Techn. Universiẗat, Diss., 2005--München.

Les processus moléculaires mis en jeu lors de maladies de surcharge lysosomale (MSL) et qui conduisent à des dysfonctions neuronales sont peu connus. Afin de mieux comprendre comment s’opèrent ces dysfonctions neuronales associées à la mucopolysaccharidose de type VII (MPS VII), une MSL causée par la déficience en l’activité enzymatique de la ß-glucuronidase, nous avons généré des neurones humains MPS VII à partir cellules souches pluripotentes induites (iPSC). Grâce à la reprogrammation des fibroblastes de patients MPS VII, nous avons généré et caractérisé des neuroprécurseurs dérivés d’iPSC (NSC) et des neurones. Les iPSC MPS VII ont été positives pour les tests de pluripotence (activité de la phosphatase alcaline, expression des marqueurs de pluripotence SSEA3, TRA-2-49 et Nanog par immunofluorescence et expression des gènes de pluripotence SOX2, Oct4 et Lin28 par qRT-PCR, formation des corps embryonnaires et génération de cellules dérivées des trois feuillets embryonnaires in vivo par la formation de tératomes) et présentaient un caryotype normal. Les NSC dérivés d’iPSC exprimaient les marqueurs Nestin et SOX2, et ont été utilisés pour générer des neurones. Les neurones MPS VII exprimaient des marqueurs neuronaux comme MAP2, formaient des synapses et présentaient une activité calcium-dépendante.Afin d’identifier les dysfonctions moléculaires présentes dans la MPS VII, nous avons comparé les NSC et les neurones, avec ou sans milieu conditionné contenant l’enzyme recombinante humaine de la ß-glucuronidase (rhGUS), enzyme actuellement utilisée en phase 1/2, de chez Ultragenyx. Cette enzyme est internalisée par les cellules, rejoint leurs lysosomes et corrige les dysfonctions lysosomales de la MPS VII, restaurant ainsi un phénotype cellulaire physiologique (phénomène aussi appelé ‘enzyme replacement therapy’ (ERT)). Ces diverses conditions nous permettent d’éviter la variabilité clonale des iPSC, et de mieux identifier les déficiences neuronales, corrigées par l’ERT, qui sont associées à la MPS VII. / The molecular pathways linking lysosomal storage diseases (LSD) to neuronal dysfunction are poorly understood. To better understand neuronal dysfunction associated with mucopolysaccharidosis type VII (MPS VII), a LSD due to deficiency in ß-glucuronidase activity, we generated human MPS VII neurons from induced pluripotent stem cells (iPSC). Starting from MPS VII patient fibroblasts, iPSC-derived neural stem cells (NSC) and neurons were generated and characterized. MPS VII iPSC were positive for pluripotency tests (alkaline phosphatase activity, expression of pluripotency markers SSEA3, TRA-2-49 and Nanog by immunostaining and pluripotency gene SOX2, Oct4 and Lin28 expression by qRT-PCR, embryonic bodies formation and generation of cells derivated from the three germ layers in vivo by teratoma formation) and had a normal karyotype. IPSC-derived NSC expressed the markers Nestin and SOX2, and were used to generate neurons. MPS VII neurons expressed mature neuronal markers as MAP2, formed synapses and displayed a calcium-dependent activity. To identify molecular defects in MPS VII, we compared NSC and neurons, with or without conditioned medium containing a recombinant human ß-glucuronidase (rhGUS), enzyme currently used in phase 1/2, from Ultragenyx. This enzyme is taken up by cells, reaches their lysosoms and corrects MPS VII lysosoms dysfunctions, restoring cells to healthy phenotype (phenomena also called enzyme replacement therapy (ERT)). Our assays allow us to circumvent clonal variability associated with iPSC, and to better identify neuronal defects, corrected by ERT, which are associated with MPS VII disease.

No description available.

Inaugural dissertation - Friedrich-Wilhelms-Universität, Berlin. / Cover title. Vita. No more published as inaugural dissertation. Complete work issued in the journal Oriens Christianus, 1901. British Museum attributes the Abhandlung to Ignatius VII, patriarch of the Jacobites. Includes bibliographical references.

African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in the southern African sub-region, where outbreaks regularly occur. Thereis anecdotal evidence suggesting that trans-boundary movement of infected animals may have played a role in precipitating widespread outbreaks in the past, however, since the1970s outbreaks have generally been more localised, particularly in those countries where control of animal movement is strictly regulated. The origin and relatedness of regional ASF outbreaks was investigated here by means of a two-step genetic characterisation approach whereby p72 gene sequencing was used to delineate genotypes, prior to intragenotypic resolution of viral relationships by central variable region (CVR) characterisation of the 9RL ORF. In this manner, regional virus heterogeneity and epidemiological links between outbreaks could be assessed for the first time through phylogenetic analysis of the C-terminal end of the p72 gene of viruses recovered from domestic pig outbreaks in southern Africa between 1973 and 1999. The phylogeny revealed the presence of 14 distinct p72 genotypes of which 6 (genotypes XVII–XXII) were considered novel. Eight of these were country-specific with the remaining six having a trans-boundary distribution. CVR products were heterogeneous in size ranging from 377 bp to 533 bp across the 14 southern African genotypes. Within-genotype CVR comparisons revealed the presence of a genotype XIX virus with an extended field presence in South Africa (1985–1996) and permitted discrimination between three genotype VII viruses that were identical across the p72 gene.

Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (p. 137-153). Also available online through Digital Dissertations.

Depuis plus d’un siècle, le règne de Jayavarman VII ne cesse de fasciner les chercheurs par son indéniable originalité. Premier roi ouvertement bouddhiste, sa puissance politique et militaire s’accompagne aussi d’une profonde mutation des idées et d’une créativité artistique bouillonnante qui confère une dimension hors du commun à son époque. Au rythme des découvertes qui s’accumulent et constituent un matériau archéologique d’une ampleur inégalée, les publications se multiplient, mais elles demeurent souvent spéculatives, échafaudées sur des hypothèses d’écoles qui, faute d’être contestées, se transforment en dogmes, adoptés et répétés au fil du temps, pour finalement donner l’impression inexacte que ce règne est parfaitement connu. C’est ce constat qui nous a amené à aborder l’étude du règne de Jayavarman VII sous des approches différentes, à partir d’une analyse de son historiographie axée sur une exégèse critique des publications. Cette thèse se fonde sur l’étude des matériaux archéologiques, attribués, en quantités considérables, au règne de Jayavarman VII, et appréhendés comme les éléments d’un système relevant d’un même déterminisme, celui de l’organisation de l’espace par l’autorité royale. Cette approche permet de mettre en lumière les principes directeurs de l’aménagement du territoire qui reflètent des impératifs dictés par la gestion administrative et sociale d’un pays, mais peuvent également satisfaire à d’autres exigences telles que le respect d’un modèle cosmico-religieux, les orientations personnelles du souverain ou encore le facteur économique. Derrière le souverain khmer et bouddhiste, Jayavarman VII, deus ex machina de son royaume, instigateur et catalyseur de toute politique, c’est le bouddhisme qui se révèle être le véritable inspirateur de ses actes, permettant de mieux comprendre certains des aspects atypiques de son règne, qu’il s’agisse de l’exercice du pouvoir ou de la composition architecturale des grands monuments. / For more than a century, the reign of Jayavarman VII hasn't ceased to fascinate researchers by its irrefutable originality. First king openly Buddhist, his political and military power was also accompanied by a profound change in the ideas and by an exciting artistic creativity ensuring an extraordinary dimension to his era. Following the progress of the discoveries as they build up and represent an archeological material of unprecedented scale, publications multiply, but they remain often speculative, based upon theories, that as they stay unchallenged, consolidate and transform into dogmas, adopted and repeated over time, finally giving the wrong impression this reign is perfectly well known. This observation brought us to address this reign under a different angle, after retracing the historiography focused on a critical exegesis of the publications. This thesis is based upon the study of archeological material, all attributed to the reign of Jayavarman VII and treated as the items of a system following the same determinism, the organization of space by the royal authority. This approach has high lightened the guidelines of the land use planning that reflect the needs of the administrative and social management of a country, but also other requirements such as the respect of a cosmic religious concept, the king's personal preferences or the economic factor. Behind Jayavarman VII, who appears as the “Deus ex machina” of his kingdom, the initiator and catalyst of all policy, it is Buddhism that proves to be the real inspiration of his actions and leads us to understand better certain atypical aspects of his reign, regarding the exercise of power or the architectural composition of the great monuments.

O Fator VII recombinante (FVIIr) tem sido a principal escolha terapêutica dos pacientes hemofílicos que desenvolvem inibidores contra os fatores VIII e IX utilizados como tratamento. Atualmente, o produto utilizado é produzido em células de camundongo (BHK-21), o qual oferece desvantagens considerando a complexidade das modificações pós-traducionais desta proteína e a inserção de glicosilações de origem murina altamente imunogênicas aos seres humanos. Dessa maneira a produção de proteínas para uso terapêutico em linhagens celulares humanas surge como uma alternativa promissora. Dentro desse contexto, o objetivo principal deste trabalho foi clonar e expressar o FVII de coagulação sanguínea em 3 linhagens celulares humanas (HepG2, Sk-Hep, HKB-11), compará-las com a linhagem murina BKH-21, e selecionar a melhor produtora da proteína recombinante. As células foram modificadas com o vetor lentiviral p1054-CIGWS, contendo os genes do FVII e do marcador GFP. Após a modificação das células foi observada uma eficiência de transdução de 80% nas células BHK-21-FVIIr, 73% nas células HepG2-FVIIr, 32% nas células HKB-11-FVIIr e 95% Sk-Hep-FVIIr. Análises da expressão gênica por PCR em Tempo Real mostraram que as três linhagens humanas modificadas apresentaram expressão do RNAm relativo ao FVIIr, sendo que a linhagem celular HepG2 foi a que teve maior expressão de FVIIr, seguida da Sk-Hep-1 e HKB-11. Quando submetidas ao tratamento com vitamina K por um período de 10 dias em cultura, a expressão do gene FVIIr foi semelhante para as três linhagens (HepG2: 164563 URE, HKB-11: 119122 URE e Sk-Hep: 124919 URE). O FVII é uma proteína que para sua ativação, possui como principal modificação pós traducional a -carboxilação vitamina K dependente, que ocorre por meio do ciclo da vitamina K com a participação de 3 enzimas, -carboxilase, VKORC1 e calumenina (inibidor). A expressão gênica dessas enzimas foi avaliada antes e após o tratamento com a vitamina K. Foi possível observar que houve um aumento nos níveis de RNAm nas células humanas tratadas com vitamina K, sugerindo que esta é capaz de ativar as enzimas do ciclo da -carboxilação. A cinética de crescimento celular em garrafas estáticas mostrou que a as células murinas BHK-21 modificadas possuem uma velocidade específica de crescimento 25% mais elevada que das células humanas. Contudo a cinética de produção das linhagens recombinantes mostrou que as células humanas produzem cerca de 3 vezes mais FVIIr do as células BHK-21. Devida a baixa produção de FVIIr na linhagem celular murina, e ao fato de que a linhagem humana HepG2 apresenta um perfil de crescimento extremamente lento, as linhagens recombinantes Sk-Hep-1-FVIIr e HKB-11-FVIIr foram selecionadas para ensaios de cultivo em suspensão utilizando microcarregadores em frascos spinners. Ao longo de 10 dias de cultivo as células HKB-11-FVIIr mostraram uma produção acumulada de 152 g de FVIIr, o que corresponde a 304 UI. As células Sk-Hep-1-FVIIr produziram cerca de 202,6 g de FVIIr, o que corresponde a 405,2 UI. Em suma, nossos dados comprovam que as linhagens celulares humanas são eficazes para a produção de fator VII recombinante, uma vez que, utilizando nosso modelo de produção, estas mostraram-se melhores do que a de células murinas (BHK-21) utilizadas pela indústria. Assim, estas linhagens celulares humanas podem ser usadas como uma nova plataforma para a produção de FVII, bem como para outras proteínas recombinantes, de maneira mais segura e com menor risco de desenvolvimento de anticorpos inibidores / Recombinant factor VII (FVIIr) has been the main therapeutic choice for hemophilic patients who develop inhibitors antibidies to conventional treatments (FVIII and FIX). Currently, the comercial product is produced in murine cells (BHK-21) which gives disadvantages considering the complexity of post-translational modifications of these proteins. The insertion of murine residues can be highly immunogenic in humans. Thus the production of proteins for therapeutic use in human cell lines appears as a promising alternative. In this context, the aim of this study was to clone and express the blood coagulation FVII in 3 human cell lines (HepG2, Sk-Hep-1, HKB-11) and select the best cell line for production of recombinant protein. The cells were modified with the lentiviral vector p1054-CIGWS containing the FVII gene and GFP gene marker. After cells modification we observed efficiency of transduction, in which 80% of BHK-21-FVIIr cells showed GFP expression, 73% of HepG2-FVIIr cells, 32% of HKB-11-FVIIr cells and 95% SK- Hep-FVIIr. Gene expression analysis by real-time PCR showed that the three modified human cell lines exhibited RNAm expression relative to FVIIr. When cells were treated with 5 ug/mL vitamin K in culture, the gene expression of FVIIr was similar in the three cell lines (HepG2: 164 563 URE, HKB-11: 119122 and SK-Hep URE: 124919 URE). For FVII activation, the main post translational modification is -carboxylation vitamin-K-dependent which envolves three enzymes, -carboxylase, VKORC1 and calumenina (inhibitor) . Gene expression of these enzymes was evaluated before and after treatment with vitamin K. It was observed that there was an increase in mRNA levels in human cells treated with vitamin K, suggesting that the treatment is capable of activating the enzymes of the vitamin K cycle. Cell growth kinetics showed that modified murine cells BHK-21 have a higher specific growth rate, around 25% more than human cells. However the kinetics of production of recombinant cell lines showed that human cells expressing rFVII 3-fold more rFVII than BHK-21 cells. Due the low rFVII production of murine cells, and the extremely slow growth profile of human cell line HepG2, the recombinant cell lines Sk-Hep-1-rFVII and HKB-11-rFVII have been selected for cultivation tests in suspension using microcarriers in spinners flasks. Over 10 days of cultivation the HKB-11 cells showed a cumulative production of rFVII 152 ug, corresponding to 304 IU and SK-Hep-1 cells showed a rFVII production of 202.6 ug, corresponding to 405.2 IU. In summary, our data demonstrate that human cell lines are effective for producing recombinant factor VII. Using our production model, human cells were better than murine cells (BHK-21) used by the industry. Thus, these human cell lines can be used as a new platform for the FVII production, as well as for other recombinant proteins, with less risk of developing inhibitor antibodies

Spectateur meurtri par les événements dramatiques de son temps, les atrocités insupportables, la guerre franco-anglaise, la désorganisation et l’état de déréliction morale du royaume, Jean Juvénal des Ursins (1388-1473) entreprend un long dialogue avec le roi et avec la France. Fort de son éminente et double autorité d’évêque et de juriste, ses discours et écrits politiques constituent une tentative d’avertir Charles VII, puis Louis XI, des carences et des erreurs de leur gouvernement. Au-delà du commentaire de l’actualité du temps ou de l’histoire des relations entre les rois de France et d’Angleterre, c’est un puissant engagement moral qui s’affirme, enraciné dans une conception claire du monde et des devoirs du prince. Pour pousser celui-ci à l’action et le convaincre de la nécessité d’une reformation du royaume, le propos s’organise pour gagner en efficacité argumentative et en force de persuasion. La parénèse suppose le recours aux citations d’autorité mais passe également par l’écriture allégorique et les dispositifs fictionnels. Ceux-ci accueillent une grande variété de traditions appartenant à la littérature, à l’éloquence, ou à des formes d’écriture savante (épopée, motif courtois, apologue animalier, prophétie, généalogie et plaidoyer, etc.). Le souci d’une mise en scène des mœurs oratoires y est particulièrement remarquable : l’évêque se présente, inséparable de sa famille, de diverses manières, afin de lester son autorité de l’ethos du prophète ou de travailler à l’affirmation d’une nouvelle noblesse, héritière de la vieille chevalerie féodale, les gens de robe, les juristes rompus au droit romain et les agents du roi. La présente étude se propose d’attirer l’attention sur cet écrivain de talent, trop longtemps négligé par la critique littéraire. Il s’agit de lui rendre sa place parmi les écrivains politiques qui s’affirment de plus en plus dans le contexte tardo-médiéval. Après une étude des usages particuliers que Jean Juvénal des Ursins propose de l’épître (sources, travail de la citation et de la langue), seront envisagées tour à tour la construction éthique et la réflexion économique puis enfin l’articulation entre la pensée politique et sa mise en fiction. / Spectator bruised by the dramatic events of his time, the unbearable atrocities, the Anglo-French War, disorganization and moral dereliction state of the kingdom, Jean Juvénal des Ursins (1388-1473) begins a long dialogue with the king and with France. With its outstanding authority of both bishop and lawyer, his speeches and political writings are an attempt to warn Charles VII and Louis XI about deficiencies and mistakes of their government. Beyond the comments of the current events or the history of relations between the kings of France and England, it is a powerful moral commitment which asserts itself, rooted in a clear conception of the world and duties of the prince. To urge him to act and convince him of the necessity of a reformation of the kingdom, the purpose is organized to gain efficiency and argumentative persuasiveness. The exhortation supposes the use of citations of authority and passes also by the allegorical writing and fictional devices. They host a wide variety of traditions belonging to literature, eloquence, or to forms of scholarly writing (the epic, courtly love, animal fable, prophecy, genealogy and advocacy, etc.). Concern for staging the mores oratoris is particularly remarkable: the bishop appears, inseparable from his family, in various ways, to strengthen his authority by the ethos of the prophet and to work on affirmation of a new kind of nobility, heir to the old feudal chivalry, administrators, lawyers experienced in Roman law and the agents of the king. This study aims to draw attention to this talented writer, too long neglected by literary criticism. It is a question of returning him his place among the political writers who assert themselves more and more in the late-medieval context. After a study of specific uses that Jean Juvénal des Ursins features of the epistle (sources, work on citations and language) will be considered in turn the ethical construction and economic thinking and finally the relationship between the political thought and fiction.

O Fator VII recombinante (FVIIr) tem sido a principal escolha terapêutica dos pacientes hemofílicos que desenvolvem inibidores contra os fatores VIII e IX utilizados como tratamento. Atualmente, o produto utilizado é produzido em células de camundongo (BHK-21), o qual oferece desvantagens considerando a complexidade das modificações pós-traducionais desta proteína e a inserção de glicosilações de origem murina altamente imunogênicas aos seres humanos. Dessa maneira a produção de proteínas para uso terapêutico em linhagens celulares humanas surge como uma alternativa promissora. Dentro desse contexto, o objetivo principal deste trabalho foi clonar e expressar o FVII de coagulação sanguínea em 3 linhagens celulares humanas (HepG2, Sk-Hep, HKB-11), compará-las com a linhagem murina BKH-21, e selecionar a melhor produtora da proteína recombinante. As células foram modificadas com o vetor lentiviral p1054-CIGWS, contendo os genes do FVII e do marcador GFP. Após a modificação das células foi observada uma eficiência de transdução de 80% nas células BHK-21-FVIIr, 73% nas células HepG2-FVIIr, 32% nas células HKB-11-FVIIr e 95% Sk-Hep-FVIIr. Análises da expressão gênica por PCR em Tempo Real mostraram que as três linhagens humanas modificadas apresentaram expressão do RNAm relativo ao FVIIr, sendo que a linhagem celular HepG2 foi a que teve maior expressão de FVIIr, seguida da Sk-Hep-1 e HKB-11. Quando submetidas ao tratamento com vitamina K por um período de 10 dias em cultura, a expressão do gene FVIIr foi semelhante para as três linhagens (HepG2: 164563 URE, HKB-11: 119122 URE e Sk-Hep: 124919 URE). O FVII é uma proteína que para sua ativação, possui como principal modificação pós traducional a -carboxilação vitamina K dependente, que ocorre por meio do ciclo da vitamina K com a participação de 3 enzimas, -carboxilase, VKORC1 e calumenina (inibidor). A expressão gênica dessas enzimas foi avaliada antes e após o tratamento com a vitamina K. Foi possível observar que houve um aumento nos níveis de RNAm nas células humanas tratadas com vitamina K, sugerindo que esta é capaz de ativar as enzimas do ciclo da -carboxilação. A cinética de crescimento celular em garrafas estáticas mostrou que a as células murinas BHK-21 modificadas possuem uma velocidade específica de crescimento 25% mais elevada que das células humanas. Contudo a cinética de produção das linhagens recombinantes mostrou que as células humanas produzem cerca de 3 vezes mais FVIIr do as células BHK-21. Devida a baixa produção de FVIIr na linhagem celular murina, e ao fato de que a linhagem humana HepG2 apresenta um perfil de crescimento extremamente lento, as linhagens recombinantes Sk-Hep-1-FVIIr e HKB-11-FVIIr foram selecionadas para ensaios de cultivo em suspensão utilizando microcarregadores em frascos spinners. Ao longo de 10 dias de cultivo as células HKB-11-FVIIr mostraram uma produção acumulada de 152 g de FVIIr, o que corresponde a 304 UI. As células Sk-Hep-1-FVIIr produziram cerca de 202,6 g de FVIIr, o que corresponde a 405,2 UI. Em suma, nossos dados comprovam que as linhagens celulares humanas são eficazes para a produção de fator VII recombinante, uma vez que, utilizando nosso modelo de produção, estas mostraram-se melhores do que a de células murinas (BHK-21) utilizadas pela indústria. Assim, estas linhagens celulares humanas podem ser usadas como uma nova plataforma para a produção de FVII, bem como para outras proteínas recombinantes, de maneira mais segura e com menor risco de desenvolvimento de anticorpos inibidores / Recombinant factor VII (FVIIr) has been the main therapeutic choice for hemophilic patients who develop inhibitors antibidies to conventional treatments (FVIII and FIX). Currently, the comercial product is produced in murine cells (BHK-21) which gives disadvantages considering the complexity of post-translational modifications of these proteins. The insertion of murine residues can be highly immunogenic in humans. Thus the production of proteins for therapeutic use in human cell lines appears as a promising alternative. In this context, the aim of this study was to clone and express the blood coagulation FVII in 3 human cell lines (HepG2, Sk-Hep-1, HKB-11) and select the best cell line for production of recombinant protein. The cells were modified with the lentiviral vector p1054-CIGWS containing the FVII gene and GFP gene marker. After cells modification we observed efficiency of transduction, in which 80% of BHK-21-FVIIr cells showed GFP expression, 73% of HepG2-FVIIr cells, 32% of HKB-11-FVIIr cells and 95% SK- Hep-FVIIr. Gene expression analysis by real-time PCR showed that the three modified human cell lines exhibited RNAm expression relative to FVIIr. When cells were treated with 5 ug/mL vitamin K in culture, the gene expression of FVIIr was similar in the three cell lines (HepG2: 164 563 URE, HKB-11: 119122 and SK-Hep URE: 124919 URE). For FVII activation, the main post translational modification is -carboxylation vitamin-K-dependent which envolves three enzymes, -carboxylase, VKORC1 and calumenina (inhibitor) . Gene expression of these enzymes was evaluated before and after treatment with vitamin K. It was observed that there was an increase in mRNA levels in human cells treated with vitamin K, suggesting that the treatment is capable of activating the enzymes of the vitamin K cycle. Cell growth kinetics showed that modified murine cells BHK-21 have a higher specific growth rate, around 25% more than human cells. However the kinetics of production of recombinant cell lines showed that human cells expressing rFVII 3-fold more rFVII than BHK-21 cells. Due the low rFVII production of murine cells, and the extremely slow growth profile of human cell line HepG2, the recombinant cell lines Sk-Hep-1-rFVII and HKB-11-rFVII have been selected for cultivation tests in suspension using microcarriers in spinners flasks. Over 10 days of cultivation the HKB-11 cells showed a cumulative production of rFVII 152 ug, corresponding to 304 IU and SK-Hep-1 cells showed a rFVII production of 202.6 ug, corresponding to 405.2 IU. In summary, our data demonstrate that human cell lines are effective for producing recombinant factor VII. Using our production model, human cells were better than murine cells (BHK-21) used by the industry. Thus, these human cell lines can be used as a new platform for the FVII production, as well as for other recombinant proteins, with less risk of developing inhibitor antibodies