupancy of CDK9 along this gene in a UBR5 dependant manner. We propose a new function of TFIIS in the transition between initiation and elongation stages, by regulating the stability of the early CDK9-Pol II transcribing complexes. Key words: chromatin immunoprecipitation, general transcription factors, tandem-affinity purification, RNA polymerase II, Rpb4–Rpb7 heterodimer, transcription factor IIF (TFIIF), transcription factor IIS (TFIIS), UBR5 ubiquitin ligase, Positive Transcription Elongation Factor b (P-TEFb), CDK9 ubiquitination.

Grâce à un grand nombre d’études biochimiques, génétiques et structurales effectuées dans les dernières années, des avancements considérables ont été réalisés et une nouvelle vision du processus par lequel la machinerie transcriptionnelle de l’ARN polymérase II (Pol II) décode l’information génétique a émergé. De nouveaux indices ont été apportés sur la diversité des mécanismes de régulation de la transcription, ainsi que sur le rôle des facteurs généraux de transcription (GTFs) dans cette diversification. Les travaux présentés dans cette thèse amènent de nouvelles connaissances sur le rôle des GTFs humains dans la régulation des différentes étapes de la transcription. Dans la première partie de la thèse, nous avons analysé la fonction de la Pol II et des GTFs humains, en examinant de façon systématique leur localisation génomique. Les patrons obtenus par immunoprécipitation de la chromatine (ChIP) des versions de GTFs portant une étiquette TAP (Tandem-Affinity Purification) indiquent de nouvelles fonctions in vivo pour certains composants de cette machinerie et pour des éléments structuraux de la Pol II. Nos résultats suggèrent que TFIIF et l’hétérodimère Rpb4–Rpb7 ont une fonction spécifique pendant l’étape d’élongation transcriptionnelle in vivo. De plus, notre étude amène une première image globale de la fonction des GTFs pendant la réaction transcriptionnelle dans des cellules mammifères vivantes. Deuxièmement, nous avons identifié une nouvelle fonction de TFIIS dans la régulation de CDK9, la sous-unité kinase du facteur P-TEFb (Positive Transcription Elongation Factor b). Nous avons identifié deux nouveaux partenaires d’interaction pour TFIIS, soit CDK9 et la E3 ubiquitine ligase UBR5. Nous montrons que UBR5 catalyse l’ubiquitination de CDK9 in vitro. De plus, la polyubiquitination de CDK9 dans des cellules humaines est dépendante de UBR5 et TFIIS. Nous montrons aussi que UBR5, CDK9 and TFIIS co-localisent le long du gène  fibrinogen (FBG) et que la surexpression de TFIIS augmente les niveaux d’occupation par CDK9 de régions spécifiques de ce gène, de façon dépendante de UBR5. Nous proposons que TFIIS a une nouvelle fonction dans la transition entre les étapes d’initiation et d’élongation transcriptionnelle, en régulant la stabilité des complexes CDK9-Pol II pendant les étapes précoces de la transcription. / Biochemical, genetic and structural studies made over the last years bring a new view on the RNA polymerase II (Pol II) machinery and the process by which it decodes the genetic information. They provided new insights into the diversity of the transcriptional regulation mechanisms, and on the role played by the general transcription factors (GTFs). The studies presented in this thesis provide new evidence on the role of human GTFs in the regulation of different stages of transcription. In the first part of the thesis, we investigated the function of the human Pol II and GTFs in living cells, by systematically analyzing their genomic location. The location profiles obtained by chromatin immunoprecipitation (ChIP) of TAP (tandem-affinity purification) tagged versions of these factors indicate new in vivo functions for several components of this machinery, and for structural elements of the Pol II. These results suggest that TFIIF and the heterodimer Rpb4–Rpb7 have a specific function during the elongation stage in vivo. Additionally, our study offers for the first time a general picture of GTFs function during the Pol II transcription reaction in live mammalian cells, and provides a framework to uncover new regulatory hubs. Secondly, we report on the identification of a new function of the factor TFIIS in the regulation of CDK9, the kinase subunit of the Positive Transcription Elongation Factor b (P-TEFb). We identify two interaction partners for TFIIS, namely CDK9 and the E3 ubiquitin ligase UBR5. We show that UBR5 catalyzes the ubiquitination of CDK9 in vitro. Moreover, the polyubiquitination of CDK9 in human cells is dependent upon both UBR5 and TFIIS, and does not signal its degradation. We also show that UBR5, CDK9 and TFIIS co-localize along specific regions of the  fibrinogen (FBG) gene, and that the overexpression of TFIIS increases the occupancy of CDK9 along this gene in a UBR5 dependant manner. We propose a new function of TFIIS in the transition between initiation and elongation stages, by regulating the stability of the early CDK9-Pol II transcribing complexes. Key words: chromatin immunoprecipitation, general transcription factors, tandem-affinity purification, RNA polymerase II, Rpb4–Rpb7 heterodimer, transcription factor IIF (TFIIF), transcription factor IIS (TFIIS), UBR5 ubiquitin ligase, Positive Transcription Elongation Factor b (P-TEFb), CDK9 ubiquitination.