O P. vivax é a segunda espécie mais prevalente causadora de malária no mundo. Medidas de controle ineficientes exigem o desenvolvimento de novas estratégias de prevenção, como vacinas, novas drogas e novos inseticidas. O objetivo geral do trabalho foi gerar uma formulação vacinal universal com proteínas e adenovírus recombinantes capazes de induzir anticorpos contra as diferentes formas alélicas da proteína circumsporozoíta (CSP) do P. vivax. As proteínas foram produzidas em E. coli e purificadas por cromatografia de afinidade e troca iônica. A obtenção destas proteínas nos permitiu testar qual seria a melhor formulação vacinal para a indução de anticorpos contra as três formas alélicas da proteína CSP de P. vivax (PvCSP). Anticorpos específicos reconheceram esporozoítas do P. vivax por imunofluorescência. Por fim testamos o uso de dois adenovírus recombinantes, um símio e um humano, deficientes em replicação, expressando as três regiões imunodominantes da proteína PvCSP em fusão. Estes foram capazes de induzir resposta imune específica contra as proteínas PvCSP sendo testados em esquema de prime-boost heterólogo, onde camundongos foram primados com os adenovírus e nas doses-reforço receberam a mistura com as três proteínas recombinantes. / The Plasmodium vivax is the second most prevalent species of malaria in the world. Inefficient measures of control used today demand the development of new strategies for prevention, as vaccines, new drugs and new insecticides. The central objective of this thesis was to generate a universal vaccine formulation with proteins and recombinant adenoviral vectors representing the different allelic forms of the circumsporozoite protein (CSP) of the P. vivax. The recombinant proteins were expressed in E. coli and purified. These proteins allowed us to test which would be the best vaccine formulation for the induction of antibodies against the three allelic forms of CSP. The specific antibodies also recognized P. vivax sporozoites by immunofluorescence. Finally we test the use of two recombinant adenoviral vectors, a simian and a human, both replication deficient, expressing a protein containing the repeat regions of the CSP in fusion. These adenoviral vectors induced specific immune response against CSP and were successfully used in an immunization regimen of heterologous prime and boost where in the first dose the mice received recombinant adenoviral vector and in the subsequent doses, the mixture with three recombinant proteins.

L’Adénovirus (AdV) entre dans la cellule hôte par endocytose puis s’échappe de l’endosome en lysant la membrane de ces vésicules, empêchant ainsi sa dégradation via les lysosomes. Or, les membranes endommagées sont reconnues comme des signaux de danger par le système immunitaire et peuvent déclencher une réponse antivirale, telle que l’expression d’interféron (IFN). Dans nos conditions expérimentales, nous avons montré que l’infection par l’AdV n’induit pas l’expression d’IFNβ et qu’au contraire, le virus semble inhiber cette réponse. En revanche, l’entrée du virus active TBK1 (Tank Binding Kinase 1) qui est une kinase clef de la voie IFN mais qui est également impliquée dans la régulation de l’autophagie, une voie de dégradation cellulaire. Notre laboratoire a précédemment montré que l’autophagie est activée lors de l’entrée de l’AdV, par la rupture de la membrane endosomale. Nous avons donc étudié le mécanisme d’activation et le rôle de TBK1 lors de l’infection par l’AdV. Nos résultats montrent que la rupture de la membrane endosomale induite par le virus est nécessaire pour l’activation de TBK1 et que cette kinase est recrutée spécifiquement sur les sites de dommage membranaire. De plus, nous avons montré que TBK1 est impliqué dans l’activation de l’autophagie induite par l’AdV. Cependant, contrairement à ce qui est décrit pour l’autophagie dirigée contre les bactéries, cette activation de TBK1 est indépendante de NDP52 et d’autres adaptateurs conventionnels de l’autophagie. En résumé, nos travaux montrent que l’AdV est capable de contrôler la réponse IFN et que les ruptures de membrane induites par le virus activent TBK1 et l’autophagie par un nouveau mécanisme. Nos données suggèrent un rôle conservé de TBK1 dans l’activation de l’autophagie sélective contre les agents pathogènes. / Adenoviruses enter host cells by endocytosis and then escape from the endosomal compartment by lysing the endosomal membrane, thereby preventing its degradation via lysosomes. However, damaged membranes are recognized as danger signals by the cell intrinsic immune system and trigger an antiviral response, such as expression of interferon (IFN). In our experimental conditions we have shown that adenovirus infection does not induce the expression of IFNβ. On the contrary, our data suggest that the virus appears to inhibit the IFNβ response. However, adenovirus entry activates TBK1 (Tank Binding Kinase 1), which is a key kinase of the IFN pathway but is also involved in the regulation of autophagy, a cellular degradation pathway. Our laboratory previously showed that autophagy is activated upon rupture of the endosomal membrane during adenovirus entry. We therefore studied the activation mechanism and the role of TBK1 during adenovirus infection. Our results show that virus-induced endosomal membrane rupture is required for activation of TBK1 and that this kinase is specifically recruited at membrane damage sites. In addition, we show that TBK1 is involved in the activation of autophagy induced by adenovirus. TBK1 activation is independent of NDP52 and other conventional autophagic adapters, which is in contrast to membrane damaging bacteria. Thus, autophagy targeting membrane penetrating adenoviruses differs from the one induced by bacteria. In summary our work shows that adenovirus is able to control the IFN response and that membrane rupture induced by adenoviruses activates TBK1 and autophagy by a novel mechanism. In contrast our data suggest a conserved role for TBK1 in driving selective autophagy against invading pathogens.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os adenovÃrus (Ad) sÃo agentes importantes de infecÃÃes respiratÃrias agudas (IRAs) em crianÃas, especialmente entre seis meses e cinco anos de idade. Este foi um estudo retrospectivo que teve como objetivos: estabelecer o perfil epidemiolÃgico e clÃnico das infecÃÃes respiratÃrias agudas causadas por Ad em crianÃas atendidas no Hospital Infantil Albert Sabin, na cidade de Fortaleza, no perÃodo de janeiro de 2001 a dezembro de 2006; observar a freqÃÃncia de detecÃÃo de Ad em casos de IRAs em crianÃas atendidas nesse hospital; descrever as principais caracterÃsticas clÃnicas das infecÃÃes respiratÃrias causadas por Ad; pesquisar a existÃncia de padrÃo de sazonalidade do Ad na cidade de Fortaleza; determinar a taxa de isolamento do Ad em cultura de cÃlulas HEp-2 inoculadas com amostras de secreÃÃes de nasofaringe (SNF) consideradas positivas para Ad por imunofluorescÃncia indireta (IFI); e criar um banco de cepas de Ad para futuros estudos sobre diversidade antigÃnica e genÃmica desses agentes. Foram coletadas nesse perÃodo, amostras de SNF de 3070 crianÃas com atà sete dias de sintomas de IRA. Todas as amostras foram submetidas a IFI e, dessas, 54 se mostraram positivas para Ad. Quarenta e uma dessas amostras positivas foram inoculadas em monocamadas de cÃlulas HEp-2, obtendo-se isolamento viral em 32 delas. Outras 103 amostras negativas por IFI, escolhidas aleatoriamente, foram inoculadas, resultando em trÃs isolamentos adicionais, totalizando 57 casos confirmados de infecÃÃo por Ad. A freqÃÃncia de detecÃÃo de Ad foi de 1,86% em relaÃÃo ao total de casos de IRAs e 6,1% das IRAs virais. NÃo foi observado um padrÃo de sazonalidade nem correlaÃÃo com perÃodo chuvoso ou seco. A maior parte das IRAs por Ad ocorreu em crianÃas na faixa etÃria entre sete e 24 meses com 63,15% dos casos. As IRAs por Ad foram observadas principalmente em crianÃas atendidas em ambulatÃrios (50,88%), e o diagnÃstico predominante foi a infecÃÃo de vias aÃreas superiores (70,18%). Os principais sintomas e sinais apresentados pelos pacientes com IRA por Ad foram febre, tosse, coriza, anorexia, vÃmitos e/ou diarrÃia, obstruÃÃo nasal e dispnÃia. A principal conduta terapÃutica aplicada em casos de IRAs por Ad foi o uso de aerossol / salbutamol (42,11%) / Adenoviruses (Ad) are important etiological agents of acute respiratory infections (ARI) in children, particularly between six months and five years old. This was a retrospective study, whose objectives were: to perform clinical and epidemiologic profile of adenoviral respiratory infections in children attended in Hospital Infantil Albert Sabin, in the city of Fortaleza, from January 2001 to December 2006; to observe the detection frequency of Ad in cases of ARI in children attended in that hospital; to describe the main clinical features of adenoviral respiratory infections; to search for the existence of a seasonal pattern of adenoviral infections in the city of Fortaleza; to determine the isolation rate of Ad in HEp-2 cell lines inoculated with samples of nasopharyngeal secretions (NPS) considered positive to Ad by indirect immunofluorescence (IIF); and to create a collection of Ad strains for future studies on antigenic and genomic diversity of these agents. NPS samples of 3,070 children with ARI up to seven days of the onset of symptoms were collected. IIF was applied to all of the samples, and among them, 54 were positive to Ad. Forty one of those positive samples were inoculated into HEp-2 cells, resulting in 32 viral isolations. Other 103 randomly choosen negative samples were also inoculated, resulting in more three isolations, reaching the number of 57 confirmed cases of Ad infections. The detection frequency of Ad was 1.86% of total number of cases of ARI and 6.1% of cases of viral ARI. It was not observed any seasonal pattern or correlation to rainy or dry season. Most cases of adenoviral ARI occurred in children aged seven to 24 months, representing 63.15% of cases. Ad ARI was observed mainly in children attended in the out-patient facility (50.88%), and the predominant diagnosis was upper respiratory tract infection (70.18%). The main clinical features in Ad patients were fever, cough, rhinorrhea, anorexia, vomiting or diarrhea, nasal obstruction, and dyspnea. The main therapeutic management for Ad patients was use of nebulization (about 42% of patients)

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-14T19:55:31Z No. of bitstreams: 2 Tese - Hugo Delleon da Silva - 2013.pdf: 14374996 bytes, checksum: d653dbc3306ce002b867a103b5a20bb4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-16T18:33:50Z (GMT) No. of bitstreams: 2 Tese - Hugo Delleon da Silva - 2013.pdf: 14374996 bytes, checksum: d653dbc3306ce002b867a103b5a20bb4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-16T18:33:50Z (GMT). No. of bitstreams: 2 Tese - Hugo Delleon da Silva - 2013.pdf: 14374996 bytes, checksum: d653dbc3306ce002b867a103b5a20bb4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-10-10 / Human adenovirus (HAdV) is indicated as a viral biomarker of water quality. Thus, studies that show or even the occurrence of these interactions in water are of great importance, since these studies are still scarce. The aims of these studies were: (i) to detect, quantify and molecularly characterize the HAdV serotypes that can to circulate in the water supply treatment in the city of Goiania; (ii) evaluate the infectivity and recovery of genomic copies (GC) of HAdV-5 in simulated condition with solids derived from tropical soils and under controlled conditions of the pH and the presence of organic matter (OM); (iii) establish a mathematical equation to evaluate the recovery rate of virus genome copies in simulated conditions with clay soils. Thus, in the second half of 2012, we collected sample water in a volume of 5 L of 4 treated water reservoir and their respective points in the distribution network in the city of Goiania, totaling 80 samples. The samples were concentrated, quantified by qPCR and sequenced. Altogether 76.6% (100 - 109 CG/mL) and 37.5% (101 - 108 CG/mL) of the samples were positive for the reservoir and their respective points in the distribution network respectively. Therefore, Goiânia’s treated water is contaminated with a high number of HAdV type C. In the study of the interaction of HAdV-5 with the solid (sediments), a hydromorphic soil sample was divided into two parts: soil organic matter (WOM) and soil less organic matter (LOM). Then, it was added separately 5, 25 and 50 mg of soil WOM and LOM in sterile polypropylene tubes of 50 mL, added ultrapure water and adjusted the pH to 6.0 and 8.0. Then was added 1 mL of viral aliquot and the volume made up to 50 mL. The tubes in replica were shaken at 150 rpm for 1h at 24 °C followed by decantation. The viral genome was quantified (GC/mL) by Real-Time PCR and the Infectivity by Assay Plate Lysis. Water with solids promoted a reduction in the number of GC/mL and viral infectivity. The OM did not affect the recovery of GC/mL (p> 0.05). However, the OM was harmfulto to the infectivity of the virus, with a reduction of 2 log10 of Plaque Forming Units per milliliter (PFU/mL), when compared with treatments LMO. The acidic pH is unfavorable to virus inactivation, and the clay is the main element responsible for the interaction of HAdV-5. The mathematical equation is useful in assessing the recovery of viral genomic copies in clay solutions. These data can offer support in ecoepidemiological studies of viral inactivation or water treatment. / O adenovírus humano (HAdV) é indicado como um biomarcador viral para a análise de qualidade da água. Assim, estudos que evidenciam a ocorrência ou mesmo as interações destes em água são de suma importância, já que as pesquisas na área são inexpressivas. Neste contexto, foram objetivos do estudo: (i) detectar, quantificar e caracterizar molecularmente os HAdVs circulantes no sistema de abastecimento de água tratada da cidade Goiânia; (ii) avaliar a infecciosidade e a recuperação de cópias genômicas (CG) de HAdV-5 em soluções simuladas com sólidos tropicais e em condições controladas de pH e presença de matéria orgânica (MO) e (iii) estabelecer uma equação matemática para avaliar a taxa de recuperação de cópias genômicas virais em condições simuladas com solos argilosos. Assim, no segundo semestre de 2012, foram coletadas amostras de água tratada (5 L) em 4 reservatórios e em seus respectivos pontos na rede de distribuição de Goiânia, totalizando 80 amostras. As amostras foram concentradas, quantificadas por PCR em Tempo Real Quantitativa (qPCR) e parte destas sequenciadas. Ao todo, 76,6% (100 - 109 CG/mL) e 37,5% (101 - 108 CG/mL) das amostras foram positivas para os reservatórios e seus pontos na rede de distribuição respectivamente. Portanto, a água tratada de Goiânia está contaminada por um elevado número de HAdV tipo C. Quanto ao estudo com os sólidos, amostra de um solo hidromórfico foi subdividida em duas partes: solo com matéria orgânica (CMO) e solo sem matéria orgânica (SMO). Foi adicionado separadamente 5, 25 e 50 mg de solo CMO e SMO em tubos de polipropileno estéreis de 50 mL, adicionado água ultrapura e o pH das soluções ajustado para 6,0 e 8,0. Em seguida, foi adicionado 1 mL de alíquota viral e o volume foi completado para 50 mL. Os tubos em réplica foram agitados a 150 rpm por 1h a 24 ºC e decantados por 1h. O genoma viral foi quantificado (CG/mL) por qPCR e a Infecciosidade por Ensaio em Placa de Lise. A água com sólidos promoveu uma redução na recuperação das CG/mL e na infecciosidade viral. A MO não influenciou na recuperação das CG/mL (p>0,05), todavia, foi predudicial à infecciosidade viral, com diminuição de 2 log10 de Unidades Formadoras de Placa por Mililitro (PFU/mL), quando comparado com os tratamentos SMO. O pH 6,0 desfavorece a inativação e a argila é o principal elemento responsável pela interação do HAdV-5. A equação matemática é útil na avaliação da recuperação das cópias genômicas virais em soluções argilosas. Estes dados podem auxiliar em estudos eco-epidemilógicos, de inativação viral ou tratamento da água.

Orientador: Karin Werther / Banca: Eliana Reiko Matushima / Banca: Eveline dos Santos Zanetti / Resumo: Os cervídeos têm sido acometidos por doenças hemorrágicas virais, tais como, doença epizoótica hemorrágica (DEH), língua azul (LA) e doença hemorrágica por adenovírus (DHA). Como as lesões macroscópicas, entre elas, enterite hemorrágica, edema pulmonar, petéquias e sufusões em diversos órgãos, são observadas nas três doenças, há necessidade de técnicas acuradas para realizar o diagnóstico definitivo. A partir do material de arquivo (blocos de parafina) existente no Departamento de Patologia Veterinária da FCAV - Unesp, 42 cervídeos brasileiros, tanto de vida livre como de cativeiro, foram selecionados, por apresentarem sinais clínicos e/ou lesões macroscópicas sugestivas de doenças virais hemorrágicas. Das amostras analisadas, utilizando técnica de imunoistoquímica, todas apresentaram resultado negativo para adenovírus. Utilizando técnica de RT-PCR em tempo real para vírus da doença epizoótica hemorrágica, os resultados foram também negativos. A mesma técnica aplicada para vírus da língua azul revelou sete animais positivos (16,66%) confirmados com eletroforese em gel de agarose 4% e sequenciamento. Todos os casos positivos foram de animais provenientes de cativeiro, sendo três fêmeas (duas jovens, uma adulta) e quatro machos jovens. As principais alterações macroscópicas observadas nesses animais foram conteúdo intestinal hemorrágico, mucosas avermelhadas do trato gastrointestinal, úlceras em língua e petéquias em diversos órgãos. Na histologia observou-se principalmente infiltrado inflamatório, hemorragia e congestão em diversos órgãos. Os vírus da DHA e DEH não estão envolvidos nos óbitos dos cervídeos estudados. A relevância deste trabalho está no fato de ser a primeira descrição de material genético do vírus da LA em cervídeos brasileiros / Abstract: Cervids have been affected by viral hemorrhagic diseases, such as Epizootic hemorrhagic disease (EHD), Bluetongue (BT), and Adenoviral hemorrhagic disease (AHD). Once that gross lesions, among them, hemorrhagic enteritis, pulmonary edema, petechiae and suffusions in several organs, are similar, it is necessary to use accurate techniques to the definitive diagnosis. From the archival material (paraffin blocks) available in the Department of Veterinary Pathology of FCAV - Unesp, 42 Brazilian deer, both free living and captive, were selected because they had lesions suggestive of hemorrhagic viral disease. The samples analyzed, using Immunohistochemistry, were all negative for adenovirus. Using real time RT-PCR for EHD, the results were also negative. The same technique applied to BT virus revealed seven positive animals (16,66%) confirmed after agarose 4% gel electrophoresis and gene sequencing. The main macroscopic changes observed in these animals were hemorrhagic intestinal contents, reddish mucous membrane of the gastrointestinal tract, ulcers on tongue and petechiae in various organs. Mostly histological changes observed were inflammatory infiltrate, hemorrhage, and congestion in various organs. All positive cases were from captive animals, three females (two young and one adult), and four young males. The AHD and EHD virus are not involved in the deaths of the deers studied in this research. The significance of this study is due to the fact that it was the first time the genome of BT virus was identified in Brazilian cervids / Mestre

Cancer immunotherapy is an approach to cancer therapy that involves the enhancement of the cancer patient’s own innate and/or adaptive immune systems to attack their own cancer. Clinically available cancer immunotherapies rely on different strategies: infusion of ex vivo manipulated autologous dendritic cells (DCs), infusion of genetically engineered autologous cytotoxic CD8+ T lymphocytes, stimulation of T lymphocyte proliferation, or inhibition of immunosuppressive pathways to improve T lymphocyte effector function. Nonetheless, only a small percentage of cancer patients receive benefit from immunotherapies and thus further improvements in clinical outcomes are required. Among numerous other therapeutic immunotherapies strategies being developed and tested, adenovirus serotype 5-based vectors (Ad5) have been well studied in preclinical and clinical settings. Preclinical research has shown that vaccination of mice with Ad5-OVA (an Ad5 encoding a model tumor antigen, chicken ovalbumin (OVA)) results in activation and proliferation of OVA-specific CD8+ T lymphocytes capable of specific killing of tumor cells that express OVA. This dissertation evaluates the potential of polymeric nanoparticles (NP) loaded with all-trans retinoic acid (ATRA), a vitamin A derivative with potent immunostimulatory effects, to improve the immunostimulatory and therapeutic effects of Ad5-OVA in a murine E.G7-OVA tumor model, a well described model that can be used for studying the immune response to Ad5-based immunotherapies. In the first part of this work, poly(lactide-co-glycolide) NP loaded with ATRA (ATRA-PLGA-NP) were prepared and characterized. Next, the antitumor effect and the magnitude of the OVA-specific immune response due to Ad5-OVA vaccination versus ATRA-PLGA-NP (or ATRA soluble) plus Ad5-OVA combination treatment were compared in vivo. The results showed that the combination treatment using ATRA-NP, but not ATRA soluble, resulted in enhanced survival and enhanced levels of OVA-specific CD8+ T lymphocytes in peripheral blood, spleen, and tumor. Next, cRGD- and mannose-functionalized PLGA-PEG NP were developed in an attempt to actively target the tumor neovasculature and DC-rich organs, respectively. The functionalization efficacy was confirmed by ex vivo fluorescence imaging studies. In vivo studies using E.G7-OVA-challenged mice showed that treatment with ATRA-loaded cRGD-functionalized PLGA-PEG-NP + Ad5-OVA, despite not enhancing the levels of OVA-CD8+ T lymphocytes in peripheral blood, substantially enhanced survival compared to either the combination of Ad5-OVA + non-functionalized ATRA-PLGA-PEG-NP or Ad5-OVA + conventional ATRA-PLGA-NP. On the contrary, treatment with mannose-functionalized PLGA-PEG-NP + Ad5-OVA, despite optimally enhancing the levels of OVA-CD8+ T lymphocytes in peripheral blood (compared to all other treatment groups), did not lead to enhanced survival compared to either the combination of Ad5-OVA + non-functionalized ATRA-PLGA-PEG-NP, Ad5-OVA + conventional ATRA-PLGA-NP, and over Ad5-OVA treatment alone. Although not investigated further in this dissertation, it was speculated that the observed trend in survival benefit provided by ATRA-PLGA-PEG-cRGD-NP + Ad5-OVA over the other NP formulations may have been due to higher levels of ATRA within the TME due to actively targeting the tumor vasculature, corroborating previous studies which demonstrated that ATRA functions as a potent stimulator of anti-tumor cellular immune responses within the tumor. The paradoxical results obtained with mannose-functionalized PLGA-PEG-NP are less readily explained. In conclusion, it was demonstrated in this work that the co-administration of Ad5-OVA and ATRA-loaded NP formulations enhanced the tumor specific cellular immune response and the survival of tumor challenged mice compared to vaccination with Ad5-OVA alone.

Die rheumatoide Arthritis (RA) ist eine chronische, progressive und systemische Autoimmunerkrankung, in deren Zentrum das dauerhaft entzündete Synovialgewebe der Gelenke steht. Aufgrund vielfältiger Knochen- und Knorpel-destruierender Prozesse kommt es zu irreversiblen Funktionalitätsverlusten der betroffenen Gelenke. Eine tragende Rolle bei der Ausprägung der klinischen Manifestationen wird dabei der exzessiven Synthese des proinflammatorischen Cytokins IL-1 zugesprochen. Dessen Aktivität kann durch kompetitive Blockade des IL-1 Rezeptors Typ I mit dem natürlich vorkommenden, antiinflammatorischen IL-1 Rezeptorantagonisten (IL-1Ra) inhibiert werden. Der Cytokin-blockierende Therapieansatz mit Anakinra, einem rekombinant hergestellten IL-1Ra, konnte die pharmakologischen Behandlungsmöglichkeiten der RA seit 2001 wesentlich erweitern. Gleichwohl erfordern die geringen Halbwertszeiten von IL-1Ra regelmäßige subkutane Injektionen, um hinreichende therapeutische Wirkstoffspiegel im Patienten aufrecht zu erhalten. Vor diesem Hintergrund bieten somatische Gentherapiekonzepte eine vielversprechende Alternative zu den konventionellen Behandlungsstrategien bei der RA-Therapie. Ein IL-1Ra-Gentransfer ins Gelenk soll die persistierende, lokale, endogene Synthese des therapeutischen IL-1Ra-Proteins ermöglichen und lässt in dieser Hinsicht eine nachhaltige Verbesserung der klinischen Symptomatik erwarten. In dieser Arbeit wurden dafür gentherapeutische Foamyvirus-Adenovirus-Hybridvektoren (FAD) zur Expression des IL-1Ra entwickelt und die Funktionalität der Konstrukte evaluiert. Die Vektoren sollten die effizienten adenoviralen Transduktionsmechanismen mit dem Potential der foamyviralen somatischen Integration für einen direkten in vivo Gentransfer kombinieren. Das System besteht aus einem adenoviralen Hochkapazitätsvektor vom Serotyp 5, der eine selbstinaktivierende PFV-Vektorkassette unter Kontrolle des Reversen Tetracyclin Transaktivator Systems (Tet-On) enthält. In FAD-transduzierten Zellen wurde die funktionelle Induzierbarkeit der PFV-Vektorexpression nachgewiesen und die Kinetik der PFV-Partikelfreisetzung charakterisiert. Nach Induktion der PFV-Vektorkassette konnte in FAD-transduzierten Zellen ein langfristig-stabiler IL-1Ra-Gentransfer gezeigt werden. Ferner konnten protektive Effekte eines FAD-vermittelten IL-1Ra-Gentransfers im Zellkulturmodell nachgewiesen werden. Tierexperimentelle Untersuchungen zeigten eine erfolgreiche Transduktion von Synovialzellen nach intraartikulärer Applikation von FAD-Vektoren. Das Tetracyclin-regulierbare Hybridvektorsystem zur Expression des IL-1Ra, das in der vorliegenden Arbeit geschaffen wurde, könnte zukünftig die Basis für ein effektives Werkzeug zum intraartikulären Gentransfer in der klinischen Praxis bieten. / Rheumatoid arthritis (RA) is a chronic, progressive and systemic autoimmune disease, characterized by invasive synovial hyperplasia. Several inflammatory cartilage- and bone- destroying processes lead to an irreversible loss of joint functionality. The excessive synthesis of the pro-inflammatory cytokine IL-1 has been implicated as a primary mediator of pathology in RA. The activity of IL-1 is initiated upon binding to the IL-1 receptor type I and can be inhibited by the naturally occurring anti-inflammatory IL-1 receptor antagonist (IL1-Ra) protein. The cytokine-blocking therapeutic approach with anakinra, a recombinant form of IL-1Ra, has significantly improved the pharmacological treatment of RA since 2001. Nevertheless, due to the short half-life of IL-1Ra, repeated subcutaneous injections are required to maintain therapeutic concentrations in the patient. Thus, somatic gene therapy may offer a promising alternative to conventional therapeutic strategies for treating RA. Following gene delivery of IL-1Ra, it may be expected that a sustained improvement of clinical symptoms is achievable due to the endogenous cellular synthesis and local secretion of the therapeutic IL-1Ra protein. In this work, foamy virus-adenovirus hybrid vectors (FAD) were developed for the expression of IL-1Ra and the functionality of the constructs was evaluated. The hybrids combine the high transduction efficiency of adenovirus vectors with the integrative potential provided by prototype foamy virus (PFV) vectors, for direct in vivo gene transfer. In the system, a complete expression cassette for self-inactivating PFV vectors, which is under the control of the tetracycline-dependent regulatory system (Tet-On), was inserted into the backbone of a serotype 5-based high-capacity adenoviral vector. In FAD-transduced cells, the induction of the PFV vector cassette was demonstrated and the release of secondary infectious PFV vectors was characterized. After the induction of the PFV vector cassette in FAD-transduced cells, a stable long-term IL1-Ra expression was shown. Furthermore, the anti-inflammatory potential of the FAD-mediated IL-1Ra gene transfer was successfully evaluated in a cell culture model. Animal studies indicated successful transduction of cells in the synovium after intra-articular application of FAD-vectors. The tetracycline-inducible hybrid vector system for the expression of IL-1Ra, which was created in the present work, may provide the future basis for an effective tool for intra-articular gene transfer in clinical settings.

Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.

SerpinB2 (aka plasminogen activator type 2) is well described as an extracellular inhibitor of urokinase-type plasminogen activator (uPA). However, the majority of SerpinB2 is retained intracellularly, and many uPA-independent activities have been reported for SerpinB2 suggesting an alternate function. This thesis explores the role of SerpinB2 in epithelial tumour cell lines, highlights the problems associated with various expression systems and argues that SerpinB2 has no role in growth or apoptosis of tumour cells. A potential role for immune modulation and angiogenesis is suggested in in vivo models. Previous research using SerpinB2 transfected, clonally selected tumour cell lines suggested that SerpinB2 regulates the retinoblastoma tumour suppressor protein (Rb) by binding and protecting Rb from degradation. Despite the use of two techniques under numerous conditions and positive controls, no significant interaction between SerpinB2 and Rb was found. SerpinB2 was reported to bind Rb through a PENF homology motif located within the SerpinB2 C-D interhelical loop region. The PENF homology motif was postulated to represent the motif responsible for binding to the C-pocket of Rb. Epstein Barr Virus nuclear antigen 6 (EBNA6) is a known Rb binding protein, which contains two predicted PENF homology motifs. However, mutation of the two PENF homology motifs within EBNA6 did not reduce Rb binding. Furthermore, the SerpinB2 PENF homology motif is actually not well conserved between SerpinB2 proteins from multiple species, whereas other regions of the SerpinB2 C-D loop show a high level of conservation. These data do not support a role for SerpinB2 and the PENF homology motif in Rb binding. SerpinB2 has been proposed to have a role in regulating growth and apoptosis. To further investigate this proposed phenotype of SerpinB2, SerpinB2 was expressed in a range of epithelial tumour lines using transient transfection. No change in growth, apoptosis or Rb levels were found. After ≈2-3 month antibiotic selection for the SerpinB2-expressing plasmid, SerpinB2 protein was lost without the loss of the transgene, indicating selective pressure against long-term SerpinB2 protein expression. To further investigate long-term SerpinB2 expression adenovirus and lentivirus vectors were used. Infection of tumour cell lines with adenovirus vectors expressing SerpinB2 resulted in reduced cell growth, characterised by increased p53 (but not Rb) levels and G2 arrest or apoptosis. When SerpinB2 expressing lentivirus vectors were used to transduce the same tumour cell lines, high levels of long-term expression of functional SerpinB2 was achieved. However, SerpinB2-expressing cell lines showed no differences in growth, proliferation, Rb levels, or apoptosis induced by a range of agents. Growth and apoptosis observed with adenovirus SerpinB2 had all the characteristics of adenovirus-associated toxicity, which has been reported previously for specific proteins. These experiments highlighted the problems associated with SerpinB2 expression systems and suggest that SerpinB2 expression per se is not toxic nor has a role in regulating Rb, growth and apoptosis. Screening of a number of tumour cell lines identified the HPV16 transformed cervical cancer line as expressing high levels of SerpinB2. SerpinB2 was located both extracellularly and intracellularly with a cytoplasmic and nuclear distribution. A high molecular weight SerpinB2 species was identified in CaSki cells and was shown to be the N-linked glycosylated species. Sequencing showed the protein to be Type A SerpinB2 and the protein was shown to form an inhibitory complex with uPA. An abundant low molecular weight SerpinB2 species was also identified in CaSki cell supernatants and appeared to be a proteolytic fragment of SerpinB2. Treatment of CaSki with PMA, TNFα and IFNγ increased SerpinB2 levels. Lentiviral based shRNA failed to significantly down regulate SerpinB2 expression and increasing SerpinB2 levels with lentiviral expression did not change growth, apoptosis, Rb levels or E7 transcription. Lentiviral expression of SerpinB2 in (normally SerpinB2 negative) HPV16 transformed SiHa cells, also failed to show changes in Rb levels or E7 transcription. CaSki thus express wild-type and functional SerpinB2, but no evidence could found that SerpinB2 effects HPV16 E7 transcription or Rb levels. The data presented identifies CaSki as valuable source of biologically functional SerpinB2. SerpinB2 expression in breast cancer cells has been associated with positive prognosis. Tubo, a SerpinB2-negative murine breast carcinoma cell line, was transduced with lentivirus expressing SerpinB2 and grown subcutaneously in BALB/c mice. SerpinB2 expressing tumours appeared red and were larger than control tumours. Furthermore, SerpinB2 expressing tumours had a ≈2 fold higher density of blood vessels when compared to Tubo and Tubo expressing EGFP. Mice carrying tumours expressing SerpinB2 also showed reduced anti-tumour IgG2 responses. These data suggest that a role for SerpinB2 in regulating angiogenesis and antitumour immunity. In conclusion, this thesis challenges the notion that SerpinB2 regulates Rb, cell cycle, and apoptosis and suggests a potential role for SerpinB2 in tumour angiogenesis and immunity.

SerpinB2 (aka plasminogen activator type 2) is well described as an extracellular inhibitor of urokinase-type plasminogen activator (uPA). However, the majority of SerpinB2 is retained intracellularly, and many uPA-independent activities have been reported for SerpinB2 suggesting an alternate function. This thesis explores the role of SerpinB2 in epithelial tumour cell lines, highlights the problems associated with various expression systems and argues that SerpinB2 has no role in growth or apoptosis of tumour cells. A potential role for immune modulation and angiogenesis is suggested in in vivo models. Previous research using SerpinB2 transfected, clonally selected tumour cell lines suggested that SerpinB2 regulates the retinoblastoma tumour suppressor protein (Rb) by binding and protecting Rb from degradation. Despite the use of two techniques under numerous conditions and positive controls, no significant interaction between SerpinB2 and Rb was found. SerpinB2 was reported to bind Rb through a PENF homology motif located within the SerpinB2 C-D interhelical loop region. The PENF homology motif was postulated to represent the motif responsible for binding to the C-pocket of Rb. Epstein Barr Virus nuclear antigen 6 (EBNA6) is a known Rb binding protein, which contains two predicted PENF homology motifs. However, mutation of the two PENF homology motifs within EBNA6 did not reduce Rb binding. Furthermore, the SerpinB2 PENF homology motif is actually not well conserved between SerpinB2 proteins from multiple species, whereas other regions of the SerpinB2 C-D loop show a high level of conservation. These data do not support a role for SerpinB2 and the PENF homology motif in Rb binding. SerpinB2 has been proposed to have a role in regulating growth and apoptosis. To further investigate this proposed phenotype of SerpinB2, SerpinB2 was expressed in a range of epithelial tumour lines using transient transfection. No change in growth, apoptosis or Rb levels were found. After ≈2-3 month antibiotic selection for the SerpinB2-expressing plasmid, SerpinB2 protein was lost without the loss of the transgene, indicating selective pressure against long-term SerpinB2 protein expression. To further investigate long-term SerpinB2 expression adenovirus and lentivirus vectors were used. Infection of tumour cell lines with adenovirus vectors expressing SerpinB2 resulted in reduced cell growth, characterised by increased p53 (but not Rb) levels and G2 arrest or apoptosis. When SerpinB2 expressing lentivirus vectors were used to transduce the same tumour cell lines, high levels of long-term expression of functional SerpinB2 was achieved. However, SerpinB2-expressing cell lines showed no differences in growth, proliferation, Rb levels, or apoptosis induced by a range of agents. Growth and apoptosis observed with adenovirus SerpinB2 had all the characteristics of adenovirus-associated toxicity, which has been reported previously for specific proteins. These experiments highlighted the problems associated with SerpinB2 expression systems and suggest that SerpinB2 expression per se is not toxic nor has a role in regulating Rb, growth and apoptosis. Screening of a number of tumour cell lines identified the HPV16 transformed cervical cancer line as expressing high levels of SerpinB2. SerpinB2 was located both extracellularly and intracellularly with a cytoplasmic and nuclear distribution. A high molecular weight SerpinB2 species was identified in CaSki cells and was shown to be the N-linked glycosylated species. Sequencing showed the protein to be Type A SerpinB2 and the protein was shown to form an inhibitory complex with uPA. An abundant low molecular weight SerpinB2 species was also identified in CaSki cell supernatants and appeared to be a proteolytic fragment of SerpinB2. Treatment of CaSki with PMA, TNFα and IFNγ increased SerpinB2 levels. Lentiviral based shRNA failed to significantly down regulate SerpinB2 expression and increasing SerpinB2 levels with lentiviral expression did not change growth, apoptosis, Rb levels or E7 transcription. Lentiviral expression of SerpinB2 in (normally SerpinB2 negative) HPV16 transformed SiHa cells, also failed to show changes in Rb levels or E7 transcription. CaSki thus express wild-type and functional SerpinB2, but no evidence could found that SerpinB2 effects HPV16 E7 transcription or Rb levels. The data presented identifies CaSki as valuable source of biologically functional SerpinB2. SerpinB2 expression in breast cancer cells has been associated with positive prognosis. Tubo, a SerpinB2-negative murine breast carcinoma cell line, was transduced with lentivirus expressing SerpinB2 and grown subcutaneously in BALB/c mice. SerpinB2 expressing tumours appeared red and were larger than control tumours. Furthermore, SerpinB2 expressing tumours had a ≈2 fold higher density of blood vessels when compared to Tubo and Tubo expressing EGFP. Mice carrying tumours expressing SerpinB2 also showed reduced anti-tumour IgG2 responses. These data suggest that a role for SerpinB2 in regulating angiogenesis and antitumour immunity. In conclusion, this thesis challenges the notion that SerpinB2 regulates Rb, cell cycle, and apoptosis and suggests a potential role for SerpinB2 in tumour angiogenesis and immunity.