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Les glioblastomes sont des tumeurs du système nerveux central hautement létaux. Ils se caractérisent par leur grande infiltration dans les tissus avoisinants. Ils modifient les vaisseaux sanguins préexistants et ils migrent d’une façon perivasculaire. Cette cooption vasculaire est un processus entraînant l’expression d’Angiopoietine-2 (Angpt2) par des cellules endothéliales et sa liaison au récepteur Tie2. Le premier objectif de cette étude était d’examiner le potentiel thérapeutique de deux protéines qui pourraient interférer avec Angpt2, à savoir Angpt3 et la partie soluble extracellulaire du récepteur Tie2 (sTie2). Le deuxième objectif était de développer des vecteurs lentiviraux capables d’exprimer ces protéines, tout en offrant la possibilité d’identifier et détruire les cellules génétiquement modifiées. À cette fin, nous avons construit un vecteur contenant une cassette bicistronique qui exprime le marqueur amélioré de la protéine fluorescente verte (EGFP) fusionnée au gène suicide provenant du virus herpès simplex de type I-thymidine kinase (HSVtk). Les cellules du gliome GL261 transduites avec ce vecteur pourraient être suivies et tuées sur demande par l’administration de la prodrogue ganciclovir, soit in vitro, soit après l'implantation dans le cerveau des souris. Malgré l’expression des hauts niveaux d’Angpt3 et de sTie2 obtenus avec ce vecteur, nous avons observé qu’Angpt3 augmente la déstabilisation capillaire et la croissance de gliomes, alors que sTie2 n’exerce aucun effet. Globalement, cette étude a permis de comprendre l’importance de la voie de signalisation de Tie2 dans le développement des gliomes et le rôle d’Angpt3, mais suggère que ni cette molécule ni sTie2 soient des agents efficaces contre les gliomes malins. Cette étude fournit également le prototype d’un vecteur lentiviral pour la thérapie génique plus sécuritaire. / Glioblastomas are highly lethal tumors of the central nervous system characterized by large spread into the surrounding tissues. They modify and migrate along pre-existing blood vessels. This vascular cooption is a process involving the release of angiopoietin-2 (Angpt2) from endothelial cells and binding to the Tie2 receptor. The first goal of this study was to examine the therapeutic potential of two proteins that could interfere with Angpt2, namely Angpt3 and the soluble extracellular domain of Tie2 (sTie2). The second goal was to develop a lentiviral vector capable of delivering such proteins while offering the possibility to identify and destroy the genetically modified cells. To this end, we designed a bicistronic construct expressing the marker enhanced green fluorescent protein (EGFP) fused to the suicide gene herpes simplex virus 1-thymidine kinase (HSVtk). GL261 glioma cells transduced with this vector could be tracked and killed on command by the administration of the prodrug ganciclovir, either in vitro or after implantation into mouse brains. High levels of Angpt3 or sTie2 could be achieved with this vector; however, Angpt3 increased capillary destabilization and glioma growth, whereas sTie2 exerted no effect. Overall, this study helps to understand the importance of the Tie2 signaling pathway in glioma development and the role of Angpt3, but suggests that neither this molecule nor sTie2 are effective agents against malignant gliomas. This study also provides a lentiviral vector design for safer gene therapy.

Vectors derived from Human Immunodeficiency Virus type 1 (HIV-1) are being widely developed for gene therapy applications, principally because they are able to transduce both dividing and non-dividing cells and result in stable, long term gene expression. However, these vectors are difficult to produce in high titres and sufficient volumes for large scale experiments and clinical application. Therefore, an investigation into methods to improve the production of HIV-1 derived gene transfer vectors was undertaken. One factor that limits the production of recombinant virus is the amount of viral genomic RNA available for packaging into virions. Therefore, a transfer vector was modified with the aim of increasing the amount of genomic RNA produced. Substitution of the polyadenylation (pA) signal, mutation splice donor sites and removal of unnecessary sequences were all examined. pA signal readthrough was quantified to determine the effect of these modifications on the rate of pA signal readthrough. Insertional mutagenesis and vector mobilisation are recognised risk factors with all integrating vectors. Self inactivating (SIN) vectors, which contain a deletion of U3 sequences in the 3’ LTR, demonstrate a reduced rate of mobilisation. Transduction with these vectors results in a provirus containing no viral promoter elements, with transcription of the transgene being controlled from an internal promoter. However, LTR repair of SIN vectors occurs at an appreciable frequency. Therefore, the extent of this deletion was maximised and the effect on the frequency of the repair examined. The production of lentiviral gene therapy vectors by large-scale transient transfection is both time consuming and technically difficult. Therefore, methods to increase the scale of production without compromising virus titre were developed. This resulted in fewer transfections and less handling of the cells when making virus on a large scale (3-4 L). In order to process the virus on this scale in a single day (i.e. 8 hours), new concentration and purification methods were established. The protocol consisted of low speed centrifugation, 0.45 μm filtration, 750 kDa ultrafiltration, 0.8 μm filtration and ultracentrifugation. However, the use of ultracentrifugation means that this protocol is not amenable to further scale up. Therefore, the replacement of the ultracentrifugation step with anion exchange was investigated. A number of different resins and anion exchange devices were investigated, two of which show promise for large scale purification of HIV-1 derived gene transfer vectors. In an ideal world, HIV-1 derived gene transfer vectors would be produced using stable packaging cell lines engineered to produce the desired virus. However, previous attempts to produce such a cell line with the desired properties have had limited success and have generally used outdated helper systems. Therefore, in an attempt to combine the efficiency advantages of having a single helper plasmid with the safety advantages of expressing each protein separately, a single packaging construct that contained separate transcription units for each of the required proteins was produced. Transcription of cyotoxic proteins was controlled by inducible promoters. Initial results suggest that such a system is technically feasible but that further work is required to optimise the expression of helper functions. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309550 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2008

Chez les métazoaires, les cellules spécialisées se caractérisent par la sortie du cycle cellulaire alors que les cellules souches et progénitrices se caractérisent par un intense potentiel d'auto-renouvellement, lequel est perdu durant la différenciation. L'auto-renouvellement est contrôlé par une combinaison de facteurs intrinsèques et extrinsèques qui déclenchent une prolifération cellulaire équilibrée. Dans ce contexte, nous avons montré que le knock-out des facteurs de transcription MafB et c-Maf dans les monocytes, résulte en une expansion prolongée des monocytes et macrophages matures en culture, sans aucun signe de perte du phénotype différencié ou de la fonction. Etant donné que les macrophages sont impliqués dans la majorité des maladies dégénératives, les maladies inflammatoires ainsi que la biologie du cancer, l'amplification de macrophages consisterait en un atout considérable pour les applications thérapeutiques. Dans cette optique, et comme les macrophages sont également connus pour promouvoir le développement tumoral, nous avons étudié le comportement des macrophages Maf-DKO dans le contexte tumoral. Initialement, nous avons montré que les macrophages Maf-DKO sont capables d'empêcher l'installation de la tumeur ainsi que de réduire une masse tumorale établie, et ce indépendamment du model tumoral étudié. Ceci consiste en une nouvelle approche thérapeutique contre le cancer. Nous nous sommes ensuite intéressés à fournir une « preuve de principe » quant à la prolifération des monocytes humains après inhibition de l'expression des gènes MafB et c-Maf humains et à étudier leur potentiel dans des applications thérapeutiques. / In metazoans, specialized cells are typically withdrawn from the cell cycle, whereas stem cells and progenitor cells have extensive self-renewal potential that is usually lost on differentiation. Self-renewal is controlled by a combination of cell-intrinsic and extrinsic signals that trigger balanced cellular proliferation. In this context, we previously reported that the knock-out of two monocytic transcription factors, MafB and c-Maf, enables extended expansion of mature monocytes and macrophages in culture without loss of differentiated phenotype and function. As macrophages are involved in degenerative diseases, inflammatory diseases and cancer biology, amplified macrophages may provide potential therapeutic applications. In this context and since macrophages are also known to enhance tumor development, we aim to investigate Maf-DKO macrophages behavior in a tumor context Initially, we have shown that regardless of tumor model (ID8 ovarian carcinoma or B16 melanoma), Maf-DKO macrophages have the ability to prevent tumor growth and reduce established tumor mass in tumor bearing mice. The potential provides a novel therapeutic approach for cancer cell therapies. Next we aimed to provide a proof of principle for the amplification of human monocytes by the inhibition of MafB/c-Maf genes and to investigate their potential in therapeutic applications. So far, we have shown that the down-regulation of MafB and c-Maf in human monocytes results in a colony formation in semi-solid medium, reflecting that the knock down of MafB and c-Maf results in proliferative advantage.

La transmission des messages douloureux depuis les nocicepteurs périphériques jusqu’aux structures supraspinales est sous le contrôle de systèmes modulateurs, parmi lesquels figurent les voies bulbo-spinales sérotoninergiques. Cependant leur rôle n’est pas complètement élucidé puisque selon le type de récepteur sérotoninergique mis en jeu, elles pourraient soit réduire la sensation douloureuse soit au contraire l’exacerber. De plus, le contrôle exercé par la sérotonine (5 HT) pourrait dépendre du type de douleur, aiguë, inflammatoire ou neuropathique chronique. En réalité, ces ambiguïtés tiennent au fait que les études réalisées jusqu’à présent ont surtout impliqué des approches pharmacologiques souvent peu fiables. Ce constat m’a conduit à mettre en œuvre une nouvelle approche expérimentale pour tenter de cerner le véritable rôle modulateur des voies sérotoninergiques bulbo-spinales sur différents types de douleur dans des conditions physiologiques normales ou physiopathologiques chez le rat. A cette fin, j’ai réalisé ce travail de thèse en trois étapes. La première étape a consisté à préciser les caractéristiques anatomiques des voies sérotoninergiques bulbo-spinales visualisées par un marquage antérograde (PHA-L) couplé à l’immunomarquage du transporteur de la sérotonine (SERT). L’étude différentielle des deux sous-structures de la région bulbaire B3 : le noyau raphé magnus (RMg) et le noyau paragigantocellulaire latéral (LPGi) m’a permis de montrer que les neurones sérotoninergiques qu’elles contiennent se projettent respectivement dans les couches profondes (VI-VI) et superficielles (I-II) de la corne dorsale, à tous les étages de la moelle épinière. Afin de dépléter sélectivement la 5-HT dans la corne dorsale, j’ai, dans une deuxième étape, injecté bilatéralement dans la région B3 un lentivirus recombinant LV-shTPH2 pour y éteindre l’expression de la TPH2, l’enzyme limitante de la synthèse de 5-HT. Les contrôles immunohistochimiques ont montré l’inhibition bilatérale de l’expression de la TPH2 dans le RMg et le LPGi, et la diminution de l’immunomarquage de la 5-HT sélectivement dans la corne dorsale, l’expression inchangée du transporteur SERT confirmant le maintien de l’intégrité des fibres sérotoninergiques chez les rats LV-shTPH2. L’évaluation de la sensibilité des animaux ainsi déplétés en 5-HT à différents tests validés de nociception aiguë n’a pas révélé de modifications significatives par rapport aux témoins. Mais dans des conditions de forte stimulation nociceptive thermique (50°C) ou chimique (carragénine), l’augmentation de l’immunomarquage de c-Fos dans la corne dorsale chez les rats LV-shTPH2 montre que la déplétion de 5-HT facilite la sensibilisation synaptique. Des différences encore plus nettes entre les rats LV-shTPH2 et les témoins sont surtout apparues en cas de douleur inflammatoire (formaline) ou neuropathique (ligature du nerf sciatique). Les modifications induites par la déplétion de 5-HT n’étaient cependant pas univoques puisqu’une hypoalgésie a été observée dans le cas d’une douleur inflammatoire et, qu’au contraire, l’hyperalgésie associée à la douleur neuropathique était exacerbée chez les rats LV-shTPH2. Ces modulations opposées laissant à penser l’implication de projections et/ou de récepteurs sérotoninergiques distincts, nous avons réalisé, dans une troisième étape, une étude pharmacologique qui a permis de montrer que l’effet pro-hyperalgésique observé chez les rats neuropathiques déplétés en 5-HT était probablement lié à un déficit d’activation de récepteurs 5 HT7. Notre étude démontre la réalité du contrôle physiologique du relai spinal des voies nociceptives par la 5-HT, via l’implication de projections bulbo-spinales et de récepteurs sérotoninergiques dont l’identification pourrait déboucher sur de nouvelles perspectives thérapeutiques. / The transmission of pain signals from peripheral nociceptors to supraspinal structures is under the control of modulatory systems, which include bulbo-spinal serotoninergic pathways. However, their role is not yet fully understood, as they may as well reduce or exacerbate pain as a function of 5-HT receptor types involved. Furthermore, their modulatory role might also differ depending on whether pain is acute, or chronic, with inflammatory or neuropathic origin. In fact, this ambiguous knowledge results from data essentially obtained with rather unreliable pharmacological approaches. This led me to set up an innovative experimental approach for a better assessment of the real implication of bulbo-spinal serotoninergic projections in pain control mechanisms under validated physiological and pathophysiological conditions in the rat. To this goal, I performed my PhD thesis work in three steps. The first step consisted of a precise anatomical description of bulbo-spinal serotoninergic projections identified by anterograde labeling (with PHA-L) and immunolabeling of the 5-HT transporter (SERT). The differential labeling of the two sub-areas within the B3 bulbar region : the nucleus raphé magnus (RMg) and the nucleus paragigantocellularis lateralis (LPGi) allowed the demonstration that their respective 5-HT neuron groups project separately into the deep (V-VI) and superficial (I-II) laminae of the dorsal horn, at all levels of the spinal cord. For the second step, aimed at depleting 5-HT selectively within the dorsal horn, I injected bilaterally into the B3 area a recombinant lentivirus, LV-shTPH2, designed to suppress the expression of TPH2, the rate limiting enzyme for 5-HT synthesis. Immunohistochemical controls showed that the resulting bilateral inhibition of TPH2 expression in both the RMg and the LPGi was associated with 5-HT depletion but no change in SERT (as expected of unaltered 5-HT fibers) in the dorsal horn. Using various behavioral tests, I could not detect any change in acute pain sensitivity in 5-HT-depleted rats. In contrast, the increased c-Fos immunohistochemical labeling within the dorsal horn after strong thermal (50°C) or chemical (carrageenan) noxious stimulation showed that 5-HT depletion promotes synaptic sensitization. Even more striking differences between LV-shTPH2 injected rats and paired controls were observed in case of inflammatory (formalin) or neuropathic (sciatic nerve ligation) pain. However, 5-HT depletion-induced changes differed under both conditions since hypoalgesia was noted in case of inflammatory pain, whereas neuropathic pain-evoked hyperalgesia was enhanced in 5-HT depleted, LV-shTPH2-injected rats. Because such opposite modulations might have been underlain by distinct serotoninergic pathways (i.e. from the RMg versus the LPGi for instance) and/or distinct 5-HT receptors, we decided, within the time remaining for achieving my PhD work, to perform a third step, focused on the 5-HT7 receptor type. Using pharmacological approaches, we could obtain data showing that exacerbated hyperalgesia in 5-HT-depleted neuropathic rats resulted from loss of tonic 5-HT7 receptor activation by endogenous 5-HT. Altogether, our data clearly demonstrate that bulbospinal serotoninergic projections are involved in the physiological control of pain signaling at spinal cord level. Further identification of underlying neuronal pathways and receptors might open novel therapeutic perspectives.

Made available in DSpace on 2016-04-28T20:17:27Z (GMT). No. of bitstreams: 1 2007 - Antonia Regina Sessa da Silva.pdf: 384094 bytes, checksum: 91d2147f8402249dead112e338e89b66 (MD5) Previous issue date: 2007-03-30 / Brazil has currently the second bigger flock of equines of the world and the brazilian equines breeding plays an important role in the development of the sector of agribusiness. Among the infectious disease that affect the national equines breeding, the Equine infectious anemia (EIAV) has been shown of difficult control. The Equine infectious anemia is caused by a retrovirus, it has a worldwide distribution, and it is recognized as the most important disease of equines. The most common clinical signals in the acute phase, are anemia followed of jaundice in the mucosae, ventral oedema, mioglobinury, caquexy and, mainly, intermittent fever. Once it has no treatment, Ministry of Agriculture, Cattle and Supplying determines, through legal mechanisms, a politics of obligatory examination in credentialed laboratories, for the transport and commercialization of equines in the country. The examination is based on the test of Coggins, an immunodiffusion in agar gel (IDGA) of the serum of the animal tested against antigen of the EIAV. As the infection is lifetime, positive animals are euthanasiated by Veterinarians of the Ministry of Agriculture. Considering that precision of the results is critical, once that false positives animals could uselessly be sacrificed, and false negatives will be preserved as infection source, diverse problems may occur, appeared the interest of a systematic analysis of the reproducibility of kits and on possible sources of error. For this purpose, positive and negative sera, referred by the repetition of the tests for Coggins and ELISA, had been evaluated by IDGA comparatively with three kits of different manufacturers. In the experiments of reproducibility, it observed a great distinction in the quality of the precipitation line promoted for the positive sera in the tests. This phenomenon may be justified by the use of the conditions techniques legal, determined for would carry of the Ministry of Agriculture that is distinct of the recommendation of the analyzed manufacturers of two of kits. Exactly thus, the evaluation of the reproducibility of the results of kits with the referred sera showed high correlation. / O Brasil possui atualmente o segundo maior rebanho de eq??deos do mundo e a Eq?ideocultura Brasileira desempenha um importante papel no desenvolvimento do setor de agroneg?cios. Entre as doen?as infecciosas que afetam a eq?inocultura nacional, a Anemia Infecciosa dos Eq?inos (AIE) tem se mostrado de dif?cil controle. A AIE ? causada por um retrov?rus, apresenta uma distribui??o mundial, e ? reconhecida como a mais importante doen?a dos eq?inos. Entre os sinais cl?nicos mais comuns, na fase aguda da doen?a, encontram-se a anemia seguida de icter?cia nas mucosas, edema ventral, mioglobin?ria, caquexia e, principalmente, febre intermitente. Como n?o h? tratamento, Minist?rio da Agricultura, Pecu?ria e Abastecimento (MAPA) determina, atrav?s de mecanismo legais, uma pol?tica de exame obrigat?rio em laborat?rios credenciados, para o transporte e comercializa??o de eq??deos no Pa?s. O exame ? baseado na prova de Coggins, uma imunodifus?o em ?gar gel do soro do animal testado contra ant?geno do v?rus da AIE. Como a infec??o ? vital?cia, animais soropositivos s?o eutanasiados por M?dicos Veterin?rios do MAPA. Considerando que a precis?o dos resultados ? cr?tica, posto que animais falso positivos podem ser sacrificados inutilmente, e falso negativos preservados como fonte de infec??o, surgiu o interesse de um an?lise sistem?tica da reprodutibilidade dos kits e poss?veis fontes de erro no diagn?stico. Para esta finalidade, soros positivos e negativos, referenciados pela repeti??o dos testes de Coggins e ELISA, foram avaliados pela prova de IDGA, comparativamente com tr?s kits de diferentes fabricantes. Nos experimentos de reprodutibilidade, observou-se uma grande distin??o na qualidade da linha de precipita??o promovida pelos soros positivos nos testes nos diferentes kits. Este fen?meno pode ser justificado pela utiliza??o das condi??es t?cnicas legais, determinadas por portaria do MAPA, que s?o distintas das recomenda??es dos fabricantes de dois dos kits analisados. Mesmo assim, a avalia??o da reprodutibilidade dos resultados dos kits com os soros referenciados mostrou elevada correla??o.

Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional. / Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis, which may have adverse consequences, such as induction of proto-oncogenes. Such events have been described in clinical trials for the treatment of SCID-X1, chronic granulomatous disease and beta thalessemia with some retroviral vectors. Currently, there are few simple and quick methods that can reveal and quantify clonal expansion. Thus, we describe the construction of a vector library containing random markers, called \"barcodes\". The sequencing of the barcode could reveal, characterize and quantify the clonal expansion of a transduced cells population. This methodology will be valuable to test new arrangements of promoters and therapeutic genes, allowing the development of safer vectors, helping to reduce the probability of clonal proliferation events triggered by insertional mutagenesis.

La phénylcétonurie (PCU est une maladie métabolique génétique causée par une déficience d'activité phénylalanine hydroxylase (PAH). Une hypomyélinisation du cerveau a été documentée chez les patients non traités, mais sa pathophysiologie reste floue. Nous avons investigué l'influence de la phénylalanine (Phe), phénylpyruvate (PP) et phénylacétate (PA) sur les oligodendrocytes. Nous avons premièrement montré dans un modèle murin de PCU que le nombre d'oligodendrocytes n'était pas différent dans le corps calleux entre animaux PCU et sains. Ensuite, en utilisant la technique des co-cultures myélinisantes nous avons pu déterminer que Phe, PP et PA n'ont pas d'effet direct sur la synthèse des gaines de myéline. Ces données indiquent que ces trois composés n'exercent probablement pas de rôle direct dans l'hypomyélinisation du système nerveux central constatée dans le cadre de la PCU. Ces données suggèrent donc des mécanismes d'action indirects. De plus, nous avons investigué la faisabilité d'un modèle de thérapie génique pour la PCU. Celui-ci implique la transduction ex vivo d'hépatocytes ou cellules souches mésenchymateuses par un vecteur lentiviral puis leur implantation dans le foie de l'organisme receveur. Phenylketonuria (PKU) is a metabolic genetic disease characterized by deficient phenylalanine hydroxylase (PAH) enzymatic activity. Brain hypomyelination has been reported in untreated patients, but its mechanism remains unclear. We therefore investigated the influence of phenylalanine (Phe), phenylpyruvate (PP), and phenylacetate (PA) on oligodendrocytes. We fisrt showed in a mouse model of PKU that the number of oligodendrocytes is not different in corpus callosum sections from adult mutants or from control brains. Then, using enriched oligodendroglial cultures, we detected no cytotoxic effect of high concentrations of Phe, PP, or PA. Finally, we analyzed the impact of Phe, PP, and PA on the myelination process in myelinating cocultures using both an in vitro index of myelination, based on activation of the myelin basic protein (MBP) promoter, and the direct quantification of myelin sheaths by both optical measurement and a bioinformatics method. None of these parameters was affected by the increased levels of Phe or its derivatives. Taken together, our data demonstrate that high levels of Phe, such as in PKU, are unlikely to directly induce brain hypomyelination, suggesting involvement of alternative mechanisms in this myelination defect. Moreover, we investigated the feasibility of a gene therapy for phenylketonuria. This project involved the ex vivo transduction of hepatocytes and mesenchymal stem cells with lentivirus vector and the engraftment of these cells in the liver's recipient.

Arteriosklerose führt zu schwerwiegenden klinischen Komplikationen bei Herz-Kreislauf-erkrankungen, welche die führenden Todesursachen in den westlichen Industrieländern sind. Die Arteriosklerose ist typischerweise eine Erkrankung arterieller Gefäße und betrifft nicht die venöse Gefäßwand. Bei der Entstehung von Arteriosklerose spielen die Endothelzellen als Barrierezellen und Regulatoren der Gefäßfunktion eine Schlüsselrolle. Ein wichtiger Schwerpunkt der Forschung ist die Differenzierung der Endothelzellen. Arterielle und venöse Endothelzellen weisen schon im frühen Embryonalstadium unterschiedliche Phänotypen auf. Ein besseres Verständnis der arterio-venösen Differenzierung wäre von großer Bedeutung für antiarteriosklerotische Therapien. Darüber hinaus könnte eine Reprogrammierung (z.B. von Vene in Arterie) entscheidend für neue Therapieansätze bei der Senkung der Restenoserate venöser Bypässe von Patienten mit koronarer Herzkrankheit und bei AV-Shunt-Operationen von dialysepflichtigen Patienten sein. In dieser Arbeit wurden differenzierte humane primäre arterielle und venöse Endothelzellen nach Genmodulation untersucht. Der Fokus der Genmodulation wurde auf das arterielle Markergen Hey2 und auf das venöse Markergen COUP-TFII gelegt. Das arterielle Markergen Hey2 ist ein Zielgen der Notch-Signalkaskade, während der molekulare Mechanismus der Rolle von COUP-TFII bei der venösen Differenzierung noch nicht bekannt ist. Daher wurde der Einfluss des arteriellen Markergens Hey2 und des venösen Markergens COUP-TFII auf die Notch-Signalkaskade untersucht, um ein besseres Verständnis über die molekularen Mechanismen der arterio-venösen Differenzierung zu gewinnen. Da humane primäre Endothelzellen mit kommerziell verfügbaren Transfektionsmitteln schwer transfizierbar sind, wurde zunächst ein lentivirales Vektorsystem etabliert. Hiermit wurde eine erfolgreiche und stabile Genexpression bzw. Genrepression in arteriellen und venösen Endothelzellen ermöglicht. Die Genmodulationen in arteriellen Endothelzellen, die durch die Expression des venösen Markergen COUP-TFII bzw. durch die Repression des arterellen Markergen Hey2 verursacht wurden, führten zu der neuen Erkenntnis, dass das venöse Markergen COUP-TFII in arteriellen Endothelzellen als ein Repressor des arteriellen Markergens Hey2 fungiert. Diese Repression wird durch eine direkte Bindung von COUP-TFII an den Hey2-Promotor vermittelt. Die COUP-TFII Expression bewirkte keine Veränderung in der Expression von Notch4, Dll4 und Nrp1. Dies könnte bedeuten, dass (i) COUP-TFII in arteriellen Endothelzellen kein Regulator von diesen Genen ist, (ii) Kooperationspartner von COUP-TFII fehlen, die in arteriellen Endothelzellen nicht vorhanden sind, oder (iii) der molekulare Mechanismus dieser Gene aufgrund seiner wichtigen Rolle nicht durch die Modifikation eines einzigen Gens beeinflussbar ist, da die Gene der Notch-Signalkaskade redundant kontrolliert werden. In venösen Endothelzellen wurden Genmodulationen durch Expression des arteriellen Markergens Hey2 bzw. durch die Repression des venösen Markergens COUP-TFII durchgeführt. Eine Expression des arteriellen Markergens Hey2 in venösen Endothelzellen konnte nicht die Expression der Gene der Notch-Signalkaskade aktivieren. Dies bedeutet, dass die Regulation dieser Gene durch einen übergeordneten molekularen Mechanismus gesichert ist. Interessanterweise konnte die Expression von Hey2 eine Reduktion der Hey1 Expression bewirken. Dies ist ein alternativer Effekt von Hey2 im Vergleich zu arteriellen Endothelzellen. Eine Repression des venösen Markergens COUP-TFII konnte die Expression der Gene Dll4, EphrinB2 und EphB4 induzieren. Vermutlich ist die Induktion der EphB4 Expression ein Kompensationsmechanismus auf die reduzierte COUP-TFII Expression. COUP-TFII sichert den venösen Phänotyp wahrscheinlich durch die Repression von EphrinB2 und Dll4, wobei die Reduktion von Dll4 vermutlich eine größere Bedeutung hat. Da Dll4 ein Ligand und Aktivator der Notch-Signalkaskade ist, ist seine Repression entscheidend für venöse Endothelzellen. Das arterielle Markergen Hey2 ist für die normale Embryogenese von großer Bedeutung, jedoch ist Hey2 als eines der Zielgene der Notch-Signalkaskade wahrscheinlich nicht in der Lage, molekulare Mechanismen, die zu unterschiedlichen endothelialen Phänotypen führen, zu aktivieren. Um arterielle Endothelzellen zu einem Reprogramming zu bewegen, wären wahrscheinlich Genmodulationen der Mitglieder der Notch-Signalkaskade, die upstream von Hey2 liegen, nötig. Hingegen ist die Rolle des venösen Markergens COUP-TFII in der Regulation der arterio-venösen Differenzierung von entscheidender Bedeutung. COUP-TFII spielt eine direkte Rolle in der Aufrechterhaltung der venösen Identität. Die Repression von COUP-TFII in venösen Endothelzellen bewirkt, dass sich die Expression des Gens Dll4, das die Notch-Signalkaskade aktiviert, in Richtung des arteriellen Expressionsniveaus bewegt. Für eine Reprogrammierung der venösen Endothelzellen in einen arteriellen Phänotyp ist das venöse Markergen COUP-TFII eines der Zielgene.

Submitted by Jefferson Filgueira Alcindo (jefferson.alcindo@yahoo.com.br) on 2018-04-18T19:55:54Z No. of bitstreams: 1 Tese doutorado Jefferson.pdf: 1217923 bytes, checksum: 08592edaa8bb1967844d2d8dd8a6ae88 (MD5) / Approved for entry into archive by Isabel Pereira de Matos (isabel@fmva.unesp.br) on 2018-04-19T13:33:02Z (GMT) No. of bitstreams: 1 alcindo_jf_dr_araca_int.pdf: 1217923 bytes, checksum: 08592edaa8bb1967844d2d8dd8a6ae88 (MD5) / Made available in DSpace on 2018-04-19T13:33:02Z (GMT). No. of bitstreams: 1 alcindo_jf_dr_araca_int.pdf: 1217923 bytes, checksum: 08592edaa8bb1967844d2d8dd8a6ae88 (MD5) Previous issue date: 2018-04-04 / Esse trabalho teve como objetivo avaliar, em um rebanho com presença de animais com sinais clínicos da enfermidade, a aplicabilidade e eficácia das medidas de controle para a atrite-encefalite caprina e perdas produtivas associadas. Os animais foram testados sorologicamente e algumas medidas de controle foram instituídas, entre elas: segregação dos rebanhos (positivo e negativo), retirada dos cabritos ao nascimento, aleitamento dos neonatos com colostro/leite de vaca ou de cabra tratados termicamente, linha de ordenha e manejo reprodutivo de acordo com status sorológico e descarte orientado de animais reagentes. Dados produtivos tais quais produção de leite, taxa de prenhez, peso dos animais adultos e crias, taxa de morbidade, mortalidade e descarte foram avaliados. Os custos relacionados à implantação do programa e perdas associadas à produtividade também foram analisados. A soroprevalência da doença decresceu a partir da segunda sorologia, atingindo a menor taxa na quarta sorologia (40,54%) e voltando a aumentar no final do estudo (50,72%). Animais soropositivos e com duas lactações produziram 0,26 litros a menos de leite diariamente quando comparados aos animais soronegativos. O peso das crias ao nascimento também foi menor em animais que provinham de mães sororeagentes. Conclui-se que as medidas de controle são aplicáveis em rebanhos comerciais, entretanto possuem sérias implicações quanto a sua eficácia em rebanhos com soroprevalência intermediária e alta, contribuindo pouco para a redução de novos casos. O impacto produtivo refletiu-se, principalmente, na produção de leite e o custo para implantação do programa de controle foi elevado. / The objective of this study was to evaluate the application and efficacy of control measures for caprine arthritis encephalitis and associated productive losses in a herd with the presence of animals with clinical signs of the disease. The animals were tested serologically and some control measures were instituted, including: segregation of herds (positive and negative), removal of goats at birth, suckling of newborns with colostrum / cow's milk or thermally treated goat's milk, management line and reproductive management according to serological status and discarded oriented of reactive animals. Productive data such pregnancy rate, adult and calf weight, mortality rate, mortality and discards. Costs related to program implementation and losses associated with productivity were also analyzed. The seroprevalence of the disease decreased after the second serology, with a lower rate in the fourth serology (40.54%) and increasing again at the end of the study (50.72%). Seropositive animals with two lactations produced 0.26 liters less milk daily when compared to seronegative animals. The weight of the offspring at birth was also lower in animals that came from mothers who were seroreagentes. It is concluded that control measures are approached, such as their efficacy in herds with intermediate and high seroprevalence, contributing little to a reduction of new cases. The productive impact was mainly reflected in milk production and in the cost to implement the high-level control program