Dissertations / Theses: 'Adenoviru'

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Silva, Luciana Helena Antoniassi da 1977. "Desenvolvimento de adenovírus recombinantes expres-sando as glicoproteínas F e G do metapneumovírus aviário (aMPV) e do vírus respiratório sincicial bo-vino(bRSV)." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316630.

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Orientadores: Clarice Weis Arns, Fernando Rosado Spilki
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os membros da família Paramyxoviridae são vírus que causam infecções em humanos e animais de importância econômica global. Entre os membros desta família incluem patógenos de importância mundial para os humanos, como o vírus respiratório sincicial humano (hRSV), o metapneumovírus humano (hMPV) e vírus de importância em Medicina Veterinária, como o vírus respiratório sincicial bovino (bRSV) e o metapnemovírus aviário (aMPV). Os membros da família Paramyxoviridae, subfamília Pneumovirinae são vírus envelopados, não-segmentados dotados de genoma de RNA de fita simples com sentido negativo. Na primeira parte do estudo, desenvolvemos um adenovírus recombinante expressando a proteína F do aMPV. A expressão da proteína F foi determinada por Western Blot. Os níveis de transcrição do gene F foram avaliados por RT-PCR em tempo real, em células HEK-293 e células HEP-2. Foi realizada a imunização experimental de Ad-aMPV-F e foi analisada a indução de resposta de anticorpos em camundongos BALB/c. Os títulos de anticorpos neutralizantes foram detectados após a imunização com Ad-aMPV-F. Na segunda parte do trabalho o objetivo foi à construção de adenovírus recombinantes expressando a proteína F do bRSV. A proteína F parece ser um antígeno ideal para fins de diagnóstico. Utilizando anticorpo anti-V-5, uma banda de ~90 kDa foi detectada no sobrenadante de cultura de células HEK-293 infectadas com Ad-bRSV-F. Na terceira parte do estudo, o objetivo foi à construção de dois vetores adenovirais expressando as proteínas G do aMPV e bRSV, a expressão destas proteínas em células HEK-293 infectadas foi analisada pela expressão do gene repórter, da proteína verde fluorescente (GFP)
Abstract: The members of the family Paramyxoviridae are viruses that cause infectious in human and animals of importance to global economics. Among the member of this family include pathogens of importance global for humans such as human respiratory syncytial virus (hRSV), the human and metapneumovirus (hMPV) and of viruses importance in veterinary medicine, such as bovine respiratory syncytial virus (bRSV) and avian metapnemovírus (aMPV). The members of the Paramyxoviridae are enveloped, non-segmented viruses, with negative-sense single stranded genomes. In the first part of the study, we developed a recombinant adenovirus expressing the F protein of AMPV. The expression of F gene was determined by Western Blot. The levels of transcription were evaluated by RT-PCR in real time in HEK-293 cells and HEP-2 cells. Immunization experiment was carried out Ad-AMPV-F was analyzed and the induction of antibody response in BALB/c mice. The neutralizing antibody titers detected after immunization with Ad-AMPV-F. In the second part, the objective was to construct recombinant adenoviruses expressing the F protein of bRSV. Protein F appears to be an ideal antigen for diagnostic purposes. Using the anti-antibody AdV-5, a single band of ~ 90 kDa was detected in the culture supernatant in 293 cells infected with Ad-bRSV-F. In the third part of the study, the objective was to build two adenoviral vectors expressing the G protein of aMPV and bRSV and the expression of these proteins in infected HEK-293 cells were analyzed for expression of the reporter gene, green fluorescent protein (GFP)
Doutorado
Microbiologia
Doutora em Genética e Biologia Molecular

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TRIPODI, LORELLA. "INTESTINAL MICROBIOTA IS A MAJOR DETERMINANT IN THE RESPONSE TO ONCOLYTIC VACCINE IN A MOUSE MODEL OF MELANOMA." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/884815.

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Cancer immunotherapy has achieved tremendous results, however the outcome of therapies targeting immune inhibitory pathways, specifically CTLA-4 and the axis between programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) has many genetic and environmental sources of variability. Many studies demonstrated the influence of gut microbiome on immune checkpoint inhibitors (ICIs) outcome. Besides ICIs, oncolytic vaccines (OVs) are a promising therapeutic alternative in cancer immunotherapy with possible relevant contribution to treatment of several types of tumors; OVs are, in fact, able to convert immunologically “cold” tumors into “hot” ones. OVs represent an optimum candidate to combine with ICIs, increasing their response blockade both in immunogenic and poorly immunogenic tumors. We hypothesized that manipulation of intestinal gut microbiota could also affect OVs therapeutic efficacy; at this aim, we determined whether efficacy of the oncolytic adenovirus Ad5D24-CpG (Ad-CpG) therapy could be affected by the gut microbiome in a syngeneic mouse model of melanoma. Sterilization of the gut microbiota with highdose vancomycin impaired efficacy of Ad-CpG therapy, reducing the tumor-infiltrating IFN-gamma CD8 T-cell. Cohousing mice pre-treated with vancomycin and a control group, with consequent microbiota restoration, prior to treatment with Ad-CpG, ablated the negative effect of antibiotic, confirming that Ad-CpG-reduced efficacy was mediated by the intestinal microbiota. Considering the ability of Bifidobacterium as a positive regulator of antitumor immunity in vivo, by promoting pro-inflammatory signals in innate immune cells, we evaluated tumor regression in syngeneic mouse model of melanoma treated with a combination of Ad-CpG and Bifidobacterium spp. cocktail. The group receiving the combined regimen showed the best tumor control and an enrichment of bacteria belong to Firmicutes phylum, evaluated by fecal microbiome profiling by 16S rRNA. Our data indicates that gut microbiota affects the immune responses elicited by oncolytic adenovirus Ad-CpG and Bifidobacterium supplementations maximize its activity.

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Rauma-Pinola, T. (Tanja). "Adenovirus endocytosis and adenoviral gene transfer in cardiovascular and dermatologic disease models." Doctoral thesis, University of Oulu, 2004. http://urn.fi/urn:isbn:9514274342.

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Abstract Adenoviral gene transfer is a valuable tool in molecular biology research. In order to be an efficient and safe vector, adenovirus structure and infection mechanism as well as molecular biology of the used transgene need to be well studied. The aim of this study was to evaluate the role of adenovirus as a gene transfer vector from several perspectives. Adenovirus uses receptor-mediated endocytosis in order to enter the target cell. The effect of Rab5 GTPase on adenovirus entry and gene transfer efficiency was examined first. Next, adenovirus was used as an investigatory tool in the cardiovascular research, focused on clarifying the role of adrenomedullin (AM) in heart and vascular remodeling. Finally, a model of adenoviral gene transfer into skin fibroblasts was used. The role of Rab5 GTPase in the adenovirus endocytosis was examined in HeLa cells using Cy3-labeled adenovirus, and gene transfer efficiency using β-galactosidase encoding adenovirus. Rab5 increased both adenovirus uptake and gene transfer, whereas dominant negative Rab5S34N decreased both endocytosis and gene transfer. The data indicate that Rab5 is needed in mediating the adenovirus uptake into the target cell. In the rat heart, adenovirus-mediated AM gene transfer transiently improved systolic function both in vivo and in vitro. AM caused activation of translocation of protein kinases C ε and δ, whereas phosphorylation of p38 mitogen activated protein kinase was decreased in the left ventricle. AM significantly attenuated the development of angiotensin II-induced cardiac hypertrophy. In rats with myocardial infarction, AM enhanced dilatation of left ventricle and thinning of anterior wall. The role of AM in neointima formation was evaluated in rat artery after endothelial injury. Intravascular AM gene transfer decreased neointimal growth and increased neointimal myofibroblasts apoptosis. These results show that AM regulates left ventricular systolic function and remodeling in the heart, and plays a role in pathological vascular remodeling. Adenovirus-mediated lysyl hydroxylase (LH) gene transfer into skin fibroblasts of type VI Ehlers-Danlos syndrome patient and rat skin increased functional LH production, elevated LH activity, and human LH mRNA production both in vitro and in vivo. LH gene replacement therapy may thus lead to possibilities to improve skin wound healing in Ehlers-Danlos syndrome patients.

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Rauschhuber, Christina. "Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for Gene Therapy." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-128560.

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Wiles, Karen Anna, and n/a. "Coxsackie and Adenovirus Receptor (CAR) expression is a potential limiting factor in adenoviral oncotheraphy." University of Otago. Dunedin School of Medicine, 2007. http://adt.otago.ac.nz./public/adt-NZDU20070619.161353.

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Novel approaches to cancer treatment in the context of Gene Therapy have been gaining popularity as an alternative to conventional therapies which have proven to lack specificity, resulting in tumour cell resistance, tumour progression and mortality. As a consequence the use of adenoviruses has been widely developed not only as a replication deficient vector for gene therapy but also as a replication competent oncolytic agent designed to selectively target and kill tumour cells. Unfortunately their success in clinical application has been limited, and it has been suggested that a lack of the primary viral attachment receptor 'CAR' could be a barrier to infection by limiting access to target cells. If Ad/CAR binding is the rate limiting step for successful Ad therapy, it is essential to establish a CAR expression profile in normal and tumour tissue, and in tumour progression, to enable more effective targeted therapy. Furthermore, in the context of using adenovirus as an anticancer strategy by exploiting its replicative lysis, it is important to explore whether Ad success is affected by CAR expression and to identify factors downstream of CAR that may be influential in this process. In the first experimental chapter, an in vivo immunohistochemical analysis of tissue array slides determined CAR expression in a range of normal and tumour tissue. CAR was differentially expressed dependent on cell of origin, with normal stem cells and basal cells displaying very high CAR, signifying its importance in early development and differentiation. Epithelial cells were also high in CAR but its expression was negligible in mesenchymal, lymphoid and neural cells. This trend was also reflected in most tumour tissue albeit with a general decrease in CAR compared to corresponding normal tissue of the same organ. An exception was the blastic tumours which displayed high CAR reflecting their embryonic state of derivation. CAR expression also decreased in high grade, poorly differentiated tumours of the prostate, stomach and breast compared to their well differentiated counterparts. In the second experimental chapter, a more comprehensive study of breast cancer biopsy specimens was undertaken, to determine both the expression of CAR and the tumour suppressor gene p53 in relation to tumour grade. The rationale being that both loss of CAR expression and p53 mutation (resulting in loss of function), have been associated with tumour progression. It is possible that CAR and p53 interact directly or indirectly and may be modulated by each other. This study revealed a decrease in both CAR and hormone receptor expression and an increase in p53 'mutational' status with increasing tumour grade. These three factors when compared independently to tumour grade are statistically significant associations, implying that CAR expression and hormone responsiveness decrease with tumour progression and p53 function is compromised or lost via mutation. There was also a significant association between CAR expression and hormone receptor status, however a significant association between CAR expression and p53 status within the tumour grades was not found. Treatment outcome with Ads will also depend on defining factors downstream of CAR attachment that affect adenovirus 'permissivity', which is ultimately measured by viral replication and cell death, relying on the bystander effect to eradicate all tumour cells. The in vitro analysis revealed statistically significant associations between CAR receptor expression, 'infectivity' (virus infection) and permissivity. Cell lines that were more susceptible to Ad5 were generally of epithelial origin, had high CAR, and were easily infected. However there were exceptions and CAR was not the sole determinant in adenovirus cell entry nor in its ability to replicate and kill the cell. Permissivity was also related to p53 status. Thus, although CAR expression may indeed be a limiting factor, it is apparent that a combination of other events contributes to a deficient infection, especially in the deregulated tumour environment. The results presented in this thesis clearly demonstrate that there is more to the story of 'CAR' which hints that its role in viro-oncotherapy is not limited solely to its function as an attachment receptor for adenovirus but may also involve its function as a cell adhesion molecule and signal transducer. The further elucidation of these aspects of CAR�s potential role in the scheme of tumour biology may alter the course and strategy of cancer therapy in the future.

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Alkahlout, Amal S. "Establishment of Isoform-specific Coxsackievirus and Adenovirus Receptor Knockout Epithelial Cell Lines to Understand the Mechanism of Adenoviral Infection." Wright State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=wright1590945950982052.

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Ferreira, Fernando António da Costa. "Distribuição nuclear, dinâmica e função das topoisomerases I, IIα e IIβ na replicação de genomas : estudo experimental no adenovírus serótipo 2-." Doctoral thesis, Universidade Técnica de Lisboa. Faculdade de Medicina Veterinária, 2008. http://hdl.handle.net/10400.5/224.

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Projectos de Investigação no âmbito dos quais o trabalho foi realizado:2001/05 - Cell biology of human topoisomerase IIα (Fundação para a Ciência e a Tecnologia – Programa Operacional Ciência, Tecnologia e Inovação/BCI/36194/2000. 2005/07 - Topoisomerases: at the border between DNA replication and chromatin structure (Fundação para a Ciência e a Tecnologia – Programa Operacional de Ciência e Inovação/BIA-BCM/63368/2004)
Tese de Doutoramento em Ciências Veterinárias
Neste trabalho foi estudado o papel das topoisomerases celulares (I, IIα e IIβ) na replicação, usando os adenovírus como modelo. Os adenovírus apresentam um genoma de ADN de cadeia dupla e são responsáveis por infecções respiratórias, gastro-intestinais, oftalmológicas, neurológicas e genito-urinárias. São organismos ubiquitários, infectam passáros e a maioria dos mamíferos, incluindo o Homem. Com efeito, a importância destes vírus tem vindo a aumentar por surgirem associados a novos quadros nosológicos (imunodeficiências, transplantes de órgãos) e por poderem vir a funcionar como vectores terapêuticos em doenças genéticas e na viroterapia do cancro. No presente trabalho (1) analisámos a distribuição das topoisomerases I, IIα e IIβ no núcleo de células infectadas, com utilização de tecnologias modernas de microscopia, (2) caracterizámos funcionalmente os locais de acumulação destas enzimas, (3) testámos se a replicação e a transcrição virais eram necessárias ao seu recrutamento, (4) estudámos a dinâmica das topoisomerases I e IIα in vivo por FRAP, (5) realizámos a analise mutacional da topoisomerase I e (6) quantificámos as concentrações e actividades catalíticas das topoisomerases antes e depois da infecção. Por fim, (7) abordámos a conexão funcional entre estas proteínas celulares e a replicação do vírus por depleção selectiva de cada topoisomerase (siRNA), evitando os efeitos genotóxicos dos fármacos antitopoisomerase. Os resultados obtidos permitem concluir que ambos os tipos de topoisomerases (I e II) são utilizados pelo adenovírus durante a sua replicação, mas que o seu papel é diferencial, com relevo inesperado para as topoisomerases IIα e IIβ. Estes resultados sugerem que as topoisomerases poderão ser potenciais alvos na terapêutica de patologias infecciosas e potenciais factores preditivos na terapêutica viral do cancro.

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Duarte, Patrícia. "Desenvolvimento da linhagem celular LEY79SF para produção de adenovírus livre de partículas competentes de replicação." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-09022010-110303/.

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A presença de Ad com competência para replicar (RCA, replication-competent adenovirus) nas preparações é um dos maiores problemas para a produção de Ad em larga escala. RCAs são gerados pela recombinação entre seqüência do vetor e seqüência homóloga do gene E1 presente nas células helper. Objetivo: desenvolver uma nova linhagem auxiliar para produção de Ad livre de RCA - LEY79 - derivada da linhagem de retinoblastoma humano Y79, tratando-se da primeira linhagem empacotadora de adenovírus com inativação mutacional da proteína supressora de tumor pRb, que crescem em suspensão. Células Y79 foram infectadas com o retrovírus pCLDE1A/E1BSN, selecionadas com G418. A eficiência de produção de AdeGFP na linhagem LEY79 foi testada e comparada com a HEK293A. Células Y79 foram adaptadas em meio livre de soro. Esperamos com a linhagem LEY79SF inovar no campo de processos para a produção de Ad recombinante.
The presence of Ad with the ability to replicate (RCA, replication-competent adenovirus) in preparations is a major problem in the large-scale production of Ad. RCAs are generated by recombination between the vector sequence and sequence of the homologous gene in E1 helper cells. Objective: To develop a new helper cell line for the production of RCA-free Ad., called LEY79, derived from the Y79 of human retinoblastoma line, the first line Packer adenovirus with mutational inactivation of the tumor suppressor protein pRb, which are adapted to grow in suspension. Y79 cells were infected with the retrovirus pCLDE1A/E1BSN, selected with G418. The efficiency of production of AdeGFP in the LEY79 was tested and compared with the HEK293A. Y79 cells were adapted to grow in serum-free medium. We hope that use of the the LEY79SF cell line will promote innovation in the processing and production of recombinant Ad.

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Guissoni, Ana Carla Peixoto. "Análise proteômica de células a-549 infectadas por adenovírus espécie f sorotipo 40 (HAdV-40)." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/7528.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Human adenovirus (HAdVs) are causative agents of different clinical syndromes such as gastroenteritis, respiratory diseases, eye diseases and cystitis. Adenovirus infection can modify the cellular homeostasis through the interaction with the host cell by inducing proteins of several metabolic pathways. The resulting knowledge of this virus-cell interaction may aid the elucidation of the pathogenic mechanisms caused by adenovirus and the host response against viral infection. To study this interaction, a methodology that has been widely used is proteomics, a tool used in this study, which aimed to identify induced proteins due to viral infection. In this context, we used cells A-549 infected with human adenovirus of type F, serotype 40 (HAdV-40). Infected cells and non-infected cells were used for the osmotic lysis, which were quantified by the Bradford method and then digested with trypsin. Peptides were separated on the LC system in two dimensions. The ionization of the peptides was performed by nano-eletronspray source and through analysis of ToF-MSE system aiming the protein identification. A sum of 336 proteins were identified, 206 of them induced and 130 suppressed by the infection with HAdV-40. The main pathways affected by viral infection were: energy, cellular organization, stress response and apoptosis. It was observed alteration of cell metabolism with increase of the glycolytic pathway, β-oxidation and respiratory chain. Also, the results suggest cytoskeleton reorganization and apoptosis induction. The data can to contribute knowledge about adenovirus pathogenesis considering the proteins related to distinct metabolic pathways induced by viral infection.
Adenovírus humano (HAdVs) são agentes causadores de diversas síndromes clínicas como gastroenterite, doenças respiratórias, oculares e cistite. A infecção por adenovírus pode levar a alterações na homeostase celular a partir da interação com a célula hospedeira pela indução de proteínas de diversas vias metabólicas. O conhecimento resultante desta interação vírus-célula pode auxiliar na elucidação da patogenia destes vírus e na resposta do hospedeiro contra a infecção viral. Para o estudo desta interação, uma metodologia que tem sido bastante utilizada é a proteômica, ferramenta esta usada no presente estudo, que tem como finalidade a identificação de proteínas induzidas a partir da infecção viral. Foram utilizadas células A-549 infectadas por adenovírus humano da espécie F, sorotipo 40 (HAdV-40). Para o procedimento, a partir de células infectadas e controles, procedeu-se à extração de proteínas por lise osmótica, as quais foram quantificadas pelo método de Bradford e a seguir digeridas com tripsina. Os peptídeos foram separados em sistema de cromatografia líquida em duas dimensões. A ionização dos peptídeos foi realizada em fonte nano-eletronspray e a análise destes através do sistema ToF-MSE, possibilitanto a identificação das proteínas. Foram identificadas 336 proteínas, sendo 206 induzidas pela infecção por HAdV-40 e 130 reprimidas. As principais vias afetadas pela infecção viral foram: energia, organização celular, resposta ao estresse e apoptose. Observou-se alteração do metabolismo da célula com o aumento da via glicolítica, β-oxidação e da cadeia respiratória. Além disso, os resultados sugerem reorganização do citoesqueleto e indução de apoptose. Esses dados podem contribuir para o conhecimento da patogenia dos adenovírus considerando as proteínas relacionadas com vias metabólicas distintas induzidas pela infecção viral.

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Arcieri, Luis Ernesto Farinha. "Desenvolvimento e avaliação de ferramentas de imunização baseadas na região globular da fibra adenoviral modificada com o domínio C4 da glicoproteína gp120 do HIV." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-25112008-121908/.

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A glicoproteína gp120 do HIV apresenta domínios conservados, dentre os quais está o C4. Este domínio está envolvido no reconhecimento da molécula CD4 na superfície das células alvo, e anticorpos dirigidos contra ele neutralizam o vírus. Em trabalho anterior, este domínio foi introduzido dentro da região globular da proteína fibra do adenovírus. Como a fibra adenoviral é estimulante do sistema imune, decidimos testar a capacidade de indução de anticorpos anti-C4 por essa proteína modificada. Assim, foram construídos vetores plasmídicos e adenovirais portando o gene da região globular da fibra adenoviral contendo o domínio C4, que usados na imunização de camundongos induziram anticorpos capazes de reconhecer a proteína gp120. Também construímos um vetor baculoviral expressando essa proteína híbrida, que purificada por HPLC, foi utilizada para imunizar camundongos que também produziram anticorpos capazes de reconhecer a proteína gp120. Nossos dados sugerem que a região globular da fibra adenoviral é uma boa plataforma para a exposição de epítopos de imunização.
HIV glycoprotein gp120 has conserved domains, one of them being the C4 domain. This region is involved in the recognition of the CD4 marker in target cells and antibodies that recognize this domain can block HIV infection. Previously, the C4 domain was introduced in the adenovirus fiber knob. As the adenovirus fiber stimulates de immune system, we decided to test the production of anti-C4 antibodies by this hybrid protein. We constructed plasmid and adenovirus vectors carrying the fiber knob modified with the C4 domain. Immunization of mice with these vectors showed the production of specific antibodies that recognized de gp120 glycoprotein. Also, we constructed a baculovirus vector expressing the hybrid protein, which was purified by HPLC. Mice immunized with this protein also produced antibodies capable of recognizing gp120. Our data suggest that the fiber knob is a good carrier protein for epitope immunization.

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Baldini, Maria Helena Mazzoni. "Pesquisa de doenças hemorrágicas em cervídeos do Refúgio biológico Bela Vista da Itaipu Binacional, Foz do Iguaçu-PR." Universidade do Estado de Santa Catarina, 2016. http://tede.udesc.br/handle/handle/2358.

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Capes
Neotropical deer are poorly studied species, particularly those belonging to the genus Mazama because of the access difficulty to these animals. The conservation status of Mazama nana is not well known and their main threats are poaching, habitat destruction and exposure to domestic animal diseases. Among the important diseases in cervids, haemorrhagic syndromes are very common and lead to a high rate of morbidity and mortality. Diseases leading to signs of widespread bleeding are known as haemorrhagic diseases and in deer, there are three causative viral agents currently recognized: an adenovirus (Adenoviral haemorrhagic disease virus) and two orbiviruses (Bluetongue disease virus, BTV, and Epizootic haemorrhagic disease virus, EHDV). The Bela Vista Biological Refuge of Itaipu Binacional has been successfully reproducing the species Mazama nana, popularly known as the brazilian dwarf brocket deer, since 1990. While the death of animals with compatible signs with bleeding disorders has been occurring ever since, the etiological agent has not yet been identified. Thus, this project aimed to identify the viral agent that has been causing haemorrhagic disease in the herd of Brazilian dwarf brocket deer from the Bela Vista Biological Refuge. Therefore, we evaluated autopsy samples, including liver, heart, lymph nodes and esophagus as well blood from 32 live animals in the search for viral genetic material, using namely the RT-PCR test for BTV, semi-nested RT-PCR for EHDV and the conventional PCR for adenovirus. In addition, we applied the Agar Gel Immunodiffusion technique (AGID) for detecting antibodies against BTV. Four samples, belonging from animals that died with haemorrhagic disease signs, were positive for RT-PCR test against BTV, and from this material, it was possible to isolate and serotype the virus. At the end of the study, we identified three serotypes thus far unknown in Brazil, the 14 BTV3, BTV14 and BTV18 plus a serotype not yet identified. The prevalence of seropositive animals to BTV was 3.12%. All semi-nested RT-PCR tests for EHDV and conventional PCR for AHDV had negative results. Therefore, we concluded that the BTV is the causative agent of the haemorrhagic disease of the brazilian dwarf brocket deers from the Bela Vista Biological Refuge and at least three serotypes are involved in the epidemiology of the disease
Os cervídeos neotropicais são pouco estudados, particularmente as espécies pertencentes ao gênero Mazama, em razão da dificuldade de acesso aos animais silvestres e suas amostras. O status de conservação da espécie Mazama nana não está bem definido. As principais ameaças a esses animais são a caça ilegal, a destruição de seu habitat e a proximidade a animais domésticos, que podem introduzir doenças comuns em populações de ruminantes silvestres. Dentre as doenças importantes em cervídeos, as síndromes hemorrágicas são muito frequentes e levam a um alto índice de morbidade e mortalidade. As doenças que provocam sinais e quadro de hemorragia generalizada em cervídeos são conhecidas pelo nome genérico de doenças hemorrágicas, e são três os agentes virais reconhecidos atualmente: um adenovírus (vírus da doença hemorrágica por adenovírus) e dois orbivirus, (o vírus da língua azul BTV e o vírus da doença epizoótica hemorrágica EHDV). O Refúgio Biológico Bela Vista da Itaipu Binacional vem reproduzindo com sucesso a espécie Mazama nana desde 1990, conhecida popularmente como veado-bororó-do-sul, porém, fazendo uma revisão histórica, percebe-se que desde que a criação foi iniciada, ocorreram mortes de cervídeos com sinais compatíveis com as doenças hemorrágicas. Entretanto, o agente etiológico nunca foi identificado. O presente projeto teve por objetivo identificar o agente viral que tem causado a doença hemorrágica no plantel de veados-bororó-do-sul do Refúgio Biológico Bela Vista, situado no município de Foz do Iguaçu, Paraná. Foram avaliadas amostras obtidas por meio de 12 necropsia, incluindo fragmentos de fígado, coração, linfonodos e esôfago, além de amostras de sangue de 32 animais vivos para a pesquisa de material genético viral pelo teste de RT-PCR em tempo real para o vírus da língua azul e semi-nested RT-PCR para o vírus da doença epizoótica hemorrágica, além de PCR convencional para o adenovírus de cervídeos. A técnica de IDGA foi utilizada para a pesquisa de anticorpos contra o vírus da língua azul. Quatro amostras, provenientes de animais que vieram a óbito com sinais de doença hemorrágica, foram positivas para o teste de RTq-PCR contra língua azul (BTV), e a partir desse material foi possível isolar e sorotipificar o vírus. Ao final do estudo, foram identificados três sorotipos até então desconhecidos em território brasileiro, o BTV3, BTV14 e BTV18. A prevalência de animais soropositivos para BTV foi de 3,12%. Todos os testes de semi-nested RT-PCR para o vírus da doença epizoótica hemorrágica (EHDV) e de PCR convencional para adenovírus tiveram resultados negativos. Com este estudo foi possível determinar que o BTV é o agente causador da doença hemorrágica no plantel de veados-bororós-do-sul do Refúgio Biológico Bela Vista e que pelo menos três sorotipos estão envolvidos na epidemiologia da doença

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Rovira, i. Rigau Maria. "Adenovirus oncoselectius pel tractament de càncer de pàncrees. Combinació d'estratègies de direccionament a tumor i bioselecció de microRNAs potenciadors de l'activitat adenoviral." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/456987.

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L’adenocarcinoma ductal pancreàtic (PDAC) és una neoplàsia molt agressiva a conseqüència de la seva elevada capacitat metastàtica i la gran resistència que presenta als tractaments convencionals de quimioteràpia i radioteràpia. Només un percentatge molt reduït de pacients és elegible per a la resecció quirúrgica del tumor, l’únic tractament amb opcions de cura. La major part dels casos són diagnosticats en estadis avançats de la malaltia en els que la supervivència a 5-anys se situa al voltant del 7%. La millora del pronòstic dels pacients amb càncer de pàncrees ha estat pràcticament nul·la en els últims 30 anys, de manera que existeix una clara necessitat de desenvolupar nous mètodes per facilitar la detecció precoç del tumor i teràpies més efectives pel seu tractament. Els adenovirus oncolítics estan esdevenint una teràpia prometedora pel tractament de neoplàsies agressives com el PDAC, obtenint resultats prometedors en assajos clínics. Tanmateix, els virus administrats fins al moment no han permès aconseguir respostes antitumorals complertes i és necessari disposar d’adenovirus més potents, però també de replicació selectiva en tumor. En el primer bloc d’aquesta tesi, ens hem fixat en algunes de les desregulacions que presenten les cèl·lules tumorals per tal de dissenyar estratègies de direccionament a tumor i obtenir adenovirus oncoselectius més segurs. Concretament, l’increment de l’expressió de metal·loproteases de matriu, la reactivació en els tumors de vies relacionades amb el desenvolupament embrionari i la pèrdua de l’expressió de miRNAs específics de teixit han estat el racional per a generar modificacions genètiques que permetessin regular l’entrada dels adenovirus a la cèl·lula i l’expressió del gen E1A a nivell transcripcional i post-transcripcional. D’aquesta manera, s’ha determinat que la combinació de diversos mecanismes de control de la replicació viral permet obtenir adenovirus més segurs per una administració sistèmica tot mantenint una activitat antitumoral significativa. En el segon bloc de la tesi, ens hem centrat en conferir més potència als adenovirus oncolítics. Vam hipotetitzar que les desregulacions en els perfils d’expressió de miRNAs de les cèl·lules tumorals podrien tenir un impacte negatiu en el cicle adenoviral, limitar la formació de nova progènia viral i per tant, reduir l’eficàcia antitumoral dels adenovirus oncolítics. Amb l’objectiu de contrarestar aquestes possibles limitacions i obtenir adenovirus amb una activitat antitumoral augmentada, es va dur a terme la bioselecció d’una biblioteca de miRNAs humans en adenovirus per tal d’identificar miRNAs potenciadors de l’activitat adenoviral en PDAC. A través d’aquest sistema high-throughput, es va determinar que el miR-99b i el miR-485 milloraven l’expressió dels gens virals i la formació de virions infectius en cèl·lules de PDAC gràcies a la regulació, directa o indirecta, de factors cel·lulars amb expressió diferencial en cèl·lules neoplàsiques i cèl·lules no tumorals. Així doncs, la identificació de miRNAs que milloraven el fitness viral va permetre obtenir adenovirus oncoselectius més potents front a PDAC.
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive neoplasia due to its high metastatic capacity and its resistance to chemotherapy. A small number of patients are eligible for tumor resection, the only curative treatment, and most of the cases are diagnosed at an advanced stage of the disease, in which the 5-year survival is around 7%. Therefore, there is a clear need for the development of better diagnostic methods and more effective treatments for this neoplasia. Oncolytic adenoviruses are becoming a promising therapy for the treatment of aggressive cancers, such as PDAC. Promising results have been obtained in clinical trials, although complete antitumoral responses have not been reached and more potent but also more selective viruses are required. In this thesis, we have focused in some deregulations present in cancer cells in order to design tumor targeting strategies for adenoviruses. Specifically, the overexpression of matrix metalloproteases, the reactivation of embryonic pathways and the loss of tissue specific miRNA’s expression have been the rational for the genetic modifications that allow the control of viral replication at transductional, transcriptional and post-transcriptional levels. We have determined that the combination of different strategies is useful for obtaining safer adenovirus for a systemic administration while maintaining a significant antitumoral activity. We have also focused in conferring more potency to oncolytic adenoviruses. We hypothesized that deregulations of miRNA profiles in cancer cells may have a negative impact on the adenoviral cycle, reducing the antitumoral efficiency of the virus. With the objective to counteract these limitations, we performed a bioselection of a human miRNA adenoviral library, aiming to identify miRNAs that confer potency to the adenoviruses against PDAC. We identified that miR-99b and miR-485 improved viral gene expression and the formation of infective particles in PDAC through the direct or indirect regulation of cellular factors differentially expressed in neoplastic cells and non-tumoral cells. Therefore, the identification of miRNAs that improved viral fitness gave rise to more potent oncoselective viruses against PDAC.

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Lyons, Mark. "Haematocompatibility of adenovirus." Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413185.

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Mesquita, JacÃ Ricarte Lima de. "Perfil clÃnico-epidemiolÃgico de InfecÃÃes respiratÃrias agudas causadas por adenovÃrus em crianÃas atendidas em hospital de referÃncia da cidade de Fortaleza - CE." Universidade Federal do CearÃ, 2007. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=911.

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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 atÃ 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)

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Silva, Hugo Delleon da. "Adenovírus humano em água tratada e avaliação da sua recuperação em solução com sólidos tropicais." Universidade Federal de Goiás, 2013. http://repositorio.bc.ufg.br/tede/handle/tede/3360.

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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.

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Umeda, Luana de Cássia. "Métodos clássicos e moleculares para avaliação da qualidade virológica de lodo de esgoto e de água de reúso: determinação da eficiência e limites de detecção." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-09112012-115558/.

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Os vírus entéricos humanos são encontrados no esgoto e em subprodutos dos processos de tratamento. Recentemente vem sendo recomendados como indicadores de qualidade microbiológica em normas da legislação brasileira e também nas de outros países, mas ainda com parâmetros a definir. O objetivo do estudo é a avaliação e a comparação entre métodos clássicos e moleculares aplicados à detecção de vírus entéricos em amostras de água de reúso e de lodo, visando subsidiar a legislação brasileira. Ensaios de semeadura experimental de protótipos de rotavírus e de adenovírus foram realizados nas matrizes ambientais e os vírus detectados por métodos clássicos (cultivo celular e reação de imunoperoxidase) e moleculares (PCR/nested-PCR, RT-PCR e ICC-PCR), determinando-se os limites de detecção de cada método para cada matriz. A pesquisa de rotavírus e adenovírus presentes naturalmente em 25 amostras de água de reúso e em 25 de lodo possibilitou a comparação dos métodos propostos. O ICC-PCR mostrou ser o método mais factível a ser aplicado na área de saneamento.
Human enteric viruses are common contaminants of raw sewage and subproducts of sewage treatment processes. In recent years, those viruses were recommend as new microbiological indicators in different matrices in Brazilian legislation and others countries, although some questions should be elucidated. At present, the aim was to evaluate and compare the efficiencies of standard and molecular virological methods for detection of human enteric viruses in sludge and reclaimed water samples. Rotavirus and adenovirus were experimentally spiked in the proposed matrices and virus recovery and detection limits established for each method and matrice. Viruses naturally presented in 25 samples of sludge and 25 samples of reclaimed water were assayed by all methods and results evaluated and compared for statistical significance. From all methods evaluated, ICC-PCR showed to be the most suitable for virus surveillance in sludge and reclaimed water.

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Röger, Carsten [Verfasser], Henry [Akademischer Betreuer] Fechner, Jens [Akademischer Betreuer] Kurreck, Claus-Thomas [Akademischer Betreuer] Bock, and Roland [Akademischer Betreuer] Lauster. "Antivirale Therapie gegen Adenoviren mittels löslicher Coxsackievirus- und Adenovirus-Rezeptor-Varianten / Carsten Röger. Gutachter: Jens Kurreck ; Claus-Thomas Bock ; Roland Lauster. Betreuer: Henry Fechner." Berlin : Technische Universität Berlin, 2015. http://d-nb.info/1074912373/34.

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Dicks, Matthew Douglas James. "Novel adenovirus vaccine vectors." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:383aaf3d-284a-4bd7-877c-3270bd7c2e4f.

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Replication defective adenoviruses have emerged as promising vectors for delivery of vaccine antigens. The development of new vaccine vectors has recently focused on serotypes t, which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This thesis describes the construction and optimisation of ChAdOX1, a new vector based on chimpanzee adenovirus Y2S, which has recently been manufactured to clinical grade for a Phase 1 human trial. Comparative immunogenicity studies between vectors of different serotype were performed in mice, with careful consideration of the infectious titer of vector preparations, since this parameter can confound studies based solely on viral particle estimation. Aft intramuscular administration, HAdV-S (Human adenovirus C) based vectors elicited superior transgene product specific T cell and antibody responses compared to a selection of chimpanzee adenovirus vectors (from Human adenovirus EJ including ChAdOX1. The construction of ChAdOXl in a bacterial artificial chromosome (BA C), enabled precise, and flexible modification of the genome by recombmation mediated genetic engineering. (recombmeering). Reverse genetics was performed to identify vector determinants of immunogenicity. Chimeric ChAdOXl vectors were created by replacement of native virus associated RNA (VA-RNA) and fiber sequences with the corresponding sequences from HAdV-5 Using these chimeric vectors, the importance of innate immunity and vector transduction in determining vector immunogenicity was investigated. Though the mechanisms responsible ultimately remain unclear, superior transgene product specific immune responses with HAdV-5 correlated with higher levels of transgene expression in vivo after vector administration. The current study has conclusively demonstrated that neither VA-RNA sequences, nor the fiber protein, are responsible for differences in immunogenicity between vectors, contrary to hypotheses based on previous studies.

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Teixeira, Lais Helena. "Geração e análise da imunogenicidade de proteínas recombinantes baseadas nas diferentes formas do antígeno circumsporozoíta de Plasmodium vivax visando o desenvolvimento de uma vacina universal contra malária." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-11072014-110149/.

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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.

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Matkovic, Urska. "Construction of Adenoviral vectors for cancer gene therapy and evaluation of toxic effects of adenoviral infection on Adrenocortical cells." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425193.

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Aims of the work were construction and evaluation of recombinant adenoviral vectors for gene therapy of esophageal, hepatocellular and adrenocortical carcinomas in vitro and in vivo. Since adenoviruses have tropism for adrenal gland, human adrenocortical cells were used to assess the toxicity of replication competent adenovirus type 5 and replication incompetent adenoviral vectors. In this regard, the effects of adenoviral infection on adrenocortical gene expression, cell proliferation, cell cycle, cell death and steroidogenesis were investigated.

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Emerson, Corey. "Structural Characterization of an Engineered Adenovirus Vector and Complement-mediated Inactivation of Adenovirus." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1619961226521366.

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22

Pelliccia, M. "STRATEGIES FOR ENHANCING VIRAL GENE TRANSFER AND THE THERMOSTABILITY OF VIRAL VECTORS IN VACCINE APPLICATIONS." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/265518.

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At the most basic level viruses are biological nano-containers constituted by genetic material enclosed in a protein shell, capsid. A peculiar feature of viruses, both bacterial and some eukaryotic viruses, lies in the high packaging density of the genome in order to fit itself in the small capsid and hence the high internal osmotic pressure. Virus is a relatively stable particle equipped with fascinating mechanical properties of the capsid that are crucial for the virus lifecycle. Viruses have only one purpose: infect a host cell for reproducing themselves in order to generate new viral progeny (Roos et al. 2007). Therefore, the first and foremost consideration arising from the concept of virus reflects its pathogenesis and virulence that can ultimately result in many important infectious diseases such as common cold, influenza, hepatitis, rabies, measles, cancer and AIDS. As a consequence, pathogenic viruses represent a heavy hurdle for the global health and there is a strong need for developing robust strategies such as vaccines or antiviral drugs against virus infections (Baram- Pinto et al. 2010). On the other hand, viruses in the course of evolution have become efficient specialized gene delivery agents. Therefore they represent powerful tools in biomedicine for gene therapy and vaccine purposes (Schaffer et al. 2008). For successful gene therapy and immunization programs, the efficiency and stability of viral vectors are fundamental aspects (Jorio et al. 2006). To address this challenge, in the present research project we have investigated the interaction between viruses and nanomaterials. In the last years materials on the nanoscale for their unique properties have provided a broad range of potential biomedical uses (Verma et al. 2008) and for that reason we decided to explore their application with viruses. More specifically, we have examined three types of sulfonate- functionalized gold nanoparticles (AuNPs), namely, MUS:OT, MUS and MUS:brOT NPs, which are less than 5 nm in size, negatively charged and poorly cytotoxic (Verma et al. 2008). The NPs are coated with self-assembled monolayer (SAM) of thiolated organic molecules and one of the ligand is a sulfonated molecule, MUS (Verma et al. 2008). The MUS ligand itself was tested in our experiments as well. As virus models we focused on human recombinant adenovirus type 5 (Ad), one of the most promising viral vector as vaccine and gene therapy carrier and two picornaviruses of the genus enterovirus, namely, EV1 and CVB3, important human pathogens associated with several infectious diseases (e.g. myocarditis, aseptic meningitis, encephalitis, paralysis)(Kossila et al. 2002)(Marjomäki et al. 2014a). In spite of their medical impact, there are no therapeutic treatments available against picornavirus infections and the only vaccine products are against three types of poliovirus and hepatitis A virus (Merilahti et al. 2012). Two sets of experiments were carried out: (1) Short-term incubation of Ad with nanomaterials for 1 h at 37°C prior transducing HeLa cells or before in vivo administration in zebrafish and mice. The results demonstrated that Ad shortly pre-treated with nanomaterials showed a significant increase in the gene expression in vitro and in vivo The NPs’enhanced adenovirus transduction aims to reduce Ad vector doses in vivo thereby minimizing the adverse reactions of the immune response due to high vector dosage; (2) Long-term thermostabilization studies of Ad, EV1 and CVB3 in vitro in the presence and in the absence of our nanomaterials and other substances such as sugars (sucrose, glucose, glycerol) and Polyethylene glycol (PEG) molecules at 37°C or room temperature for extensive periods of time. Our results showed the capability of the nanomaterials and sucrose to increase substantially the heat stability of the viruses. In order to elucidate the thermal inactivation mechanism of viral particles and the stabilizing effect provided by some compounds on viruses we set out to formulate an analytical theory. This line of research fits in the context of developing more thermo-stable viral vector preparations for vaccine purposes that do not require the maintenance of the challenging cold chain system in order to preserve the effectiveness of viral vaccines during the storage, shipment and administration to the patients and hence to ensure the success of global immunization programs (Alcock et al. 2010).

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23

Monaghan, Alan. "Mechanisms of adenovirus DNA replication." Thesis, University of St Andrews, 1995. http://hdl.handle.net/10023/13935.

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The development of a cell-free system in which adenovirus DNA synthesis can be initiated in vitro by using the viral genome or plasmids containing the origin of replication as template has led to the identification of the sequences important for origin function and the isolation and purification of the proteins required for viral DNA replication. In vitro studies on adenovirus types 2 and 5 have shown that their replication requires the formation of a large nucleoprotein complex. This is composed of three virally encoded proteins: adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, and two cellular proteins nuclear factor I and nuclear factor III. While the presence of DNA helicases in other eukaryotic DNA replication systems have been well characterised, this was not the case for adenovirus DNA replication. Initial attempts to identify DNA helicase activity associated with any of the adenovirus replication proteins were unsuccessful. However, a novel DNA unwinding activity was found associated with the DNA binding protein (Ad.DBP). We examined the interaction of DBP with partial DNA duplexes and demonstrated that it could displace oligonucleotides annealed to single-stranded M13 DNA. In addition, DBP could also unwind small fragments of fully duplex DNA. Unlike a DNA helicase, DBP promoted DNA unwinding was nucleoside-5'-triphosphate and Mg2+ independent and exhibited no directionality. The activity required saturating amounts of DBP and was both efficient and cooperative in nature. The helix-destabilising activity was shown to be situated in the C-terminal domain of the protein. These properties suggest a role for DBP in DNA replication in which DBP destabilises duplex DNA during origin unwinding and replication fork movement. The second part of the thesis dealt with the characterisation of the putative "active site" of the adenovirus DNA polymerase. This experimental approach was prompted by data from earlier studies which indicated that DBP could increase the processitivity of the polymerase as well as its sensitivity to nucleotide analogue inhibitors. The "active site" was labelled with pyridoxal-5'-phosphate (PLP), a substrate binding site directed reagent for DNA polymerases. Treatment of Ad.5 DNA polymerase with PLP followed by reduction of the enzyme-PLP adduct resulted in irreversible inactivation of the polymerase activity while the 3'-5' exonuclease associated with Ad.5 DNA polymerase was minimally affected. Substrate protection studies indicate that PLP inhibition is complex. Neither template-primer nor substrate dNTP alone showed any protective effect from PLP mediated inhibition. However, the presence of both template -primer and complementary dNTP significantly protected against PLP inhibition. Comparative tryptic mapping of labelled enzyme, modified in the presence and absence of substrates by PLP reaction, on a C-18 reverse phase column, indicated the protection of one peptide from pyridoxylation in the presence of substrates. Amino acid sequence analyses found no sequence to be present in this peak.

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Webster, Ailsa. "Characterisation of the adenovirus protease." Thesis, University of St Andrews, 1992. http://hdl.handle.net/10023/14300.

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The substrate specificity, classification, expression and control of the adenovirus encoded protease, the activity of which is required for the production of virions, is described. The use of synthetic peptides has shown that the protease cleaves sites of the form (M,L,I)XGG-X or (M,L,I)XGX-G and these consensus sequences have been used to identify potential cleavage sites in all the known substrates of the protease. Putative cleavage sites have also been found in a number of other adenovirus proteins including the major coat proteins, the hexon and the penton, that had not previously been considered as substrates of the protease. The octapeptide, MSGGAFSW, has been used to develop a specific assay for the protease where digestion is monitored by HPLC reverse phase chromatography. Purification of the protease from adenovirus particles and the preparation of antipeptide sera against the L3 23-kDa protein have been used to confirm that the latter is the protease and that it is not proteolytically activated. Inhibitor studies reveal that the enzyme is inhibited by the thiol directed reagents iodoacetate, PCMB and DTDP, and activated by DTT or cysteine suggesting that it is a member of the cysteine class of proteases. Analysis of the sequence of the enzyme, however, shows that it is not a papain-like enzyme and it is suggested that it might be a member of the recently identified subclass of cysteine proteases that are related to trypsin. The 23-kDa protease has been expressed using E.coli and baculovirus expression systems and a purification schedule for the protein was developed using anion exchange and hydrophobic interaction chromatography. The purified enzyme, however, was not able to cleave the peptide substrate MSGGAFSW and the conclusion was drawn that the protein is probably synthesised as an apoenzyme. One of the substrates of the protease, the pre-terminal (pTP) protein was expressed in insect cells using a recombinant baculovirus. Coinfections of cells with recombinant 23-kDa and pTP baculoviruses resulted in efficient processing of the pTP to the intermediate terminal protein (iTP) in situ. The partially purified baculovirus expressed 23-kDa protein was not, however, found to digest the pTP in vitro, supplying further evidence that the adenovirus protease requires a cofactor.

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McFall, Emily. "Exploring Novel Methods to Achieve Systemic Delivery of SMN for Treatment of Spinal Muscular Atrophy." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31803.

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Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron protein (SMN), leading to progressive deterioration of α-motor neurons, onset of muscle atrophy and, in severe disease, death. We investigated whether reducing the size of Adenovirus (Ad) vectors, through use of a short fibre protein, could enhance delivery of a transgene to muscle and motor neurons after systemic delivery in vivo. Unfortunately, the biodistribution of the smaller Ad vector was unaltered compared to wildtype Ad, with most of the virus localizing to the liver. However, we determined Ad-derived SMN was efficiently packaged into cellular exosomes, suggesting a novel approach to protein delivery. We showed that exosomes naturally contain SMN both in vitro and in vivo and that exosomes can be used to deliver SMN to recipient cells. Further testing is required to establish if SMN-containing exosomes can function as an SMA therapeutic.

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Nash, Leslie. "Exosomes: A Novel Biomarker and Approach to Gene Therapy for Spinal Muscular Atrophy." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/38910.

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Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced levels of the survival motor neuron (SMN) protein. SMA results in degeneration of motor neurons, progressive muscle atrophy, and death in severe forms of the disease. Currently, there is a lack of inexpensive, readily accessible, accurate biomarkers to study the disease. Furthermore, the current FDA approved therapeutic is neither 100 % effective nor accessible for all patients, thus more research is required. Tiny cell derived vesicles known as exosomes have been evaluated in an attempt to identify novel biomarkers for many disease states and have also shown therapeutic promise through their ability to deliver protein and nucleic acid to recipient cells. The research presented herein investigates whether (1) the level of SMN protein in exosomes isolated from the medium of cells, and serum from animal models and patients of SMA is indicative of disease, to serve as a biomarker for monitoring disease progression and therapeutic efficacy; (2) SMN-protein loaded exosomes can be utilized to deliver SMN protein to SMN-deficient cells; (3) adenoviral vectors are effective at creating SMN protein-loaded exosomes in situ for body wide distribution of SMN protein. This research has shown SMN protein is naturally released in extracellular vesicles, and the level of exosomal SMN protein is reflective of the disease state. Exosomes can also be modified to hold enhanced levels of SMN protein and deliver them to both the cytoplasm and nucleus of SMN-deficient cells. Furthermore, adenoviral vectors expressing luciferase-tagged SMN1 cDNA, targeted to the liver, results in SMN protein-loaded exosomes and detectable luciferase activity, body-wide. Thus, exosomes present as an effective biomarker and potentially a novel approach to treat SMA.

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Nominato, Luís Fernando Resende da Silva. "Avaliação da transferência gênica por vetor viral na glândula lacrimal e resposta na neovascularização corneana." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/17/17150/tde-23042018-151114/.

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Objetivos: Os objetivos deste estudo foram: 1) determinar a eficácia da transferência gênica do vetor de adenovírus sorotipo 5, carreando o gene do receptor do fator de crescimento endotelial vascular (VEGF) solúvel humano (sVEGFR1) para a glândula lacrimal (GL); 2) investigar se a expressão de sVEGFR1 interfere na neovascularização da córnea (NVC), induzida por queimadura alcalina; 3) avaliar a segurança do procedimento. Métodos: Trinta e dois ratos Wistar foram submetidos à queimadura central da córnea direita com solução de hidróxido de sódio (NaOH) 1 M. Os animais foram divididos em três grupos e injetados diretamente em sua GL direita 25 ?l de vetores virais AdVEGFR1 (1x1010 pfu) (12 animais), 25 ?l do vetor AdNull (1x1010pfu) (10 animais), ou 25 ?l de solução salina (Controle). Após sete dias, a NVC foi observada e fotografada na lâmpada de fenda. A secreção lacrimal foi medida com fenol. A presença do sVEGFR1 na GL foi testada por qPCR (quantitative polymerase chain reaction) e a coloração, por imunofluorescência. O qPCR foi também utilizado para comparar o RNA mensageiro (RNAm) de ilterleucina-1beta (IL-1?), ilterleucina-6 (IL-6) e fator de necrose tumoral alfa (TNF-?) na GL e no gânglio do trigêmeo (GT). Resultados: O vetor AdVEGFR1 transfectou 83% dos ratos. O sVEGFR1 estava presente nas células acinares da GL. A NVC foi prevenida em nove de doze animais do grupo AdVEGFR1, em comparação com o grupo Ad-Null (3:10) e o grupo Controle (1:10) (p=0,0317). A secreção lacrimal e o RNAm das citocinas na GL e no GT foram semelhantes nos três grupos (p>0,05). Conclusões: A transferência gênica do vetor adenoviral para a GL demonstrou expressão local do sVEGFR1 humano, e evitou a NVC na maioria dos olhos expostos a queimaduras alcalinas, se mostrando seguro para a estrutura e função da GL.
Purpose: The aims of this study were: 1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (sVEGFR1) gene transfer to the lacrimal gland (LG); 2) to investigate whether expression of sVEGFR1 acts in corneal neovascularization (CNV), induced by alkali burn and; 3) to evaluate the safety of the procedure. Methods: AdVEGFR1viral vectors (25 ?l, 1x1010pfu) were injected in the right LG of rats and compared with AdNull vector (25 ?l, 1x1010pfu) or 25?l saline (Control) before cornea alkali burn with 1 M NaOH. After seven days, CNV was observed and photographed in the slit lamp. Tear secretion was measured with phenol red thread. The animals were tested for human VEGFR1 mRNA and protein in the LG by qPCR and immunofluorescence staining, respectively. qPCR was also used to compare the mRNA of IL-1?, IL-6, and TNF-? in LG and ipsilateral trigeminal ganglion (TG). Results: Ad-VEGFR1 transfected 83% of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals of Ad-VEGFR1 group, compared to Ad-Null (3:10) and Control (1:10) (p=0.0317). The tear secretion and the cytokines mRNA in LG and TG were similar all three groups (p>0.05). Conclusion: Adenoviral vector gene transfer to LG as the has shown local expression of human sVEGFR1, as It prevented CNV in most of the eyes exposed to alkali burn and was safe for LG structure and function.

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Krohne, Steewen [Verfasser], and Thomas [Akademischer Betreuer] Dobner. "The role of SUMO modification during productive adenovirus infection and the identification of PIAS4 as a transcriptional repressor of early adenoviral genes / Steewen Krohne ; Betreuer: Thomas Dobner." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1172880549/34.

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Pfitzner, Søren [Verfasser]. "The late stages of human adenovirus infection - Characterising human adenovirus nuclear reorganisation and egress / Søren Pfitzner." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2021. http://d-nb.info/1236695232/34.

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Leja, Justyna. "Oncolytic Adenovirus Therapy of Neuroendocrine Tumors." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-146966.

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Neuroendocrine tumors (NETs), originally described as carcinoids, represent a rare and heterogeneous group of neoplasms associated with intensive secretion of hormones, bioactive peptides and amines. Most of the patients are diagnosed at a late stage of disease, often with liver metastases. Surgery remains the main treatment to control metastatic disease, but is not curative. Oncolytic virotherapy represents a promising approach to treat cancer and different strategies have been exploited to restrict viral replication to tumor cells. We developed an oncolytic adenovirus based on serotype 5, Ad5[CgA-E1A], where the chromogranin A (CgA) promoter controls expression of the E1A gene and thereby virus replication. We found that Ad5[CgA-E1A], selectively replicates in NET cells and it is able to suppress fast-growing human BON carcinoid tumors in nude mice. The activity of Ad5[CgA-E1A] was not completely blocked in liver cells. We further repressed virus replication in hepatocytes by targeting E1A with miR122, an miRNA specifically expressed in the liver. miRNAs bind to mRNA and induce its cleavage or translational blockage. By insertion of tandem repeats of miR122 target sequences in 3’UTR of E1A gene, we observed reduced E1A protein expression and replication arrest in miR122 expressing liver cells. The oncolytic potency of the miR122-targeted virus was not affected in NET cells. Since some NET and neuroblastoma cells express high levels of somatostatin receptors (SSTRs), we introduced in the virus fiber knob cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site of somatostatin for SSTRs. The FWKT-modified Ad5 transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to a greater extent than Ad5, indicating that the fiber knob modification may prolong the systemic circulation time. NETs represent a huge therapeutic challenge and novel diagnostic markers are needed for early detection and effective treatment of NETs. We have profiled primary tumors and liver metastases of ileocaceal NETs, using Affymetrix microarrays and advanced bioinformatics. We have identified six novel marker genes and show high similarity between primary lesions and liver metastases transcriptome by hierarchical clustering analysis.

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Gustafsson, Dan. "Adenovirus species B interactions with CD46." Doctoral thesis, Umeå universitet, Virologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-52075.

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Adenoviruses (Ad) are double-stranded (ds) DNA, non-enveloped viruses. There are seven species (A-G) of human Ads with 52 knownserotypes to date. Human Ads cause a broad range of pathologies, ranging from upper respiratory tract infections to persistent urinary tract infections. The main determinant for Ads tropism in vitro is the protruding, antenna-like, fiber protein. The fiberknob is responsible for the main interaction with the attachment receptor of the host cell. Most Ad species use the coxsackie- adenovirus receptor (CAR) as their main attachment receptor. Most species B Ads, however use CD46. CD46 is a cell surface complement regulatory protein, which is expressed on all nucleated cells in humans. Species B Ads exhibit a low seroprevalenc in the human population, making these Ads promising vector candidates for gene therapy. We have studied human Ad species B members, serotypes 7 and 11 (Ad7 and Ad11), as well as their interaction with CD46. Our first experiments showed that all species B Ads use CD46 as their main attachment receptor, with the exception of Ad3 and Ad7. Second, we performed mutational studies of recombinant Ad11p fiberknobs. These studies showed that arginine 279 in the Ad 11 fiberknob is necessary for CD46 binding. Finally we studied the effect of Ad11 binding to CD46. The results indicate that CD46 is rapidly downregulated on the cell surface after Ad11 binding. These results may provide a further understanding of the basic biology and pathology of species B Ads and may also be useful in construction of gene therapy vectors based on species B Ads.

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Morrison, Megan. "Studies on adenovirus E4orF6 binding proteins." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=30824.

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The adenovirus type 5 E4orf6 protein plays many critical roles in the viral lifecycle. This thesis attempts to identify three binding partners of E4orf6: p14, p19, and pp84. Mapping studies revealed residues 1--43 of E4orf6 were unnecessary for p14 and p19 binding. Mdm-2 and p14 ARF were selected as potential candidates for pp84 and p19 respectively. No interaction was observed between E4orf6 and Mdm-2. Binding of E4orf6 and p14ARF was observed, however attempts to clarify the interaction were inconclusive. After purification, microsequencing analysis identified p14 as Elongin C, p19 as Elongin B, and pp84 as VACM-1/Cul5. These proteins are known to exist in a complex containing E3-ligase activity, necessary for ubiquitin-proteasome mediated protein degradation. As E4orf6 acts with E1B-55kDa to induce p53 degradation, it may do so by recruiting this complex. Future studies will elucidate more about the role of E4orf6 in p53 degradation, and may reveal new functions of this viral protein.

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Brown, Jason Lee. "Expression systems for adenovirus late proteins." Thesis, University of Warwick, 2000. http://wrap.warwick.ac.uk/60074/.

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During the past few decades a new approach has emerged for the treatment of human disease. In that short period, the concepts and techniques of gene therapy have progressed from being entirely fanciful to experimental clinical application. A major stumbling block for gene therapy is the inefficiency of gene transfer and the transient nature of therapeutic gene expression. Attempts to deliver therapeutic genes using replication-defective adenoviruses have been hampered by a strong host immune response to the vector, leading to clearance of transduced cells and loss of transgene expression. Current adenovirus vectors have an additional disadvantage of being able to carry only approximately 10 kb of exogenous DNA. The work here describes attempts to improve upon existing adenoviral vectors by addressing these limitations In order to reduce the host immune response to the vector, additional deletions in the residual viral coding regions are required to prevent expression of immunogenic proteins. Deletion of the major late transcription unit (MLTU), which encodes virtually all of the viral structural proteins, would achieve this and would also increase the transgene carrying capacity of the vector. In order to create such a vector, a cell line would be required to complement the growth of the deleted vector by providing late gene functions in trans. The work presented here describes the successful cloning and subsequent analysis of the Ad5 MLTU with expression driven by the major late promoter (MLP). The expression plasmids constructed express one or more late proteins from each late gene segment in a transient assay. The plasmids also carry the EBNA-I and oriP sequences from Epstein-Barr virus which allow the plasmids to be stably maintained in eukaryotic cells. Stable cell lines were constructed using these plasmids but no late protein expression from the MLTU could be detected. Attempts to activate expression from the major late promoter by providing viral transactivating factors in trans also proved to be ineffective. To address these problems, an alternative inducible promoter was chosen. The sheep metallothionein Ia promoter was cloned upstream of the MLTU and this construct was then cloned into an episomal expression vector. This plasmid was also used successfully to regulate the expression of late proteins during transient transfection studies. However, a stable cell line constructed using the same plasmid did not show any expression of late proteins. The reasons for the inability of any cell line constructed to express late proteins are still undetermined. Possible reasons discussed are plasmid rearrangement, promoter down-regulation and possible blocks to post-transcriptional processing and translation of the complex MLTU transcript. Suggested future studies include testing of these possibilities in order to gain further insight into the regulation of expression from the MLTU construct, ultimately leading to the construction of a cell line capable of complementing the growth of adenovirus vectors with late gene deficiencies.

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Riley, Steven James. "Improving adenovirus vectors for gene therapy." Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323303.

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Tauheed, Uzair. "Identification of adenovirus new splice sites." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-185576.

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RNA splicing is a process where introns are removed and exons are joined together. Human adenovirus type 2 pre-mRNAs undergoes intensive alternative splicing and produce more than 40 differently spliced mRNAs. This thesis work is focused on the identification of new splice sites in adenovirus. By virtue of Illumina mRNA sequencing technology we have identified 255 splice sites. Splice site analysis of the introns revealed the presence of three types of splice sites GT-AG (61.2%), GC-AG (25.9%) and AT-AC (12.9%). Among 255 splice sites, 224 were new. Significantly, more than 50% of the new splice sites were located in the major late transcription unit on the positive strand of adenovirus DNA. Three new splice sites; 17452-29489 (GC-AG) located on the negative strand of adenovirus DNA in the E2 region, 9668-20346 (AT-AC) and 9699-30505 (GC-AG) on the positive strand of adenovirus DNA in the major late transcription unit were further confirmed by PCR analysis.
Adenovirus replication and transcriptome

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REA, DELPHINE. "Interactions entre cellules dendritiques et adenovirus." Paris 6, 2000. http://www.theses.fr/2000PA066403.

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Leur pouvoir exceptionnel de capture et d'appretement des antigenes et de maturation fonctionnelle font des cellules dendritiques (cd) les seules cellules presentatrices d'antigene aptes a initier les reponses immunitaires t a partir de lymphocytes naifs. La possibilite d'introduire des antigenes dans les cd et de manipuler leurs fonctions autorise le developpement de nouvelles strategies d'immunotherapie specifique. Dans cette these, nous demontrons que les adenovirus recombinants du sous-groupe c sont extremement prometteurs pour vehiculer des molecules vaccinales dans les cd ex vivo. En effet, ils accroissent les capacites immunostimulatrices des cd humaines et cooperent avec les facteurs de maturation des cd. Cependant, le transfert genique necessite un contact prolonge avec un nombre de particules virales eleve du fait de l'absence de car (coxsackie and adenovirus receptor), le recepteur d'attachement de la fibre des adenovirus du sous-groupe c a la surface des cd. Le remplacement de cette fibre par celle d'adenovirus appartenant a d'autres sous-groupes nous permet d'ameliorer le tropisme dendritique des vecteurs du sous-groupe c. Les vecteurs porteurs d'une fibre d'adenovirus du sous-groupe b, dont le recepteur cellulaire est a ce jour inconnu, se revelent en effet 100 fois plus efficaces que les vecteurs non modifies pour transferer et exprimer des genes dans les cd. Cette superiorite est liee a une penetration intracellulaire accrue et plus rapide, et a une meilleure expression des genes transferes lors de la maturation des cd. De plus, elle s'accompagne d'une meilleure presentation des antigenes codes par les genes transferes aux lymphocytes t. Les adenovirus du sous-groupe c porteurs d'une fibre d'adenovirus du sous-groupe b sont donc des vecteurs d'avenir en immunotherapie basee sur l'administration de cd genetiquement modifiees ex vivo et permettront peut-etre d'adresser directement les antigenes aux cd in vivo.

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Henaff, Daniel. "Adenovirus biology : receptors and intracellular trafficking." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20138/document.

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Les adénovirus ont une double nature, soit comme pathogène omniprésent qui peuvent occasionnellement causer des maladies soit comme vecteurs utilisés de transfert de gène. À nos connaissances, les 30 premières minutes depuis la liaison au récepteur jusqu'à l'arrivée au pore nucléaire sont identiques pour le pathogène comme pour le vecteur. L'objectif de ma thèse était de comprendre les mécanismes impliqués dans la liaison au récepteur, l'internalisation, l'échappement et le trafic endosomal vers le MTOC. J'ai d'abord étudié le mécanisme impliqué dans l'hémagglutination des virus à tropisme pour CAR et à tropisme pour SA. J'ai identifié la présence de CAR sur les érythrocytes humains et montré qu'il était le principal responsable de l'agglutination induite par les virus à tropisme pour CAR. De plus, j'ai montré que la présence de CAR sur les érythrocytes pouvait piéger le virus dans le sang et ainsi empêcher l'infection au niveau du foie. Dans un deuxième temps, j'ai participé à la caractérisation du rôle de la protéine VI et la translocation du virus au MTOC. Nous avons montré que Nedd4 était impliqué dans le ciblage du virus au MTOC via l'ubiquitination de la protéine VI. Enfin, j'ai travaillé sur le neurotropisme de CAV-2 et caractérisé sa localisa tion subcellulaire au niveau des synapses. J'ai montré qu'une partie de CAR était localisée dans des radeaux lipidiques à la synapse et que CAV-2 entrait via la voie de recyclage des vésicules synaptiques
Adenoviruses have a dual nature as ubiquitous pathogens that occasionally cause life-threatening disease and their use as gene transfer vectors. To the best of our current knowledge, the first 30 min from binding to nuclear pore docking of both wild-type virus and vector are identical. The goal of my thesis is to understand different mechanisms involved in receptor binding, internalization, endosomal escape and trafficking to the MTOC. First I studied the mechanism involved in hemagglutination of CAR-tropic and SA-tropic viruses. I identified the presence of CAR on human erythrocytes and showed that it was the main responsible for the agglutination mediated by CAR-tropic viruses. Moreover, I show that CAR on erythrocytes can sequester virus in the bloodstream and block liver infection. In a second part I participated to the characterization of the role of the protein VI and the translocation of HAd to the MTOC. We showed that Nedd4 was involved in the targeting of the virus to MTOC through ubiquitination of this protein VI. Finally, I worked on the neurotropism of CAV-2 and characterize its subcellular localization at the synapse. I showed that a part of CAR was localized in lipid raft at the synapse and enter through the synaptic vesicle-recycling pathway

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Cueto, Ana Paula Stricker. "AVALIAÇÃO DA CITOTOXICIDADE E ATIVIDADE ANTIVIRAL DA PRÓPOLIS FRENTE AO CALICÍVIRUS FELINO (FCV), ADENOVÍRUS CANINO 2 (CAV-2) E VÍRUS DA DIARRÉIA VIRAL BOVINA (BVDV)." Universidade Federal de Santa Maria, 2010. http://repositorio.ufsm.br/handle/1/5941.

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Viruses are etiologic agents of a great number of diseases of human beings and animals. However, there are only a little amount antiviral medicines available that is almost exclusively used in human health. Several studies have demonstrated that propolis has biological activities against virus, bacteria and fungi. Its chemical composition is diverse varying according to season, plants available and collection. The antiviral activity of ethanolic extracts of propolis have already been described for some human viruses. The aim of this study was to evaluate the antiviral activity of propolis ethanolic extracts against two viruses causing respiratory disease in small animals, the feline calicivirus (FCV) and canine adenovirus 2 (CAV-2), and also the bovine diarrheal disease virus (BVDV).Two samples of ethanolic extracts of propolis were used; one obtained in the lab and another obtained commercially. In order to perform the experiments the MTT test ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to determine the 50% cytotoxic concentration (CC50), the 50% inhibitory concentration (IC50) and, the terapeuthic index (TI=IC50/CC50). The propolis extracts were added to the cell cultures in different moments of the viral infection. They were included before virus inoculation, after virus inoculation, and before and after virus inoculation. The two propolis samples showed similarities in the qualitative analysis but they had small differences in the quantitative analysis. The CC50 of the extracts varied among 251 and 343μg/ml and the commercial extract showed less toxicity. Both extracts demonstrated better antiviral activity when added before virus inoculation and the ITs varied from 6 to 13. The best results were obtained with the propolis extracted in the lab. The BVD was the virus showing the greater sensitivity to the ethanolic extracts in the conditions applied. Taken all the data together, it can be concluded that the ethanolic extract of propolis has a potential for future use as a medicine in the therapy of respiratory disease caused by CAV-2, and, also, that the action mechanisms on BVDV should be better evaluated in detail regarding the hepatitis C virus (HCV).
Os vírus são os agentes etiológicos de um grande número de enfermidades em seres humanos e animais, no entanto, há um pequeno número de fármacos antivirais disponíveis para o uso que é feito quase exclusivamente na medicina humana. Estudos demonstram que a própolis apresenta variada atividade biológica frente a vírus, bactérias e fungos. Sua composição química é bastante diversa, podendo variar conforme a época, vegetação e área de coleta. A atividade antiviral de extratos aquosos e etanólicos de própolis já foi descrita para alguns vírus humanos. O presente trabalho tem como objetivo avaliar a atividade antiviral do extrato de própolis frente a dois vírus causadores de doença respiratória em pequenos animais, o calicivírus felino (FCV) e o adenovírus canino tipo 2 (CAV-2), e também ao vírus da diarréia viral bovina (BVDV). Foram testadas neste experimento duas amostras de extrato etanólico de própolis, uma delas obtida comercialmente e a outra extraída no laboratório. Para realizar este estudo utilizou-se o método colorimétrico do MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), através do qual determinou-se a concentração citotóxica a 50% (CC50), concentração inibitória a 50% (IC50) e o índice terapêutico (IT=IC50/CC50). Os extratos de própolis foram adicionados ao cultivo celular em diferentes momentos da infecção viral. Estes foram incluídos antes da inoculação viral, depois da inoculação viral, e antes e depois da inoculação viral. As duas amostras de própolis apresentaram similaridades na análise qualitativa e pequenas diferenças na análise quantitativa. A CC50 destes extratos variou de 251 a 343μg/ml sendo que o extrato comercial apresentou-se ligeiramente menos tóxico. Ambos os extratos demonstraram melhor atividade antiviral quando adicionados anteriormente à inoculação viral apresentando ITs que variaram de 6 a 13. Os melhores resultados foram obtidos com o própolis extraído no laboratório. O BVDV foi o vírus que demonstrou maior sensibilidade ao extrato etanólico de própolis nas condições experimentais aplicadas. Levando-se em consideração os resultados obtidos, pode-se concluir que o extrato etanólico de própolis apresenta potencial para futura utilização como fármaco no tratamento de doenças respiratórias causadas pelo CAV2 e que o mecanismo de ação sobre o BVDV deve ser avaliado com maiores detalhes com vistas ao vírus da hepatite C (HCV).

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Hrycak, Camilla Patrizia [Verfasser], and Ulf [Akademischer Betreuer] Dittmer. "Einfluss adenovirus-spezifischer Immunantworten auf die Immunisierung mit adenovirus-basierten Vektoren / Camilla Patrizia Hrycak ; Betreuer: Ulf Dittmer." Duisburg, 2019. http://d-nb.info/1177681749/34.

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Del, Papa Joshua. "Assessing the Oncolytic Capacity of Conditionally Replicating Adenovirus Armed with p14 Fusion Associated Small Transmembrane Protein and the Adenovirus Death Protein." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39485.

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Intratumoral injection of oncolytic viruses provides a direct means of tumor cell elimination for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this thesis, I explore the efficacy of conditionally replicating HAdV vectors expressing either the p14 Fusion Associated Small Transmembrane (FAST) protein (CRAdFAST) or p14 FAST protein in combination with the adenovirus death protein (CRAdFAST-ADP). The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect, while ADP aids in cell lysis and HAdV spread at late times in infection. I first explored the efficacy of CRAdFAST in the 4T1 immune competent mouse model of cancer. Treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene in vitro, but did not reduce the tumor growth rate in vivo. The 4T1 model was significantly resistant to HAdV infection and propagation, so I next explored CRAdFAST efficacy in human A549 cell culture and a xenograft mouse model of cancer. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion which led to the formation of large acellular regions within the tumor, and significantly reduced the tumor growth rate compared to control vector. Finally, to assess the use of a newly constructed CRAdFAST vector co-expressing the adenovirus death protein (ADP), a new model was explored comprised of CMT-64.6 mouse lung carcinoma cells which are syngeneic with Balb/C mice. This model was significantly more sensitive to HAdV infection and CRAdFAST induced fusion than the 4T1 cell line. In this model, expression of ADP and p14 FAST from a CRAdFAST-like vector (CRAdFAST-ADP) resulted in significant oncolytic synergy in vitro but not in vivo. My results indicate that expression of p14 FAST protein, and potentially ADP, from an oncolytic HAdV can improve vector efficacy for the treatment of cancer, but improved in vivo models will be required to analyze the full preclinical potential of these oncolytic HAdV vectors.

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Ramsden, James Daniel. "Angiopoietins in goitre : candidates for anti-angiogenic gene therapy?" Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273701.

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Glover, Colin P. J. "Optimization of gene transfer systems to facilitate studies investigating transcriptional pathways mediating neuronal cell death." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274633.

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43

Barbu, Andreea Roxana. "In vitro Studies of β-cell Death and Survival. Modulation by Adenoviral Vectors and Bcl-2 Overexpression." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3973.

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Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon. In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.

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David, Taynah Ibrahim Picolo. "Construção e caracterização de vetores adenovirais portadores do cDNA para interferon-beta humano." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-11052017-142613/.

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O melanoma representa menos de 5% de todos os cânceres de pele, porém, quando em estádio metastático possui prognóstico ruim. Entretanto, o genótipo dos melanomas pode prover uma oportunidade para intervenção terapêutica pelo fato de 90% dos casos de melanoma possuem p53 selvagem e grande parte destes possuem deleção na região cromossômica codificadora de interferon beta. Em prévios estudos, desenvolvemos o vetor adenoviral AdRGD-PG que fornece expressão do transgene em resposta à p53 através do promotor PG e ainda o tripeptídeo RGD, que possibilita que o adenovírus transduza uma maior gama de células pela alteração de seu mecanismo de entrada. Temos utilizado este vetor para entrega da versão murina de interferon beta em modelos de terapia gênica e imunoterapia de melanoma murino, revelando uma significativa habilidade do interferon beta em inibir a proliferação celular in vitro e in vivo e promover resposta imune antitumoral. No presente trabalho, os esforços se aplicam em adaptar essa estratégia em modelo de melanoma humano para observar se a mesma interação é encontrada. O vetor AdRGD-PGhIbeta, portador do cDNA de interferon beta humano (hIbeta) foi construído e expressão do transgene observada após transdução das linhagens estabelecidas de melanoma humano SK-MEL-05 e SK-MEL-147 (ambas p53 selvagem). Foi observado um robusto efeito antitumoral in vitro onde transferência de hIbeta promoveu acumulo de células hipodiploides (mais que 80% da população celular 96 horas após transdução) e evidências de morte por apoptose (exposição de fosfatidilserina e atividade de caspases 3/7) em ambas as linhagens. Nas duas linhagens, o efeito bystander foi demonstrado quando a presença de poucas células transduzidas (ex., 10%) foi suficiente para promover o acumulo significativo de células hipodiploides (mais que 40% neste exemplo). Em modelo de terapia gênica in situ utilizando células SK-MEL-147, também foi observado forte efeito antitumoral da hIbeta com total remissão do tumor de todos os animais tratados sem recidiva durante noventa dias. A presença de hIbeta na circulação dos animais foi confirmada 48h após o tratamento com AdRGD-PG hbeta mas presente em somente dois de sete animais 90 dias após o tratamento, sugerindo que o tratamento inicial e não um efeito off target foi responsável pela resposta. Com a finalidade de investigar efeitos colaterais do sequestro do vetor adenoviral pelo fígado, observamos a concentração circulante das enzimas aminotransferase de aspartate e aminotransferase de alanine (AST e ALT, respectivamente), que se mostrou não alterada quando comparadas entre animais que receberam injeção do vetor tratamento, vetor controle e solução salina. Com nossos resultados concluímos que vetores adenovirais carreando interferon-beta humano são capazes de transduzir a linhagem de melanoma SK-MEL-147 in vitro e in vivo, promovendo efeito bystander e remissão tumoral sem indução de efeitos adversos
Melanoma represents less than 5% of all cases of skin cancer, although, when metastatic, prognosis is dire. However, the genotype of melanomas might provide an opportunity for therapeutic intervention since 90% of melanoma cases possess wild-type p53 and a great portion of these possess deletion of the chromosomal region encoding interferon beta. In previous studies, we developed the adenoviral vector AdRGD-PG that supplies expression of the transgene in response to p53 through the PG promoter and that utilizes the RGD tripeptide, allowing the adenovirus to transduce a wider range of cells due to the alterated mechanism of entrance. We have used this vector to deliver the murine version of interferon beta in murine models of melanoma gene therapy and immunotherapy, revealing a significant ability of interferon beta to inhibit cellular proliferation in vitro and in vivo and promote an anti-tumor immune response. In the present project, we aimed to adapt this strategy for a human melanoma model in order to reveal if the same impact will be observed. The AdRGD-PGhIbeta vector encoding the human interferon beta (hIbeta) cDNA was constructed and expression of the transgene confirmed after transduction of the established human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild-type p53). A striking anti-tumor effect was observed in vitro where the transfer of hIbeta promoted an accumulation of hypodiploid cells (over 80% of the cellular population 96 hours after transduction) and evidence of death by apoptosis (exposure of phosphatidylserine and activity of caspases 3/7) in both cell lines. In these cell lines, a bystander effect was demonstrated when the presence of few transduced cells (ex., 10%) was enough to promote significant accumulation of hypodiploid cells (over 40% in this example). In a model of in situ gene therapy using SK-MEL-147 cells, hIbeta induced a strong anti-tumor effect including total tumor remission in all treated animals without relapse during ninety days. The presence of hIbeta in the circulation of the animals was confirmed 48h after treatment with AdRGD-PGhIbeta, but was present in only two of the seven animals 90 days post-treatment, suggesting that the initial treatment, not off target effects, was responsible for the response. With the goal of investigating collateral effects of adenoviral sequestration by the liver, we assayed the circulating concentration of aspartate aminotransferase and alanine aminotransferase (AST and ALT, respectively), which showed no alteration when compared with animals that received the treatment with a control vector or saline solution. We conclude that our adenoviral vector carrying human interferon-beta is capable of transducing the human melanoma cell line SK-MEL-147 in vitro and in vivo, promoting a bystander effect and tumor remission without inducing adverse effects

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Danielsson, Angelika. "Adenovirus-mediated Gene Therapy of Prostate Cancer." Doctoral thesis, Uppsala universitet, Enheten för klinisk immunologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-114132.

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Adenovirus-mediated gene therapy is a potential complement to standard cancer treatments. Advantages are that vectors can be used to target tumors and that replicating viruses lead to increased therapeutic dosage. In this thesis, an oncolytic serotype 5 adenovirus (Ad5), Ad[i/PPT-E1A, E3], was developed where viral replication is controlled by the insulator-shielded (i) prostate-specific PPT promoter. The adenoviral E3 region was inserted for its immune regulatory and lysis functions. Ad[i/PPT-E1A, E3] had improved cytotoxic abilities both in vitro and in a prostate cancer xenograft mouse model compared to a virus lacking the E3 region. To further improve adenoviral vectors, the histone deacetylase inhibitor (HDACi) FK228 was studied. FK228 has been proposed to enhance the effect of adenoviral therapy by upregulation of CAR, the primary receptor for Ad5 infection. In the present study, we observed that FK228 promotes transgene expression even better when administered after viral transduction, indicating a post-transductional enhancement of transgene expression. Another interesting finding was that FK228 reduced transgene expression from the PPT promoter in the prostate cancer cell line LNCaP. This is explained by the fact that different HDACi have the ability to provoke a neuroendocrine phenotype of LNCaP. A potential drawback with adenoviral gene therapy is the rapid clearance of the virus from the circulation. Viral particles have been coated with polyethylene glycol (PEG) to evade immune recognition, a strategy that works well in mouse models. However, less is known about the effects of adenoviral PEGylation in human blood. We have studied cell interactions and immune responses to PEGylated and uncoated Ad5 vectors in human whole blood using a blood loop model with constant blood flow. Limited effects of PEGylation were observed in human blood, which were associated with the neutralizing ability of the donor blood. An important finding that donors with high neutralizing ability in whole blood do not necessarily have neutralizing antibodies against the virus strongly implies that neutralization should be measured in whole blood.

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Ball, Inna [Verfasser]. "Tracking adenovirus infections in reptiles / Inna Ball." Gießen : Universitätsbibliothek, 2015. http://d-nb.info/1068922222/34.

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Marcellus, Richard Charles. "Regulation of apoptosis by adenovirus type 5." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0023/NQ50216.pdf.

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48

Drake, Sian Louise. "Adenovirus vectors for manipulating human immune cells." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/22482/.

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Natural Killer (NK) cell mediated immune surveillance is important in preventing and controlling malignancies. However, tumours ultimately evade NK cells, enhancing their survival and progression. The immunosuppressive cytokine TGF-β is an established, potent inhibitor of NK cell mediated anti-tumour immunity. Genetically manipulating NK cells to resist the actions of TGF-β is a potential route by which to enhance NK cell-mediated immunotherapy. However, NK cells are notoriously difficult to manipulate with conventional viral vectors or transfection techniques and alternative methodologies are required to achieve this. I have explored the ability of several virus vectors to transduce primary human NK cells, with a chimaeric adenovirus (Ad) vector proving the most promising. Replacing the Ad5 fibre with that from Ad35 (forming Ad5f35) generated a vector capable of efficient transduction of primary human NK cells and the NK cell lines, YT, NKL and NK92. Ad5F35 utilises CD46 as an entry receptor and NK cell transduction by Ad5f35 was CD46 dependent. The Ad5f35 vector provides a route to genetically manipulate NK cells. Transfection experiments in non-lymphoid cells showed that expression of a dominant negative TGF-β receptor II or inhibitory SMADs (SMAD7) inhibit the TGF-β signalling pathway. Using recombination-based methods in E.coli, an Ad5f35 vector was constructed to deliver the dominant negative TGF-β receptor II into mammalian cells. High expression and inhibitory activity was achieved in non-lymphoid cells, but expression in NK cells was low and activity reduced. Nevertheless, the Ad5f35 system clearly has potential for future applications in NK cells, including the development of NK cell based cellular therapies.

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Forrester, Natalie Alison. "Regulation of cellular signalling pathways by Adenovirus." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3251/.

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It is well established that adenoviruses inactivate the host cell DNA damage response to enhance viral DNA replication. This is achieved, in part, by the ability of viral E1B55K and E4ORF6 proteins to hijack host cell cullin-containing E3 ubiquitin ligase complexes and target key cellular proteins such as Mre11, p53 and DNA ligase IV for degradation via the ubiquitin-proteasome pathway. To assess the generality of this viral response, studies were undertaken using a panel of representative serotypes from adenovirus species A to E to determine how they interact with the host cell DNA damage response. Notably, serotypes from species B and D were unable to promote the degradation and/or relocalization of Mre11 and p53, although DNA ligase IV degradation was fundamentally conserved. Furthermore, species B and D serotypes induced the sustained overexpression of transcriptionally inactive p53, and induced both ATM and ATR kinase activity. As these events would typically be viewed as detrimental to virus survival, these data suggest that different adenovirus serotypes have evolved novel strategies in order to subvert the cellular DNA damage response during infection. Adenoviruses have long been utilised as useful tools for identifying, and characterizing, the function of fundamental cellular proteins such as tumour suppressors and those involved in the DNA damage response. Therefore, studies were also carried out to identify novel Ad12E1B54K-interacting proteins through mass spectrometric analysis, and subsequently to examine the functional significance of these interactions. Several putative novel Ad12E1B54K-interacting proteins were identified using this approach, one being the transcriptional intermediary factor 1γ (TIF1γ), a transcriptional regulator that has recently been identified as a tumour suppressor. Further studies determined that TIF1γ was relocalized to nuclear tracks in an E4ORF3-dependent manner early during adenovirus infection, and was subsequently degraded in a ubiquitin-mediated proteasome-dependent manner. Uniquely, TIF1γ degradation was shown to be E1B55K/E4ORF6-independent and E4ORF3-dependent. Data presented in this thesis also suggest that E4ORF3 does not utilise host cell cullin-based E3 ubiquitin ligases in order to promote TIF1γ degradation. The ability of E4ORF3 to target cellular substrates for degradation represents a novel way in which adenoviruses are able to target cellular substrates. Significantly, TIF1γ degradation was conserved during infection with Ad serotypes from species A to C which may highlight its importance for productive viral infection. It also appears that TIF1γ may have an as yet unidentified role in the DNA damage response since it was also found to interact with components of the ATR kinase pathway.

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Thoma, Clemens Matthias Manuel. "Improving intraperitoneal adenovirus virotherapy for ovarian cancer." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:841e4334-f408-4da3-b8e6-1d29350c5304.

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The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.

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Dissertations / Theses on the topic 'Adenoviru'

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