Ce travail de thèse porte sur l’étude de la réplication chez les archées, qui constituent le troisième domaine du vivant avec les bactéries et les eucaryotes. L’organisme modèle que nous avons utilisé est l'archée halophile Haloferax volcanii car les outils génétiques disponibles permettent d’exprimer des protéines fusionnées à la Protéine Fluorescente Verte (GFP) dans cet organisme mésophile et aérobe et ainsi de localiser les protéines d’intérêt dans des cellules vivantes. Nous nous sommes ainsi intéressés à la localisation cellulaire de quatre protéines de la réplication qui ont été fusionnées à la GFP et exprimées sous contrôle de leur propre promoteur : (i) la protéine ‘Flap Endonuclease 1’ (FEN1), qui intervient dans la maturation des fragments d’Okazaki, (ii) la protéine ‘Origin Recognition Complex’ (ORC1) impliquée dans la reconnaissance des origines de réplication, (iii) la protéine ‘Proliferating Cellular Nuclear Antigen’ (PCNA), anneau de processivité des ADN polymérases réplicatives, et (iv) la protéine de fixation à l’ADN simple-brin ‘Replication Protein A’ (RPA2) essentielle à la réplication chez H. volcanii. Seule la protéine PCNA n’a pu être exprimée en fusion avec la GFP, suggérant que la protéine fusion n’est pas fonctionnelle. GFP::Orc1 et GFP::Fen1 ont été exprimées dans la cellule mais ne présentent pas de localisation spécifique reflétant un rôle de ces protéines dans la réplication de l’ADN. En revanche des foyers de fluorescence de la protéine fusion GFP::Rpa2 ont été observés, dont le nombre augmente significativement dans des cellules exposées à l’aphidicoline, drogue inhibant la synthèse de l’ADN et induisant ainsi un stress réplicatif. Cependant une localisation différente de la protéine GFP::Rpa2 a été observée lorsque les cellules sont exposés à la phléomycine, qui induit notamment des cassures double-brin de l‘ADN. Dans ces cellules, GFP::Rpa2 forme un foyer de fluorescence massif qui colocalise avec l’ADN compacté dans la grande majorité des cellules observées. Nos résultats suggèrent donc que la localisation spécifique observée pour GFP::Rpa2 reflète son rôle dans la réparation de l’ADN et/ou le redémarrage des fourche de réplication arrêtées. / The aim of this thesis project was to improve our understanding of DNA replication in archaea, the third domain of life with bacteria and eukarya. The model organism chosen for these studies is the halophilic archaea Haloferax volcanii, a mesophilic aerobe for which genetics tools allow studying in living cells the localization of proteins fused to the Green Fluorescent protein (GFP). Four proteins involved in DNA replication were fused to the GFP and expressed under the control of their own promoter: (i) the ‘Flap Endonuclease 1’ (FEN1), involved in Okazaki fragments maturation, (ii) the ‘Origin Recognition Complex’ (ORC1), involved in DNA replication origin recognition, (iii) the ‘Proliferating Cellular Nuclear Antigen’ (PCNA), processivity factor of replicative DNA polymerases, and (iv) the ‘Replication Protein A’ (RPA2), single-stranded DNA binding protein essential for DNA replication in H. volcanii. Only the PCNA fusion to the GFP was not successful, suggesting that the GFP hinders essential roles of PCNA in DNA replication. Fen1 and Orc1 were successfully fused to the GFP and expressed in living cells, but specific localization in cells related to growth phase, reflecting different replication dynamics, were not observed. In contrast, we could observed fluorescent foci formed by the fully functional GFP::Rpa2 protein that actively responded to DNA damage in H. volcanii cells. The number of these fluorescent foci per cell was constant during cell growth but it significantly increased in cells exposed to aphidicoline, which inhibits DNA synthesis during replication. When cells were treated with phleomycine, a DNA damaging agent mainly causing double-strand breaks, formation of a massive fluorescent focus coinciding with DNA compaction was observed. Our results suggest that the specific cellular localization of GFP::Rpa2 observed reflects Rpa2 roles in DNA repair and/or DNA replication fork restart.

Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.

O transplante intrauterino de células-tronco (TIUCT) é um método de tratamento de doenças genéticas, congênitas, hematológicas e imunológicas em um feto durante a gestação. Em pesquisa básica este modelo permite o estudo da dinâmica de migração, enxertia e estado funcional de diferentes tipos de células-tronco (CT). Estas células podem ser transplantadas em diferentes momentos do período gestacional, que pode ser dividido em três momentos do desenvolvimento fetal, sendo estes, diferentes funcionalmente. A escolha deste momento para o transplante influenciará tanto no comportamento celular quanto no resultado. Para o TIUCT são utilizadas as CT mesenquimais derivadas da medula óssea ou fetais ou hematopoiéticas. Para esta pesquisa utilizamos células-tronco derivadas da polpa dentária imatura humana (CTIPDh) as quais apresentam potencial pluripotente e propriedades imunomodulatórias. Nosso principal objetivo foi avaliar a capacidade migratória, bem como de proliferação e endereçamento (homing) das CTIPDh durante o terceiro período gestacional do desenvolvimento fetal no modelo canino. Todos os procedimentos experimentais foram elaborados sob protocolo anestésico apropriado e aprovados pelo comitê de ética da FMVZ da USP. Foram transplantadas via intraperitoneal (IP) 1x106 CTIPDh GFP+ em cada feto, durante procedimento cirúrgico de laparotomia exploratória com ultrassonografia guiada intraoperatóriamente em quatro fetos com idade gestacional aproximada de 45 dias, e outros dois fetos os quais não receberam o transplante, utilizados como controle. Avaliamos os fetos pré e pós-transplante através do ultrasson. Após sete dias, realizamos a ovário-salpingo-histerectomia (OSH) para a colheita dos fetos. Em seguida coletamos seus órgãos e tecidos os quais foram fixados em paraformoldeído a 4% e criopreservados a temperatura de -80oC. Analisamos a biodistribuição das CTIPDh dentro dos órgãos e tecidos em criocortes de 5µm sob microscopia Confocal. Constatamos o homing das CTIPDh nos órgãos derivados das linhas germinativas endodermais, ectodermais e mesodermais. No estômago e intestinos as CTIPD/GFP+ foram identificadas tanto no espaço intraglandular, como na camada muscular da mucosa; no fígado no parenquima hepático; no coração especialmente no tecido muscular do miocárdio; no cérebro nos vasos da substância branca, e cerebelo entre células de Purkinje. Na placenta estas células foram encontradas especialmente junto aos vasos. Quantificamos as CTIPD GFP+ utilizando a citometria de fluxo. Comparativamente dentre os órgãos analisados, obtivemos resultados expressivos do homing celular no miocárdio (~50%), no baço e fígado. Nossos resultados foram confirmados através das análises de imunohistoquímica e imunofluorescência utilizando os anticorpos Anti-núcleo (HuNu), Anti-CTIPD e Anti-GFP humanos. Concluímos que as CTIPDh apresentam grande potencial migratório e proliferativo após o TIUCT em fetos caninos. Estas células indiferenciadas demonstraram homing, especialmente nos tecidos: hematopoiéticos fetais (placenta, fígado e baço), tecido epitelial e glandular de órgãos, bem como de nichos perivasculares de CT. Estes dados sugerem que as CTIPD através do TIU, é uma alternativa viável, segura e promissora para o tratamento de doenças genéticas, congênitas, hematológicas e imunológicas. / Intra-uterine stem cells transplantation (IUSCT) is a method for the treatment of genetic, congenital, hematological, and immunological diseases. In basic research it provides a model for studying the dynamics of migration, graft and functional status of different types of stem cells. The cells can be transplanted in different moments of gestational period, which can be divided into quarters that are not functionally equivalent. The choice of the cells and quarter where the stem cells will be applied can influence cells behavior and results of transplantation. Fetal and adult hematopoietic or bone marrow derived mesenchymal stem cells (MSCs) were mainly used for IUSCT. We previously obtained human immature dental pulp stem cell (IDPSCs), which showed pluripotent potential and immune-compatible properties. The goal of our study was to evaluate migration capacity, proliferation and homing of IDPSCs after IUSCT during the third fetal period in dogs. All experimental procedures were approved by the Ethical Committee of the School of Veterinary Medicine and Animal Science of São Paulo University and were performed under appropriate anesthesia. 1x106 of undifferentiated GFP-positive human IDPSCs were transplanted following laparotomy and intraperitoneal injection under intra-operative ultrasound control into 5 fetuses at the 45 days of gestation. Five fetuses, which did not receive IDPSCs, were used as a control. Ultrasound analyses were performed daily before collection of the fetuses. After 7 days ovarian hysterectomy was performed, fetuses were collected; organs and tissues were isolated and fixed in 4% paraformaldehyde or cryopreserved. Biodistribution of IDPSCs within the organs and tissues were analyzed on cryosections (5µm) under Confocal Microscopy. Homing of IDPSCs was observed in organs derived from three germ lines, endoderm, ectoderm and mesoderm. In stomach and in intestine GFP IDPSCs were found in intraglandular space as well as in muscularis mucosae. In liver they appeared in hepatic parenchyma; in heart in myocardium and in brain in bold vessels, in cerebellum within Purkinje cells. Using Flow cytometry assay GFP IDPSCs graft was quantified. Among the different organs an expressive homing was observed in myocardium of heart (~50%), in spleen and liver. The IDPSCs were also found in canine placenta, especially in blood vessels. These data were confirmed using anti-human nucleus (HuNu), anti-GFP and anti-IDPSCs anti-bodies. Human IDPSCs showed high migration and proliferation potential after IUSCT in dog fetuses. Undifferentiated IDPSCs demonstrated homing in fetal hematopoietic (placenta), epithelial (gastric glands) and perivascular stem cells niches. Our data suggest that IDPSCs is a new promising source for genetic, congenital, hematological, and immunological treatment for those diseases through IUSCT.

O transplante intrauterino de células-tronco (TIUCT) é um método de tratamento de doenças genéticas, congênitas, hematológicas e imunológicas em um feto durante a gestação. Em pesquisa básica este modelo permite o estudo da dinâmica de migração, enxertia e estado funcional de diferentes tipos de células-tronco (CT). Estas células podem ser transplantadas em diferentes momentos do período gestacional, que pode ser dividido em três momentos do desenvolvimento fetal, sendo estes, diferentes funcionalmente. A escolha deste momento para o transplante influenciará tanto no comportamento celular quanto no resultado. Para o TIUCT são utilizadas as CT mesenquimais derivadas da medula óssea ou fetais ou hematopoiéticas. Para esta pesquisa utilizamos células-tronco derivadas da polpa dentária imatura humana (CTIPDh) as quais apresentam potencial pluripotente e propriedades imunomodulatórias. Nosso principal objetivo foi avaliar a capacidade migratória, bem como de proliferação e endereçamento (homing) das CTIPDh durante o terceiro período gestacional do desenvolvimento fetal no modelo canino. Todos os procedimentos experimentais foram elaborados sob protocolo anestésico apropriado e aprovados pelo comitê de ética da FMVZ da USP. Foram transplantadas via intraperitoneal (IP) 1x106 CTIPDh GFP+ em cada feto, durante procedimento cirúrgico de laparotomia exploratória com ultrassonografia guiada intraoperatóriamente em quatro fetos com idade gestacional aproximada de 45 dias, e outros dois fetos os quais não receberam o transplante, utilizados como controle. Avaliamos os fetos pré e pós-transplante através do ultrasson. Após sete dias, realizamos a ovário-salpingo-histerectomia (OSH) para a colheita dos fetos. Em seguida coletamos seus órgãos e tecidos os quais foram fixados em paraformoldeído a 4% e criopreservados a temperatura de -80oC. Analisamos a biodistribuição das CTIPDh dentro dos órgãos e tecidos em criocortes de 5µm sob microscopia Confocal. Constatamos o homing das CTIPDh nos órgãos derivados das linhas germinativas endodermais, ectodermais e mesodermais. No estômago e intestinos as CTIPD/GFP+ foram identificadas tanto no espaço intraglandular, como na camada muscular da mucosa; no fígado no parenquima hepático; no coração especialmente no tecido muscular do miocárdio; no cérebro nos vasos da substância branca, e cerebelo entre células de Purkinje. Na placenta estas células foram encontradas especialmente junto aos vasos. Quantificamos as CTIPD GFP+ utilizando a citometria de fluxo. Comparativamente dentre os órgãos analisados, obtivemos resultados expressivos do homing celular no miocárdio (~50%), no baço e fígado. Nossos resultados foram confirmados através das análises de imunohistoquímica e imunofluorescência utilizando os anticorpos Anti-núcleo (HuNu), Anti-CTIPD e Anti-GFP humanos. Concluímos que as CTIPDh apresentam grande potencial migratório e proliferativo após o TIUCT em fetos caninos. Estas células indiferenciadas demonstraram homing, especialmente nos tecidos: hematopoiéticos fetais (placenta, fígado e baço), tecido epitelial e glandular de órgãos, bem como de nichos perivasculares de CT. Estes dados sugerem que as CTIPD através do TIU, é uma alternativa viável, segura e promissora para o tratamento de doenças genéticas, congênitas, hematológicas e imunológicas. / Intra-uterine stem cells transplantation (IUSCT) is a method for the treatment of genetic, congenital, hematological, and immunological diseases. In basic research it provides a model for studying the dynamics of migration, graft and functional status of different types of stem cells. The cells can be transplanted in different moments of gestational period, which can be divided into quarters that are not functionally equivalent. The choice of the cells and quarter where the stem cells will be applied can influence cells behavior and results of transplantation. Fetal and adult hematopoietic or bone marrow derived mesenchymal stem cells (MSCs) were mainly used for IUSCT. We previously obtained human immature dental pulp stem cell (IDPSCs), which showed pluripotent potential and immune-compatible properties. The goal of our study was to evaluate migration capacity, proliferation and homing of IDPSCs after IUSCT during the third fetal period in dogs. All experimental procedures were approved by the Ethical Committee of the School of Veterinary Medicine and Animal Science of São Paulo University and were performed under appropriate anesthesia. 1x106 of undifferentiated GFP-positive human IDPSCs were transplanted following laparotomy and intraperitoneal injection under intra-operative ultrasound control into 5 fetuses at the 45 days of gestation. Five fetuses, which did not receive IDPSCs, were used as a control. Ultrasound analyses were performed daily before collection of the fetuses. After 7 days ovarian hysterectomy was performed, fetuses were collected; organs and tissues were isolated and fixed in 4% paraformaldehyde or cryopreserved. Biodistribution of IDPSCs within the organs and tissues were analyzed on cryosections (5µm) under Confocal Microscopy. Homing of IDPSCs was observed in organs derived from three germ lines, endoderm, ectoderm and mesoderm. In stomach and in intestine GFP IDPSCs were found in intraglandular space as well as in muscularis mucosae. In liver they appeared in hepatic parenchyma; in heart in myocardium and in brain in bold vessels, in cerebellum within Purkinje cells. Using Flow cytometry assay GFP IDPSCs graft was quantified. Among the different organs an expressive homing was observed in myocardium of heart (~50%), in spleen and liver. The IDPSCs were also found in canine placenta, especially in blood vessels. These data were confirmed using anti-human nucleus (HuNu), anti-GFP and anti-IDPSCs anti-bodies. Human IDPSCs showed high migration and proliferation potential after IUSCT in dog fetuses. Undifferentiated IDPSCs demonstrated homing in fetal hematopoietic (placenta), epithelial (gastric glands) and perivascular stem cells niches. Our data suggest that IDPSCs is a new promising source for genetic, congenital, hematological, and immunological treatment for those diseases through IUSCT.

In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.

This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.

This master thesis is focused on volumetric data rendering and on highlighting and visualization of the selected cells of the model organisms. These data are captured by a confocal deconvolution microscope. Input data form one large volumetric block containing separate slices. This data block is rendered by an applicable method and then are identified and visualized the cells marked by the GFP (Green Fluorescent Protein) process or by chlorophyle fluorescency. The principal aim of this work is to find out the preferably optimal effective method enabling this highlighting, most preferably working without a manual check. Due to the data structure, this ambition seems hardly realizable, so it suffices to find out a manual working method. The last step is to embed the results of this work into FluorCam application, the confocal deconvolution microscope data visualizer.

Els factors de transcripció de la família Snail són fonamentals en la "transició epiteli-mesènquima", procés morfogènic essencial en el desenvolupament embrionari i en els fenòmens metastàsics tumorals.En els mamífers l'activitat d'Snail és modulada per dos mecanismes. (i) En el promotor humà es troben regions definides de resposta a factors repressors, predominants en les cèl·lules epitelials, i elements diferenciats de resposta a inductors de la "transició epiteli-mesènquima". (ii) L'activitat d'Snail és condicionada també per la seva localització subcel·lular, modulada per mecanismes no transcripcionals: la fosforilació d'Snail determina si és o no exclós del nucli. Al citosol no pot actuar com a repressor transcripcional però pot interaccionar amb la xarxa microtubular, que estabilitza i en condiciona el dinamisme. Això coincideix amb l'activació de la GTPasa RhoA i la reorientació dels filaments de vimentina, fets associats a l'adquisició de capacitat migratòria. L'efecte com a repressor transcripcional i la modulació del dinamisme microtubular són possiblement esdeveniments coordinats necessaris per al rol biològic d'Snail en mamífers. / Snail family of transcription factors is fundamental to the "epithelial-mesenchymal transition", morphogenic process essential to embryonic development and metastatic phenomena in tumors.Snail's activity is modulated in two ways in mammals. (i) The human promoter harbors definite regions that respond to repressor factors, which prevail in epithelial cells; and differentiated elements that respond to known inducers of the "epithelial-mesenchymal transition". (ii) Snail's activity is also conditioned by its subcellular localization, mechanism not dependent on its transcriptional control: Snail phosphorylation determines whether Snail is excluded or not from the nucleus. When in the cytosol, Snail is unable to act as a transcriptional repressor, but however binds to the microtubular meshwork, which becomes stabilized and whose dynamism is conditioned as a result. This fact coincides with the activation of the RhoA GTPase and reorientation of vimentin filaments, both phenomena being related to the acquisition of cell motility. The transcriptional repressor and the microtubule dynamics effects are probably two coordinated events necessary to Snail's biological role in mammals.