Le cancer est l'une des principales causes de morbidité et de mortalité dans le monde avec environ 14 millions de nouveaux cas et 8,2 millions de décès liés aux cancers selon l'OMS (Organisation Mondiale de la Santé). Le cancer du poumon est le premier cancer diagnostiqué chez l'homme et compte pour 20% du nombre de mort par cancer par an dans le monde. Parmi les cancers pulmonaires, on distingue les cancers à petites cellules et les cancers non à petites cellules. Ce travail a porté sur l'étude des adénocarcinomes pulmonaires lépidiques, un type de cancer non à petites cellules, chez l'homme et l'animal. Chez l'homme, il fait partie des tumeurs pulmonaires rares, pas ou peu lié au tabagisme. Il est similaire cliniquement, radiologiquement et histologiquement à l'adénocarcinome pulmonaire ovin, un cancer du poumon induit par le β-rétrovirus JSRV (Jaagsiekte Sheep RetroVirus) affectant les petits ruminants domestiques. JSRV transforme les cellules épithéliales du parenchyme via son enveloppe oncogénique. Ces tumeurs humaines et animales ne sont pas associées au développement de métastases extra-thoraciques ou pleurales. Le but de notre travail a été d'étudier les évènements moléculaires spécifiques des adénocarcinomes lépidiques humains et animaux par i. l'analyse transcriptomique de l'expression des gènes dans ces deux cancers, ii. la caractérisation de l'adénocarcinome pulmonaire ovin par l'expression des mucines et iii. l'étude d'un rôle putatif des séquences rétrovirales endogènes dans l'induction de l'adénocarcinome lépidique humain. Premièrement, notre étude a porté sur l'analyse en parallèle des mécanismes moléculaires spécifiques des adénocarcinomes lépidiques prédominants par l'analyse des profils d'expression des gènes impliqués dans les voies de signalisation BMI1, NOTCH, Hedgehog et WNT importantes dans le développement du poumon normal et au cours des cancers pulmonaires et dans la voie de l'angiogenèse, permettant la dissémination des cellules tumorales. L'augmentation de l'expression de CDH1 (E-cadherin), acteur crucial de la voie BMI1, et la diminution de l'expression des ligands et récepteurs activateurs de l'angiogenèse VEGF/VEGFR (Vascular Growth Factor), PDGF/PDGFR (Platelet-derived Growth Factor) et ANGPT/TIE (Angiopoietin/Tyrosine kinase with immunoglobulin like and EGF like domains) ont été observées dans ces deux cancers. Lors de l'analyse de l'expression des ARNm et des protéines, nous avons mis en évidence le blocage de la voie principale de l'angiogenèse dans les cancers lépidiques. En effet, l'absence d'expression du ligand VEGFA (Vascular Endothelial Growth Factor A) et de son récepteur VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) a été observée. Ce blocage n'était pas compensé par la surexpression de gènes impliqués dans les voies alternatives d'angiogenèse. Le blocage de la voie de l'angiogenèse explique l'absence de métastases extrathoraciques dans les adénocarcinomes lépidiques chez l'homme et l'animal. Deuxièmement, les adénocarcinomes pulmonaires lépidiques chez l'homme sont subdivisés en deux entités majoritaires : les adénocarcinomes lépidiques prédominants non mucineux et les adénocarcinomes mucineux invasifs. Dans le contexte des analogies entre ces cancers humains et l'adénocarcinome induit par le virus JSRV, nous avons analysé l'expression des mucines dans les cancers animaux. Nous avons montré que sur 37 cancers pulmonaires induits par JSRV, 62% étaient non mucineux, qu'ils sur exprimaient MUC1 (mucin 1, cell surface associated) et que 50% exprimaient MUC5B (mucin 5B, oligomeric mucus/gel-forming). Par ces résultats, nous avons montré que les adénocarcinomes pulmonaires ovins se rapprochaient principalement des adénocarcinomes lépidiques prédominants / Cancer is the main cause of morbidity and mortality worldwide with 14 million new cases and approximately 8.2 million deaths according to the WHO (World Health Organization). Lung cancer is the most diagnosed cancer in men and account for 20% of cancer death in the world annually. Among lung cancers, there are small cell lung cancers and non-small cell lung cancers. My work focused on the lepidic adenocarcinoma, a subtype of nonsmall cell lung cancer, in humans and animals. In humans, it is a rare lung cancer and it is not or rarely linked to tobacco smoking. Lepidic adenocarcinomas share striking clinical, radiological and histopathological similarities with ovine pulmonary adenocarcinomas induced by the β-retrovirus JSRV (Jaagsiekte Sheep RetroVirus) affecting sheep and goats. JSRV transforms epithelial cells of the distal lung through its oncogenic envelope. These human and animal tumors are not associated with pleural or extra thoracic metastases. My work was to study specific molecular events in human and animal lepidic adenocarcinomas by i. a transcriptomic analysis of the gene expression in these two lung cancers, ii. ovine pulmonary adenocarcinoma characterization by mucin expression and iii. the study of a potential role for endogenous retroviral sequences in the induction of human lepidic adenocarcinomas. First, my work focused on the parallel analysis of expression patterns in genes implicated in BMI1, NOTCH, Hedgehog and WNT pathways important for lung development and for lung cancer development and the angiogenesis pathway whose activation leads to cancer cell dissemination. CDH1 (E-cadherin) up regulation and the down regulation of main angiogenic ligand and receptors VEGF/VEGFR (Vascular Endothelial Growth Factor), PDGF/PDGFR (Platelet-derived Growth Factor) and ANGPT/TIE (Angiopoietin/Tyrosine kinase with immunoglobulin like and EGF like domains) were observed. Then, when analyzing lepidic adenocarcinomas mRNA and protein expression, we highlighted the blockade of the main angiogenesis pathway. Indeed, the VEGFA ligand and its receptor VEGFR2 were absent in lepidic adenocarcinomas. Moreover, this blockade was not compensated with angiogenic alternative pathways. These results correlate with the absence of extra thoracic metastases in human and animal lepidic adenocarcinomas. Secondly, human lepidic predominant adenocarcinomas are divided in two groups, non-mucinous lepidic adenocarcinomas and invasive mucinous adenocarcinomas. To characterize ovine pulmonary adenocarcinomas and determine to which one of these two human lepidic adenocarcinomas they are close to, we analyzed mucine expression in animal lung cancer. On 37 JSRV-induced lung cancer, 62% were nonmucinous, 100% expressed MUC1 (mucin 1, cell surface associated) and 50% expressed MUC5B (mucin 5B, oligomeric mucus/gel-forming). These results highlighted that ovine pulmonary adenocarcinomas are closer to non-mucinous lepidic adenocarcinomas

In my doctoral project, I studied the evolution of retroviruses and long-term interactions with their hosts. Retroviruses infect a broad range of species including possibly all vertebrates. They are unique in their ability to efficiently create endogenous retroviruses (ERVs) - viral copies integrated into the host genomes and consequently inherited by successive generations as usual genomic locus. ERVs represent a significant portion of vertebrate genomes and play an important role in a variety of cellular processes and pathologies; however, their sequences are still largely unexplored. The results of my work contributed to the uncovering of ancient evolutionary history of retroviruses. In this regard, I employed the ERV sequences, as they represent "genetic fossils" of viral infections that occurred throughout entire retroviral evolution. By discovery and analysis of ancient ERV lineages, I shed light on the deep history of retroviruses and revealed how the past infections shaped the evolution of vertebrate antiviral defense. In addition to the investigation of retroviral evolution, I also studied process of ongoing endogenization and fixation of newly emerged ERVs in a mammalian host population. In this part of my work, I focused on a unique model of ERV that have been recently invading mule deer genome.

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.

Einleitung: Der seit über 30 Jahren ausgiebig erforschte Transkriptionsfaktor p53 besitzt offenbar Funktionen, die über seine bekannte und gut untersuchte Aktivität als Tumorsuppressor hinausgehen. So scheint er auch an der Regulation der menschlichen Lebenserwartung - über die Vermittlung einer allgemeinen physischen Robustheit - sowie der weiblichen Fertilität beteiligt zu sein. Insbesondere Primaten zeichnen sich durch eine vergleichsweise lange Lebenserwartung und eine lange reproduktive Phase aus. Ob p53 hier eine Rolle spielen könnte, ist unbekannt. Unsere Arbeitsgruppe entdeckte vor einigen Jahren das humane endogene Retrovirus-K (HERV-K) Protein Np9, dessen Gen sich in mehreren Kopien nur bei Menschen, Schimpansen und Gorillas findet. Weitere Untersuchungen wiesen außerdem darauf hin, dass Np9 an den Tumorsuppressor p53 zu binden vermag. Ziele der Untersuchungen: Es stellte sich also die Frage, ob die Funktion von p53 durch die Bindung an Np9 moduliert werden kann. Eine derartige Modulation des multifunktionellen Transkriptionsfaktors wäre natürlich auf Hominiden beschränkt. In der vorliegenden Arbeit sollten einige Teilaspekte der Interaktion von p53 und Np9 näher untersucht werden. Material und Methoden: Für die Bindungskartierung von p53 und Np9 wurden GST-Pulldown-Analysen durchgeführt. Die GST-Protein-Plasmide wurden in E.coli BL21 transformiert und nach Induktion mit IPTG exprimiert. Sie dienten als „Fängerproteine“ und waren dank ihres Glutathion-S-Transferase-tags in der Lage an GST-Sepharose-Kügelchen zu binden. Der putative Interaktionspartner als „Beuteprotein“ wurde in vitro translatiert und in diesem Zuge auch mit 35S radioaktiv markiert. Dann wurde er mit den an die Beads gebundenen GST-Proteinen inkubiert und anschließend die Proben auf ein SDS-Gel aufgetragen und aufgetrennt. Das Gel wurde anschließend auf eine Membran übertragen und der Blot auf einen Radioaktivfilm aufgelegt, woraufhin die Protein-Protein-Bindungen anhand des radioaktiven Beuteproteins als Banden erkennbar waren. Abschließend wurde der Blot mit GST-Antikörper inkubiert, dann am Folgetag mit Anti-Mouse-Antikörper. Mittels ECL Substrat konnte nun die Bindung der GST-getaggten Proteine an die Sepharosebeads nachgewiesen werden. Für den Electrophoretic Mobility Shift Assay wurden verschiedene Versuchsansätze pipettiert, welchen nach einer Inkubationszeit das zuvor mit 32P radioaktiv markierte Oligonukleotid zugegeben wurde. Nach erneuter Inkubation wurden die Proben auf das nicht-denaturiende EMSA-Gel aufgetragen und elektrophoretisch aufgetrennt. Dabei wurden die Protein-Oligonukleotid-Verbindungen gemäß ihrer Ladung, Größe und Konformation getrennt. Die Gele wurden im Geltrockner getrocknet und direkt mit einer Verstärkerfolie auf den Radioaktivfilm in einer Radioaktivkassette aufgelegt. Ergebnisse: Zunächst war es notwendig, die Bindung der beiden Partner biochemisch zu kartieren. Dies geschah mittels der GST-Pulldown-Analyse, in der Fragmente der Proteine exprimiert, miteinander inkubiert und schließlich kopräzipitiert wurden. Es stellte sich heraus, dass p53 mit seinem C-Terminus an Np9 bindet. Np9 hingegen band mit seinen Aminosäureresten (aa) 1-64 (ohne den C-terminus mit den aa 65-74) an p53. Das Np9-Fragment 36-74 zeigte nur eine schwache Bindung an p53. Interessanterweise band das Np9-Fragment 36-64 stärker an p53 als Volllängen-Np9 (1-74), was auf eine die Interaktion hemmende Domäne im C-Terminus von Np9 hinweisen könnte. Um zu untersuchen, ob die Bindung von Np9 an den C-Terminus von p53 die p53-DNA-Interaktion beeinflusst, wurden Electrophoretic Mobility Shift Assays (EMSAs) durchgeführt. Es konnte gezeigt werden, dass Np9-zumindest in vitro-durch Bindung an die regulatorische Domäne von p53 und in Anwesenheit des p53-aktivierenden Antikörpers PAb421 in der Lage war, die spezifische Bindungsfähigkeit von p53 an DNA zu erhöhen und somit seine Funktion als Transkriptionsfaktor zu unterstützen. Schlussfolgerungen: Die Resultate weisen also erstmals darauf hin, dass das nukleäre HERV-K Protein Np9 spezifisch in Hominiden eine p53-abhängige Tumorsuppressoreigenschaft aufweisen könnte. Weitere Untersuchungen-insbesondere in vivo-sind nun notwendig. Dies könnte auch als Forschungsgrundlage zu endogenen Retrovirusproteinen beim Pferd dienen.:1 Einleitung und Zielsetzung der Arbeit 1 2 Literaturübersicht 2 2.1 Der Tumorsuppressor p53 2 2.2 Das Kernprotein Np9 7 2.2.1 Retroviren 7 2.2.2 Endogene Retroviren 8 2.2.3 Humane endogene Retroviren 8 2.2.4 HERV-K 10 2.2.5 Das nukleäre Protein Np9 10 3 Material und Methoden 15 3.1 Material 15 3.1.1 Chemikalien 15 3.1.2 Puffer und Lösungen 17 3.1.3 Antikörper 21 3.1.4 Enzyme 22 3.1.5 Reaktionskits 22 3.1.6 Bakterienstämme 23 3.1.7 Kulturmedien 23 3.1.8 Oligonukleotide für EMSA 23 3.1.9 Größenstandards 24 3.1.10 Plasmide 26 3.2 Methoden 28 3.2.1 Nukleinsäuretechniken 28 3.2.2 Protein-Methoden 31 3.2.3 Prokaryonten 40 4 Ergebnisse 42 4.1 Interaktion zwischen Np9 und dem Tumorsuppressorprotein p53 42 4.2 GST-Pulldown 43 4.2.1 Klonierung für die GST-Pulldown-Analysen 43 4.2.2 Induktion der Proteinexpression 48 4.2.3 GST-Pulldown-Experimente 53 4.3 EMSA (Electrophoretic Mobility Shift Assay) 57 4.3.1 Radioaktive Markierung der Sonden 58 4.3.2 EMSA-Experimente 58 5 Diskussion 63 6 Zusammenfassung 68 7 Summary 70 8 Literaturverzeichnis 72 Danksagung 86 Abbildungsverzeichnis 87 Tabellenverzeichnis 88 / Introduction: The transcription factor p53, extensively investigated for over 30 years, apparently has functions which exceeds his known and well examined activity as a tumor suppressor. It seems to be involved in the regulation of the human life expectancy – by providing a general physical robustness - as well as of the female fecundity. Primates too are characterized by a comparatively long life expectancy and long reproductive phases, yet the possible influence of p53 is unknown. Our research group has discovered some years ago the Np9 protein of human endogenous retrovirus K (HERV K), which is found in several copies only with humans, chimpanzees and gorillas. Other investigations by our group suggested that Np9 might be able to interact with the tumor suppressor p53. Objective of the investigations: To study whether the function of p53 can be modulated by the interaction with Np9. Such a modulation of the multifunctional transcription factor would of course be limited to hominids. In the present work some aspects of the interaction between p53 and Np9 were analysed. Materials and methods: For the mapping of the interaction of p53 and Np9, GST pulldown assays were carried out. The GST protein plasmids were transformed in E. coli BL21 and expressed after IPTG induction. They served as bait proteins and bound to GST sepharose beads because of their Glutathione S-transferase-tags. The putative interaction partner as a prey protein was translated in vitro and radioactively marked with 35S. After being incubated with the GST-proteins bound to the beads, the samples were transferred on a SDS gel and separated. The gel was transferred to a membrane and the blot was exposed to an X-ray film. Thus, the radioactively labelled prey protein forms bands that identify the protein-protein interaction. Finally the blot was incubated with GST antibody, then on the following day with anti-mouse antibody. Using ECL-substrate it was now possible to demonstrate that the GST-tagged proteins bound to the sepharose beads. For the Electrophoretic Mobility Shift Assay different samples were prepared and, after an incubation time, the oligonucleotide radioactively marked with 32P was added. After additional incubation it was transferred on non-denaturating EMSA gel and separated by electrophoresis. Thus the protein oligonucleotide conjugates were separated according to charge, size and conformation. The gels were dried in the gel dryer, transferred to a membrane and placed against an X-ray film in a cassette. Results: Initially a biochemical mapping of the binding of the two partners had to be carried out. This was done by means of the GST pulldown assay, in which fragments of the proteins were extruded, incubated together and finally co-precipitated. It turned out that the C-terminus of p53 bound to Np9. However, Np9 bound to p53 with his amino acid residues (aa) 1-64 (lacking the C-terminal aa 65-74). The Np9 fragment 36-74 showed only a weak binding to p53. Interestingly the Np9 fragment 36-64 was binding stronger to p53 than a full length Np9 (1-74), which could point to a C-terminal domain in Np 9 inhibiting the interaction. In order to examine whether the binding of Np9 to the C-terminal of p53 affects the interaction of p53 with DNA, Electrophoretic Mobility Shift Assays (EMSAs) were carried out. It could be shown that Np9 was able to raise the specific binding ability of p53 with DNA and to support therefore its function as a transcription factor, by binding to the regulatory domain of p53 in presence of the activating p53 antibody PAB421. Conclusions: The results show for the first time that, specifically in hominids, the nuclear HERV-K protein Np9 could have a tumor suppressing quality that is dependent on p53. Further investigations, in particular in vivo, are necessary. This could be the starting point for research on equine endogenous retrovirusproteins in horses.:1 Einleitung und Zielsetzung der Arbeit 1 2 Literaturübersicht 2 2.1 Der Tumorsuppressor p53 2 2.2 Das Kernprotein Np9 7 2.2.1 Retroviren 7 2.2.2 Endogene Retroviren 8 2.2.3 Humane endogene Retroviren 8 2.2.4 HERV-K 10 2.2.5 Das nukleäre Protein Np9 10 3 Material und Methoden 15 3.1 Material 15 3.1.1 Chemikalien 15 3.1.2 Puffer und Lösungen 17 3.1.3 Antikörper 21 3.1.4 Enzyme 22 3.1.5 Reaktionskits 22 3.1.6 Bakterienstämme 23 3.1.7 Kulturmedien 23 3.1.8 Oligonukleotide für EMSA 23 3.1.9 Größenstandards 24 3.1.10 Plasmide 26 3.2 Methoden 28 3.2.1 Nukleinsäuretechniken 28 3.2.2 Protein-Methoden 31 3.2.3 Prokaryonten 40 4 Ergebnisse 42 4.1 Interaktion zwischen Np9 und dem Tumorsuppressorprotein p53 42 4.2 GST-Pulldown 43 4.2.1 Klonierung für die GST-Pulldown-Analysen 43 4.2.2 Induktion der Proteinexpression 48 4.2.3 GST-Pulldown-Experimente 53 4.3 EMSA (Electrophoretic Mobility Shift Assay) 57 4.3.1 Radioaktive Markierung der Sonden 58 4.3.2 EMSA-Experimente 58 5 Diskussion 63 6 Zusammenfassung 68 7 Summary 70 8 Literaturverzeichnis 72 Danksagung 86 Abbildungsverzeichnis 87 Tabellenverzeichnis 88

Spumaretroviren, oder Foamyviren (FV), unterscheiden sich von Orthoretroviren durch mehrere Besonderheiten in ihrer Replikationsstrategie. Das Partikel-assoziierte Hüllglykoprotein (Env-Protein) des „Prototype Foamy Virus“ (PFV) ist im Vergleich zu anderen retroviralen Hüllglykoproteinen einzigartig. Die Koexpression des PFV Env-Proteins für die PFV-Partikelfreisetzung ist essenziell und die spezifische Funktion kann nicht von heterologen viralen Env-Proteinen übernommen werden. Das Env-Protein des PFV durchläuft eine für ein Membranglykoprotein ungewöhnliche Biosynthese. Das Env-Vorläuferprotein besitzt zu Beginn eine Typ-III-Membrantopologie, bei der der N- und der C-Terminus im Zytoplasma lokalisiert sind. Während des Transports zur Zelloberfläche wird es posttranslational durch bisher unbekannte zelluläre Proteasen in mindestens drei Untereinheiten gespalten. Das N-terminale Signalpeptid bzw. Leader-Peptid (LP) hat eine Typ-II-Membrantopologie, mit dem N-Terminus im Zytoplasma und dem C-Terminus im Lumen, wohingegen die Transmembran (TM)-Untereinheit eine Typ-IMembrantopologie besitzt, bei der der N-Terminus im Lumen und der C-Terminus im Zytoplasma lokalisiert sind. Die interne Oberflächen (SU)-Untereinheit assoziiert vermutlich im Lumen mit der extrazellulären Domäne der TM-Untereinheit. Im Rahmen dieser Arbeit wurde der Beweis erbracht, dass Furin oder Furin-ähnliche Proteasen und nicht der Signalpeptidase-Komplex für beide proteolytischen Spaltungen verantwortlich sind. Durch die N-terminale Sequenzierung der SU- und der TM-Untereinheit eines aufgereinigten PFV Env-Immunoadhäsionsproteins wurden N-terminal von beiden Spaltstellen Furin- Konsensussequenzen identifiziert. Mutationsanalysen von zwei sich in diesem Bereich überlappenden minimalen Furin-Konsensussequenzen an der PFV LP/SU-Spaltstelle im wildtypischen PFV Env-Protein bestätigten die Ergebnisse der N-terminalen Sequenzierung und bewiesen, dass nur die erste Spaltstelle genutzt wird. Obwohl diese Mutanten aufgrund geringerer Partikelfreisetzung einen signifikanten Verlust der Infektiosität zeigten, wurde keine Korrelation zur Inhibierung der Spaltung beobachtet, da andere Mutanten mit normaler LP/SU-Spaltung einen ähnlichen Defekt besaßen. Virale Env-Proteine initiieren den Eintritt membranumhüllter Viren in die Wirtszelle durch die Bindung an zelluläre Rezeptoren. Dabei führen Konformationsänderungen in den Env- Proteinen zum Verschmelzen der Virusmembran mit der Zellmembran und weiterhin zur Aufnahme des Kapsids in das Zytoplasma der Wirtszelle. Die foamyviralen Env-Proteine sind in dieser Hinsicht keine Ausnahme und vermitteln die Anheftung an die Wirtszelle durch die Bindung an den bisher unbekannten zellulären Rezeptor. Der zelluläre foamyvirale Rezeptor ist vermutlich ein ubiquitäres Molekül, denn bisher konnte keine Zelllinie identifiziert werden, die gegen FV-Infektionen resistent ist. Bislang existieren nur sehr wenig strukturelle und funktionelle Informationen der extrazellulären Domänen des PFV Env-Proteins. Deshalb wurde im Hauptteil dieser Arbeit die PFV Env-Rezeptorbindungsdomäne (RBD) charakterisiert. Hierfür wurden rekombinante PFV Env-Immunoadhäsionsproteine verwendet und deren Bindungskapazitäten an Zielzellen in der durchflusszytometrischen Analyse bestimmt. Untersuchungen zeigten, dass sowohl die extrazelluläre Domäne der C-terminalen TM-Untereinheit als auch der Transport der Immunoadhäsionsproteine durch das spezifische PFV Env LP zum sekretorischen Weg für die Bindung an Zielzellen entbehrlich sind und ließen vermuten, dass die PFV Env-RBD innerhalb der SU-Untereinheit lokalisiert ist. N- und C-terminale Deletionsanalysen der PFV Env SU-Untereinheit enthüllten eine minimale kontinuierliche RBD von AS 225 bis 555. Interne Deletionen im PFV Env-Protein von AS 397 bis 483 wurden im Gegensatz zu deletierten Regionen von AS 262 bis 300 und AS 342 bis 396 ohne signifikanten Einfluss auf die Wirtszellbindung in Immunoadhäsionsproteinen toleriert. Die Analyse der Immunoadhäsionsproteine mit einzelnen substituierten Cysteinen in der PFV Env SU-Untereinheit zeigten, dass nur die Immunoadhäsionsproteine, die in der nicht essenziellen Region von AS 397 bis 483 lokalisierte Cysteine ersetzt hatten, eine Restbindungskapazität behielten. Interessanterweise zeigte die Analyse von verschiedenen N-Glykosylierungsmutanten eine bedeutende Rolle der Kohlenhydratkette an Position N391 im PFV Env-Protein entweder hinsichtlich der direkten Interaktion mit dem zellulären Rezeptor oder für die korrekte Faltung der PFV Env-RBD. Diese Ergebnisse weisen darauf hin, dass ein diskontinuierliches Sequenzmotiv von AS 225 bis 396 und AS 484 bis 555 für die Bildung der PFV Env-RBD essenziell ist und die darin lokalisierte potenzielle achte N-Glykosylierungsstelle eine entscheidende Rolle bei der Wirtszellbindung spielt. / Spumaretroviruses or foamy viruses (FVs) use a replication pathway with features distinctive from orthoretroviruses. The particle-associated envelope (Env) glycoprotein of prototype foamy virus (PFV) is unique compared to other retroviral envelope proteins since its coexpression is strictly required for the FV particle release process and its function cannot be replaced by heterologous viral glycoproteins. The PFV Env glycoprotein shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N-and C-terminus located in the cytoplasm. During its transport to the cell surface, it is posttranslationally processed by yet-unidentified cellular proteases into at least three subunits. The N-terminal signal or leader peptide (LP) has a type II membrane topology, whereas the C-terminal transmembrane (TM) subunit has a type I membrane topology. The internal surface (SU) subunit presumably associates with extracellular domains of TM on the luminal side. Here we provide strong evidence that furin itself or furin-like proteases and not the signal peptidase complex are responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping minimal furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect. Viral Env proteins initiate entry of membrane enveloped viruses into cells by binding to cell surface receptors followed by conformational changes leading to membrane fusion and delivery of the genome containing viral capsid to the cytoplasm. The Env glycoproteins of FVs are no exception and mediate attachment to host cells through binding to an yet unknown ubiquitous cellular receptor molecule because no cell type is currently known that is resistant to FV entry. Little structural and functional information on the extracellular domains of PFV Env is available. In this study we characterized the PFV Env receptor-binding-domain (RBD) by flow-cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. Analysis showed that the extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding suggesting that the PFV Env RBD is contained within the SU subunit. N- and C- terminal deletion analysis of the SU domain revealed an minimal continuous RBD spanning aa 225-555, however internal deletions covering the region from aa 397-483, but not aa 262-300 or aa 342-396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV Env SU revealed that only most of those located in the non-essential region from aa 397-483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of the carbohydrate chain attached to N391 either for direct interaction with the cellular receptor or for correct folding of the PFV Env RBD. Taken together these results suggest that a bipartite sequence motif spanning aa 225-396 and aa 484-555 is essential for formation of the PFV Env RBD, with N-glycosylation site 8 playing a crucial role for host cell binding.

Syncytin-1 is an endogenous retroviral envelope glycoprotein specifically expressed in human placenta, where the protein was adopted for its physiological function. After interaction with specific receptors, transmembrane proteins ASCT1 and ASCT2, Syncytin-1 initiates cell-cell fusion leading to formation of multinucleated syncytiotrophoblast, which is essential for feto-maternal nutrients exchange. In this diploma thesis a new cell-cell fusion quantification assay was implemented for characterisation of Syncytin-1 fusion determinants. The assay uses Syncytin-1 and ASCT2 expressed separately with fragments of luciferase in heterologous cell-culture system. The assay enables to specifically quantify cell-cell fusions based on activity of reconstituted luciferase reporter. This study discovered new facts about the role of intracytoplasmic tail of Syncytin-1 in the process of the cell- cell fusion. This specific part of protein contains a tandem motif sensitive to changes in amino acid sequence that led to loss of fusogenic potential of Syncytin-1. It was further confirmed, that the protein Suppressyn works as an inhibitor of cell-cell fusions initiated by Syncytin-1. Suppressyn however does not bind to receptors of Syncytin-1 and the mechanism of its inhibition remains unsolved. Finally, it was demonstrated...

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

Thesis advisor: Welkin Johnson / Endogenous Retroviruses (ERVs) are “fossilized” retroviruses of a once exogenous retrovirus located in the genome of extant vertebrates. Retroviral infection results in a provirus integration into the host genome. An infection of a germline cell could lead to the provirus potentially being inherited by the offspring of the infected individual. Once in the genome, the provirus becomes subject to evolutionary processes and can become either lost or fixed in a population, remaining as “fossils” long after the exogenous retrovirus has gone extinct23. Notably, 8% of the human genome consists of ERVs30. Human Endogenous Retrovirus Type K (HERV-K)(HML-2) family is of particular interest. HERV-K integrations are as old as 30-35 million years, endogenizing before the separation of humans and Old World Monkeys. However, there are human specific insertions, some as young as 150,000 – 250,000 years, making them the youngest insertion in the human genome. There are over 90 insertions in the human genome; the bulk is shared by all humans44,47. Transcripts of HERV-K genes are upregulated in multiple cancer and tumor cell lines 14,39,46, as well as in HIV-1 infected patients 7,11,29. Just as there are human specific insertions of ERV-K, there are also Old World Monkey specific insertions44. I have identified an intact endogenous retroviral envelope open reading frame on chromosome 12 of the rhesus macaque genome. This viral envelope-encoding sequence, which I refer to as rhERV-K env, retains all the canonical features of a retroviral Env protein. An alignment between rhERV-K env and a consensus sequence of HERV-K, HERV-Kcon env, shows a 70% amino acid sequence identity. For experimental purposes, reconstructed HERV-K envelopes have been incorporated into virions of Human Immunodeficiency virus (HIV-1)19,26,49, Murine Leukemia Virus (MLV)12, and Vesicular stomatitis Virus (VSV)26,41,49. While these approaches have illuminated some aspects of HERV-K Env-mediated entry, to date a cell-surface receptor has not been identified for any ERV-K Env. This could be due to its low infectivity levels12,26,49, its seemingly broad cell tropism limiting identification of null cell lines26,49, or possibly the HERV-K consensus reconstructions are not an accurate representation of the progenitor HERV-K virus. I am interested in understanding how the ERV-K retrovirus accessed the human germline (some 150,000 – 250,000 years ago). To do this, I focused specifically on the envelope proteins of HERV-K and rhERV-K, with the goal of analyzing the ERV-K entry process. The identification and inclusion of rhERV-K Env in this study is meant to circumvent the possibility that the previously described consensus reconstructions of human HERV-K Env are not representative, and may also provide a means to compare the endogenization process in the human/ape and old-world monkey lineages. I focused on developing two systems for single-cycle infection, one based on Mason-Pfizer Monkey Virus (MPMV) (which has not been done before), and a second based on MLV, which has previously been reported on. MPMV, like HERV-K, is a betaretrovirus, and I reasoned that possibly using a betaretrovirus would overcome some of the low-infectivity issues associated with prior attempts using HIV and MLV. To develop a system for examining function of the ERV-K Env proteins, I addressed 3 issues: 1. Are the HERV-K Env and rhERV-K Env proteins expressed and properly processed? 2. Can they be incorporated into virions of a heterologous virus? 3. Are ERV-K pseudotyped virions infectious? I have answered these questions in the following thesis. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

La domestication des bétails représente une étape importante dans l'histoire de l'humanité. Le mouton était l'un des premiers animaux à être domestiqués dans le croissant fertile. Ces événements de domestication, probablement initiés au début du Néolithique, ont génétiquement construit les races contemporaines du Moyen-Orient mais aussi du monde entier. L'élevage de moutons, principalement mouton de la race Awassi, représente une activité économique essentielle du Liban ; cependant, jusqu'à présent, il n'existe que très peu de données génétiques sur cette race. De nos jours, les outils moléculaires disponibles nous permettent de définir en détail la diversité génétique des populations de moutons et de retracer leur histoire évolutive. Par conséquent, l'objectif principal de mon projet de thèse était de caractériser génétiquement la race Awassi du Liban. Pour cette étude, 277 échantillons d'ADN génomique prélevés des moutons Awassi du Liban (n = 254) et de la Syrie (n = 23) ont été analysés. Au début, nous avons utilisé cinq rétrovirus endogènes (rétrovirus endogène de moutons de Jaagsiekte-enJSRV) qui sont polymorphiques par insertion dans les génomes du mouton domestique (enJSRV-18, -7, -15, -16 et -22) et ont été précédemment considérés comme très informatifs principalement pour distinguer génétiquement les moutons primitifs des races plus modernes (c.-à-d. le dernier issu de l'épisode migratoire impliquant des moutons avec des traits de production améliorés). En utilisant cette approche, nos résultats montrent une prédominance du type R2 (enjSRV-18 seulement) confirmant que le mouton Awassi du Liban est une race moderne. Comme prévu, le rétrotype R4 (à la fois enJSRV-18 et enJSRV-7), une caractéristique commune des populations de moutons du bassin méditerranéen, se trouve également dans le génome des moutons d'Awassi du Liban et plus accentué dans les troupeaux Syriens. Il est intéressant de noter que les populations de moutons d'Awassi situés dans le nord-est du Liban et ayant ainsi un accès plus restreint à la mer Méditerranée que les autres populations (c'est-à-dire en raison de la chaîne de montagne centrale qui coupe le pays sur deux), présentent une faible fréquence de R4. Bien que l'origine des animaux utilisés pour établir les troupeaux analysés au cours de cette étude soit inconnue, nos résultats fournissent également certaines preuves que le mode d'élevage (ouvert ou fermé) peut influencer les rétrotypes observés et en particulier le R4. De manière surprenante, au cours de cette étude, nous avons également dévoilé la présence de soi-disant "Solo-LTR" (c'est-à-dire généré par une recombinaison homologue) pour un autre enJSRV (enJSRV-6) qui prédomine dans deux troupeaux d'une région particulière du Liban (Nabatieh). Et comme approche complémentaire, deux marqueurs mitochondriaux ont été utilisés, le cytochrome b (Cyt-b) et D-Loop, pour étudier l'origine maternelle de cette race et sa relation phylogénétique au sein de la famille Ovis aries. Dans notre étude, le Cyt-b se révèle plus discriminant que le D-Loop. Des mouton d'Awassi analysé, quatre haplogroupes (HPG) du Moyen-Orient ont été trouvés avec l'analyse du Cyt-b : HPG A, B, C et E, ce dernier étant peu fréquent. De même, l’analyse de la super-séquence, alignement Cyt-b_D-Loop, a permis l’identification de l’HPG D, un HPG extrêmement rare et limité jusqu’à présent aux moutons à queue grasse tel que l’Awassi. Enfin, une expansion passée de la population est observée pour les HPG A, B et C (mais pas pour HPG E) avec les distributions incompatibles et des tests de neutralité négatifs significatifs. Dans l'ensemble, les résultats obtenus au cours de cette étude fournissent une caractérisation génétique complète ainsi que quelques idées sur la structure phylogéographique des populations de moutons de la race Awassi au Liban. / Livestock domestication represents a milestone in the history of mankind. Sheep was one of the first animals to be domesticated in the Fertile Crescent. These domestication events, probably initiated in the early Neolithic, have genetically built the contemporary races of the Middle East but also of the whole world. Sheep farming, mainly sheep of Awassi breed, represents an essential economic activity of Lebanon; however, so far, only very few genetic data exist on this breed. Nowadays, the molecular tools available allow us to define in details the genetic diversity of sheep populations and to trace their evolutionary history. Hence, the main objective of my PhD project was to genetically characterize the Awassi breed of Lebanon. For this study, 277 genomic DNA samples collected from Awassi sheep of Lebanon (n=254) and Syria (n=23) were analyzed. Initially, we used five endogenous retroviruses (endogenous Jaagsiekte sheep retrovirus-enJSRV) that are insertionally polymorphic within the genomes of domestic sheep (enJSRV-18, -7, -15, -16 and -22) and have been previously shown to be very informative mainly to genetically distinguish between primitive sheep from more modern breeds (i.e. the latter originating from the migratory episode involving sheep with improved production traits). Using this approach, our results show a predominance of the R2 retrotype (enJSRV-18 only) confirming that the Awassi sheep of Lebanon is a modern breed. As expected, the R4 retrotype (both enJSRV-18 and enJSRV-7), a common feature of the sheep populations present within the Mediterranean area, is also found in the Awassi sheep of Lebanon and to more extend in those of Syria. Interesting, the populations of Awassi sheep located in the northeast of Lebanon and thus having a more restricted access to the Mediterranean Sea than the other populations (i.e. due to the central mountain chain cutting the country in two) present R4 weaklier. Even though the origin of the animals used to establish the herds analyzed during this study is unknown, our results also provide some evidences that the mode of rearing (open or closed) may influence the observed retrotypes and in particular R4. Surprisingly, during this study, we also unveiled the presence of so-called “Solo-LTR” (i.e. generated by homologous recombination) for another enJSRV (enJSRV-6) that are predominant in two herds of a particular region of Lebanon (Nabatieh). As a complementary approach, two mitochondrial markers were used, the cytochrome b (Cyt-b) and D-Loop, to investigate the maternal origin of this breed and its phylogenetic relationship within the Ovis aries family. In our study, the Cyt-b turns out to be more discriminative than the D-Loop. From the Awassi sheep analyzed, four haplogroups (HPGs) of the Middle-East were found with Cyt-b analysis: HPG A, B, C and E, the latter being the least frequent. Also, the super-sequence analysis, Cyt-b_D-Loop alignment, allowed the identification of HPG D, an extremely rare HPG, limited till now to fat-tailed sheep such as Awassi. Finally, a past population expansion is observed for the HPG A, B and C (but not for HPG E) with mismatch distributions and significant negative neutrality tests. Overall, the results obtained during this study provide a comprehensive genetic characterization as well as some insights into the phylogeographic structure of the sheep populations of the Awassi breed in Lebanon.

Retroviruses are viruses which are able to integrate to genome of host cell. Nonrandom integration of provirus near or inside some cellular genes may result in their deregulation, activation or silencing. This can later lead to cell transformation and tumor formation. This thesis discusses identification of viral integration sites (VIS) and common integration sites (CIS) in tumors originating from different organs (mostly kidneys, lungs and liver) with using mostly avian retroviruses subgroup J, specifically first natural isolate HPRS-103 and laboratory made virus MAV-J, which was made by replacing gene envB by envJ. Infection was made in ovo using chicken breeds Brown Leghorn and White Leghorn and tumors were isolated from 8 to 28 weeks after infection. For molecular analyses was used inverse PCR method and sequencing. From 74 molecularly analyzed tumors there was detected 373 VIS and 6 CIS with statistical significance over 2.10-2 . Gene with the highest number of hits was FRK (14 times), then TERT (5 times), CTDSPL (5 times), EGFR/ERBB1 (3 times), MYB (3 times) and MYC (3 times). Except 6 CIS there were other genes found, which had smaller statistical significance. Keywords: retrovirus, insertional mutagenesis, subgroup J, oncogenesis, oncogenes, MAV-J, HPRS-103, proviral integration sites, tumors