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1
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.
2
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.
4
Jäger, Lorenz. "The persistence of recombinant adenoviral vectors." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-100490.
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Watkins, Sarah Jane. "Adenoviral targeting with antibody fusion proteins." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624907.
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Shahbazi, Bijan [Verfasser], and Leonhard [Akademischer Betreuer] Mohr. "Adenovirale Überexpression von Glycoprotein gp 96 in Tumorzellen = Adenoviral overexpression of Ggycoprotein gp 96 in tumor cells." Freiburg : Universität, 2014. http://d-nb.info/1114669784/34.
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Knapp, Mark. "Development of dopamine receptor-expressing adenoviral vectors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0001/MQ28801.pdf.
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Cassivi, Stephen David. "Adenoviral-mediated gene therapy in lung transplantation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0002/MQ40800.pdf.
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Finlay, Siân. "Modulation of macrophage phenotype using adenoviral transfection." Thesis, University of Aberdeen, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409252.
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The initial aim of this study was to examine the nature of the interaction between adenovirus and transfected macrophage, and to characterise the molecular mechanisms underlying macrophage response to adenoviral infection. Results showed that adenoviral transfection activated macrophages, promoting production of pro-inflammatory mediators and priming an enhanced response to other inflammatory stimuli. Activation was dependent on the nuclear factor kappa B (NFkB) signalling pathway, which was activated within two hours of transfection, and could be prevented using an inhibitory adenovirus which blocked NFkB signalling. The ultimate phenotype of the transfected macrophage was determined both by non-specific viral activation and by the properties of the transgene expressed. The second aim of this research was to investigate the effect of the local cytokine milieu on transgene expression in vitro. Results showed that transgene expression under the control of two different promoter constructs was subject to regulation by pro-inflammatory mediators, by mechanisms at least partly dependent on the NFkB signalling pathway. the results have important implications for the use of these promoters in gene therapy applications where the gene of interest is delivered into an inflammatory environment. The final stage of the project focused on the use of transfected macrophages in vivo, in a rat model of glomerulonephritis. Results showed that transfected macrophages expressing the anti-inflammatory cytokine IL-4 localised with enhanced efficiency to inflamed glomeruli after intra-renal artery injection of the mechanisms for this were examined. Better understanding of the mechanisms which promote localisation may ultimately permit the design of genetically-modified macrophages which selectively target sites of injury for delivery of anti-inflammatory cytokines.
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Fulco, Enzo Augusto Medeiros 1982. "Corticosteróides tópicos para ceratoconjuntivite adenoviral = revisão sistemática." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311821.
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Orientador: Rodrigo Pessoa Cavalcanti LiraDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-19T10:10:15Z (GMT). No. of bitstreams: 1 Fulco_EnzoAugustoMedeiros_M.pdf: 625645 bytes, checksum: 871772c97dcfc7b49d6bc56d1a72c439 (MD5) Previous issue date: 2011
Resumo: Introdução: Corticosteróides tópicos são utilizados comumente no tratamento da ceratoconjuntivite viral aguda. Tem sido sugerida a utilidade dos corticosteróides no tratamento sintomático da conjuntivite alívio dos sinais e/ou sintomas e prevenção dos infiltrados subepiteliais. Por outro lado, observou-se o relato dos possíveis efeitos colaterais, como o prolongamento da transmissão invitro do vírus e, no âmbito da medicina clínica, ensaios clínicos revelaram a eficácia duvidosa dos colírios de corticosteróides. O objetivo deste estudo foi comparar o uso dos corticosteróides tópicos, com quaisquer drogas usadas nos ensaios clínicos com o placebo. Objetivo: Avaliar se os corticosteróides tópicos são eficazes e seguros para o tratamento da ceratoconjuntivite adenoviral para melhorar os sintomas e evitar ou minimizar complicações relacionadas à doença. Desenho: Revisão sistemática. Métodos: Pesquisa documental na Cochrane Central Register of Controlled Trials (CENTRAL) (que contém o Cochrane Eyes and Vision Group Trials Register), MEDLINE, EMBASE, PubMed, nas listas de referência de relatórios de ensaio identificados e no o Science Citation Index. Foram incluídos ensaios clínicos aleatorizados comparando quaisquer apresentações de corticosteróides tópico com quaisquer outras formas de tratamentos da ceratoconjuntivite adenoviral aguda. Resultados: Foram incluídos ensaios clínicos randomizados onde os corticosteróides tópicos foram comparados com placebo no tratamento da ceratoconjuntivite adenoviral aguda. A busca digital inicial identificou quatro ensaios clínicos comparando corticosteroides e placebo no manejo da ceratoconjuntivite epidêmica somando 243 pacientes. Uma revisão sistemática foi realizada. Conclusão: Nenhum estudo mostrou melhora no alívio dos sinais ou sintomas. A prevenção dos infiltrados subepiteliais permanece controversa, mostrando mais comumente um adiamento na história natural da doença do que uma modificação nela. O uso destes colírios deve ser recomendado com cautela e novos ensaios clínicos são necessários para comprovar sua eficácia
Abstract: Introduction: Topical corticosteroids are commonly used in the treatment of acute viral keratoconjunctivitis. It has been suggested the usefulness of corticosteroids in symptomatic relief of the signs of conjunctivitis and / or symptoms and prevention of subepithelial infiltrates. On the other hand, we observed the reported possible side effects, such as the extension of transmission of the virus in vitro, and beside that, clinical trials showing the effectiveness of corticosteroids eye drops. The aim of this study was to compare the use of topical corticosteroids, with any drugs used in clinical trials with placebo. Objective: To assess whether topical corticosteroids are effective and safe for the treatment of adenoviral keratoconjunctivitis to improve symptoms and prevent or minimize complications related to the disease. Design: Systematic review. Methods: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Group Trials Register), MEDLINE, EMBASE, PubMed and the reference lists of identified trial reports. We used the Science Citation Index to look for articles that cited the relevant studies. We included masked randomized controlled trials in which any form of topical corticosteroid treatment had been compared with placebo in the management of acute adenoviral ceroconjuctivitis. Results: Were included randomized controlled trials in which any form of topical corticosteroid treatment had been compared with placebo in the management of acute adenoviral keratoconjunctivitis. The initial digital search identified 4 clinical trials comparing corticosteroids and placebo in the management of Epidemic keratoconjunctivitis totaling 243 patients. A narrative review was conducted. Conclusion: No study has shown improvement in relief of the signs or symptoms. Prevention of subepithelial infiltrates remains controversial, most commonly showing a delay in the natural history of disease than a change in it. The use of eye drops should be recommended with caution and new clinical trials are needed to prove its effectiveness
Mestrado
Oftalmologia
Mestre em Ciências Médicas
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Uusi-Kerttula, Hanni. "Development of ovarian cancer-targeted adenoviral vectors." Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/100129/.
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Ovarian cancer is the deadliest gynaecological cancer, with less than half of patients surviving five years from diagnosis. The asymptomatic nature of the early disease commonly results in diagnosis at an advanced stage where peritoneal metastases are prevalent, and the disease rapidly develops resistance to platinum-based agents. Innovative treatments are needed to combat this untreatable disease that has a poor prognosis. Adenoviruses (Ad) are versatile gene therapy vehicles that have been studied for six decades. Their wider clinical use has been limited by poor tumour-specificity, pre-existing immunity, and toxicity-inducing off-target delivery. We have generated an Ad5.3D.A20 vector that is re-targeted to an epithelial cancer-specific marker, αvβ6 integrin, and fully de-targeted from native interactions ‒ αvβ3/5 integrins, coxsackie and adenovirus receptor (CAR), and human coagulation factor 10 (FX). Ad5.3D.A20 selectively transduced αvβ6+ epithelial ovarian cancer (EOC) cells in vitro and clinical ovarian ascites-derived EOC cells ex vivo, including in the presence of neutralising anti-Ad antibodies. In vivo, Ad5.3D.A20 exhibited significantly reduced off-target accumulation and transduction of the liver, spleen and lungs, relative to Ad5. Efficacy studies are underway to investigate its oncolytic potential. Furthermore, we explored the potential use of alternative serotypes from the rare seroprevalence subgroup D. A novel Ad10 vector showed improved resistance from neutralisation and lack of FX binding. Chimaeric Ad5 vectors pseudotyped with Ad10, -15, -24, -29, -48 or -53 fiber had reduced interactions with CAR, but no binding to CD46. Our strategy for translation initially is via intraperitoneal delivery of oncolytic vectors, bypassingmany of the barriers presented by systemic delivery, but enabling transduction of disseminated metastases. These exquisitely tumour-targeted vectors may be further modulated to carry therapeutic moieties to complement their direct cell-killing activity via the stimulation of anti-cancer immunity. The generated tropism-modified vectors have significant therapeutic potential for a wide range of immuno-oncolytic applications.
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Stepto, Hannah. "Development of adenoviral and miRNA eluting stents." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/7175/.
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Cardiovascular disease (CVD) is the most common cause of death worldwide, accounting for 31% of the annual deaths per year. The term cardiovascular disease refers to many different disorders of the heart and blood vessels, the most prolific of which is coronary heart disease (CHD). The root cause of CHD is atherosclerosis, which is the build-up of plaque in the intima of the arteries, restricting the blood supply which reaches the heart. If left untreated, the plaques lining the vessel can rupture and cause thrombosis and myocardial infarction (MI). Taking into account an individual’s risk-benefit ratio, a suitable treatment is considered with the aim to prevent MI from occurring. The two main revascularisation strategies are coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI), the latter being the most frequently used; within the UK 80% of revascularisation is performed by PCI. A PCI procedure is non-invasive and involves the deployment of an intravascular stent into the diseased vessel, which acts as a permanent scaffold that mechanically re-widens the vessel. However, despite the prolific use of intravascular stents within the clinic, there are significant problems associated with this revascularisation technique, such as in-stent restenosis (ISR) and late stent thrombosis (LST). ISR is the re-narrowing of arteries after stent deployment, characterised by a neointima growth within the lumen of the vessel. ISR is a significant clinical problem, because it can lead to repeat revascularisation procedures and often patients present frequently with unstable symptoms, fulfilling the criteria for an MI diagnosis. ISR occurs as a complex wound healing response caused by the stent deployment. Denudation and tearing of the endothelium and the mechanical stress endured by the vascular smooth muscle cells (VSMC) is a stimulus for hyperplasia. VSMC de-differentiate from the contractile to synthetic states and proliferate and migrate into the lumen of the vessel, secreting ECM which forms the bulk of the neointima. The neointima growth will continue to form until the endothelium has re-established itself. As a treatment to prevent ISR, drug-eluting stents (DES) were introduced which were coated with anti-proliferative drugs, to prevent ISR from occurring. DES proved to be very successful at preventing ISR; however elevated rates of LST were associated with these stents. This elevated event rate for DES by LST is thought to be caused as a result of delayed re-endothelialisation and by inflammatory reactions to the polymer used in the coating of DES. The aim of this study was consequently to investigate methods for delivery and coating of novel therapeutics, adenoviruses and miRNA onto stent surfaces. This would allow investigation into the prevention of ISR using these therapeutics, taking advantage of the localised stent setting. It was hoped that local delivery would provide significant advantages over systemic delivery by decreasing the dosage required, avoiding systemic side-effects and increasing delivery to the targeted area, thereby enhancing the therapeutic effect. For the development of coating methods for adenovirus eluting stents, the majority of the work conducted was done using Ad5 as a model virus vector. Three different methods were investigated and evaluated in vitro; deposition onto polyelectrolyte multilayer (PEM) surfaces, by direct conjugation with a modified poly(lactic acid) (PLA) surface through covalent bond formation and collagen gel entrapment. The development of coating methods for miRNA eluting stents focussed initially on collagen gel entrapment; however it was discovered that direct application onto a PLA surface provided a system whereby excellent delivery of the miRNA could be achieved. This methodology was extensively investigated and evaluated in vitro from stent material surfaces, and in vivo in both the porcine and murine stenting models. The results presented here have extended current methodology for both miRNA and adenovirus eluting stents. To the best of our knowledge, this is the first time that miRNA eluting stents have been used in these animal models and therefore contributes significantly to the field of miRNA-based therapeutics.
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Larochelle, Nancy. "Gene transfer to skeletal muscle using adenoviral recombinants." Diss., lmu, 2002. http://nbn-resolving.de/urn:nbn:de:bvb:19-2567.
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Zhu, Qing Gauldie Jack. "Immunization via the colonic mucosa using adenoviral vectors /." [Hamilton, Ont.] : McMaster University, 2004.
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Berman, Chet Michael. "Directed evolution in human cells via adenoviral replication." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/121778.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2019Cataloged from PDF version of thesis.
Includes bibliographical references.
The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both basic biological research, and therapeutic development. Typically, researchers turn to directed evolution either in vitro, in bacteria, or in yeast, to mutate, select, and amplify biomolecules of interest (BOls) with new and highly-tailored activities. Unfortunately, BOIs evolved in these environments often fail to function when translated into the complex environment of metazoan cells. Unique metazoan biology such as sophisticated proteostasis networks, complex cell signaling pathways, distinctive post-translational modifications, cellular trafficking, and highly regulated chromatin architecture can all derail the activity of BOIs evolved in simpler systems. Current approaches to directed evolution in metazoan cells rely on labor-intensive and time-consuming screening approaches that have a high potential for false positives.
Robust approaches for directed evolution directly in human cells are profoundly needed. In this thesis, I describe the development, characterization, and application of a broadly applicable platform for directed evolution of diverse BOIs directly in human cells. The platform relies on a partially gutted adenovirus lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution of complex function. High mutagenesis rates are attained by trans-complementation of an engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell.
This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to protease expression or activation by serially passaging adenovirus carrying the BOI. As proof-of- concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.
NIH Biotechnology Training Program for funding three years of my PhD. Rubius Therapeutics and Tiffany Chen for the summer internship opportunity
by Chet Michael Berman.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemistry
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Willemsen, Kristin R. "Improving adenoviral vectors for muscle-directed gene therapy." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/28115.
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Gene therapy is a promising approach for the treatment of Duchenne Muscular Dystrophy (DMD). Adenoviral vectors (Ad) are the most commonly used vectors in gene therapy studies however, the overall large size of the Ad particles (162nm), due in part to the fiber proteins that extrude from the surface of the virion, prevent their efficient distribution in muscle. The objective of this project was to evaluate the transduction performance of Ad5 based vectors genetically modified to encode shorter fiber proteins derived from Ad serotypes 35 and 9. Optimal transduction was dependent on fiber length in some cell lines and in mdx muscle. However, fiber-modified viruses have an improved viral dispersion and improved transduction up to 10-fold in normal muscle. In addition, an optimized non-immunogenic reporter gene ideal for monitoring vector function in murine disease models was presented. The results of these experiments will contribute to the understanding of Ad transduction in muscle and aid in the design of efficient vectors for DMD therapy.
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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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Raimondi, Giulia. "Broadening Adenoviral Oncolysis in PDAC: Interrogation of Patient-Derived Organoids for personalized virotherapy and modulation of miRNA content to boost adenoviral potency." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671205.
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The general goal of this thesis has been to progress oncolytic adenovirus therapy for PDAC, by the incorporation of novel preclinical models to test for patient-specific responses and the generation of oncolytic adenoviruses with enhanced therapeutic index.
The two main objectives have been the following:
i) Evaluate patients-derived organoids (PDOs) technology as a platform to screen for personalized virotherapy in vitro
1) Establishment of a battery of PDOs from PDAC and normal pancreatic tissues, and evaluation of their applicability in the study of adenoviral infection;
2) Screening of a battery of PDOs to identify individual sensitivities to virotherapies, and the effects derived from the combination with chemotherapy;
3) Study virotherapy-responses in metastasis originated from
PDOs xenografted in mice;
(ii) Improve oncolytic adenovirus potency by modulation of miRNAs deregulated in PDAC
4) Screening of aberrantly expressed miRNAs sensitizing viral oncolysis in PDAC via CRISPR/Cas9 system;
5) Generation of a miRNA sponge-adenovirus and evaluation of its oncolytic effects in vitro and in vivo;
6) Modulation of miRNA levels with the THZ1 transcriptional inhibitor, and assessment of the effects of its combination
with oncolytic adenoviruses.
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Mück-Häusl, Martin Andreas. "Genetic engineering of adenoviral vectors for improved therapeutic applications." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-138269.
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Kirby, Ian. "Molecular and biophysical analysis of adenoviral fiber-receptor interactions." Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429491.
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Salehi, Siamak. "Development of an integrating adenoviral vector for gene therapy." Thesis, King's College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406862.
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Lakdawala, Seema Sailesh. "Impact of DNA damage proteins on the adenoviral lifecycle." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3373343.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed Oct. 19, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 172-200).
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Xu, Ning. "Adenoviral Control of RNAi/miRNA Pathways in Human Cells." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distribution], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9387.
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Okubo, Yasunori. "Osteoinduction with bone morphogenetic protein-2 expressing adenoviral vector." Kyoto University, 2001. http://hdl.handle.net/2433/150519.
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Rinne, Andreas. "Gene silencing using adenoviral RNAi vectors in adult cardiac myocytes." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=979850819.
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Schneider, Julia. "Immunologische Konsequenzen nach Transplantation einer adenoviral infizierten Leber im Rattenmodell." kostenfrei, 2008. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/1205/.
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Ashbourne, Excoffon Katherine J. D. "Adenoviral-mediated gene transfer of the human lipoprotein lipase gene." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0013/NQ56498.pdf.
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Terry, Helen Elizabeth. "The development of recombinant Adenoviral vaccines to target pneumovirus infection." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35641/.
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Respiratory Syncytial Virus (RSV) is a member of the pneumovirus genus (family Paramyxoviridae, subfamily Pneumovirinae). RSV is an important respiratory virus of both infants and the elderly, representing an underappreciated burden on health care systems. In addition, re-infections can occur despite the presence of pre-existing immunity, suggesting that immunological memory to RSV is incomplete. To date, treatment of RSV infection is limited to the provision of supportive care and no effective vaccine is available. Although several are currently under investigation, these candidates focus upon the delivery of the F and G antigens of RSV to stimulate the immune system, rather than the internal antigens, which may provide cross protection between different subtypes of RSV. Vaccine development has been greatly hindered by the lack of an appropriate animal model in which to study vaccine efficacy and pneumovirus pathogenesis. Pneumonia virus of mice (PVM) is also a member of the Pneumovirus genus and, like RSV infection of humans, causes a bronchiolitis and fatal pneumonia in its natural host, the mouse. PVM has been proposed as an appropriate model system in which to both study pneumovirus pathogenesis and vaccine efficacy. The PVM model system was adapted to investigate a potential vaccination strategy to address the lack of an available RSV vaccine. Replication deficient recombinant adenovirus serotype 5 (rAd5) vectors were constructed which expressed the F, M and N genes of PVM J3666, in addition to a control construct, which expressed the LacZ gene of E. coli. The constructs were administered via the intranasal route to BALB/c mice and were able to elicit complete protection against a lethal dose of pathogenic PVM J3666, in both short-term experiments and in a long-term experiment, up to 20 weeks post immunisation. The protection effect elicited by the constructs was observed when administered in a single dose, and in alternative mouse strains, C3H/He-mg and C57BL/6, which had differing immunity haplotypes. The rAd5 vectors generated a PVM specific IgG humoral response to PVM and Ad5 antigen which did not correlate as the primary mediator of protection. The rAd5 candidate expressing the N gene of PVM was shown to induce IFNγ secreting T-cells. The use of a peptide library of PVM N protein determined that a specific response could be identified towards the amino acids N41-90, N81-130, N161-210 and N281-330. Thus, the PVM infection model of BALB/c mice provides an immunological platform to facilitate the study of RSV and PVM pathogenesis, immunology and vaccine development.
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Lockley, Michelle. "Activity of Adenoviral E1A CR2 Deletion Mutants in Ovarian Cancer." Thesis, Queen Mary, University of London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504564.
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d1922-947 is a type 5 adenovirus deleted in a portion of its genome, EIA CR2, which releases E2F by binding retinoblastoma protein (PRb). Transactivation of E2Fresponsive genes stimulates S phase entry and viral DNA is preferentially replicated. Since over 90% of human cancers have pRb pathway abnormalities, d1922-947 replicates selectively in malignant cells. Co-immunoprecipitation in IGROVI human ovarian cancer cells and cytotoxicity in hTERT and SV 40 immortalised ovarian epithelial cells confirmed the proposed mechanism of d1922-947 activity. In IGROVI cells, d1922~947 induced S phase and viral protein expression more rapidly, and replicated more efficiently, than Ad5 WT. Cytotoxicity of d1922-947 exceeded Ad5 WT and the EIB 55K deleted adenovirus, . d11520. In female nude mice bearing intraperitoneal (IP) IGROVI xenografts, d1922- 947 increased survival above control (p<0.001), particularly when delivered in icodextrin. Hepatotoxicity, which was less severe than Ad5 WT, was seen in some nude mice treated with d1922-947 and in immunocompetent .C57B1I6 mice bearing CMT64 murine non.,.small cell lung cancer cells IP. Transformed murine ovarian epithelial cells (IDS) did not' suppoli production of late adenovirai proteins, despite efficient DNA replication, transcription and nuclear export of late viral RNAs. This could explain the .inability of IP viruses to improve survival in C57B1I6 mice bearing ID8 cells IP . . Ovarian cancer disseminates diffusely across peritoneal surfaces, so quantifying ill VIVO eIhcacy IS pro51emauc. In IllICe oeanng ll' xenograIts 01 FIreny Iucnerase expressing IGROVI cells (IGROVI-Luc), light output increased over time, whereas IP d1922-947 held bioluminescence at baseline for over two months. A Renilla relliformis luciferase expressing EIA CR2 deleted adenovirus, dlCR2 Ren, was created, which had comparable ,anti-tumour efficacy to dI922-.947. Light emission by dlCR2 Ren correlated with EIA expression and, after a single IP injection in murine models of ovarian cancer, light emission and therefore viral' persistence was demonstrated for 2 weeks.
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Kamel, Wael. "Adenoviral small non-coding RNAs : A Structural and Functional Charaterization." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-283080.
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Since their discovery in 1953, adenoviruses have significantly contributed to the understanding of virus-host cell interactions, including mechanistic details of cellular processes such as cell cycle control and alternative RNA splicing. Among the first characterized adenoviral genes were the virus-associated RNAs (VA RNAI/II), which are produced in massive amount during a lytic infection. The VA RNAs perform multiple functions and are required for a successful adenovirus life cycle. More recently, it was shown that the VA RNAs are processed into small viral miRNAs, so-called mivaRNAs, which interfere with the function of the cellular RNAi/miRNA machinery. In papers I and II, we focused on a structural and functional characterization of the mivaRNAs using two approaches. Firstly, we created a model system where the predicted miRNA-like function of mivaRNAI could be investigated, without interfering with other VA RNA functions. This was accomplished by construction of recombinant adenoviruses, in which the seed sequence of mivaRNAI was altered. The results showed that in cell culture experiments the mivaRNAI seed sequence mutants grew as the wild type virus, suggesting that the mivaRNAs are not required during the lytic phase of an adenovirus infection. Secondly, we showed that the VA RNAs from different human adenoviruses (Ad4, Ad5, Ad11 and Ad37) undergo the same type of Dicer-dependent processing into mivaRNAs, which subsequently are loaded onto the RNA induced silencing complex (RISC), albeit with different efficiencies. In paper III, we demonstrated that the promoter proximal region of the adenovirus major late promoter (MLP) produces a novel non-canonical class of small RNAs, which we termed the MLP-TSS-sRNAs. Surprisingly the MLP-TSS-sRNA maintains the m7G-cap structure while bound to Ago2 containing RISC. These complexes are functional suppressing expression of target mRNAs with complementary binding site. Most importantly, the MLP-TSS-sRNA limits the efficiency of viral DNA replication probably through a targeting of the E2B mRNAs, which are transcribed in the antisense orientation. In conclusion, the MLP-TSS-sRNA represents the first viral small RNA, which has been shown to have a function as a regulator of an adenovirus infection.
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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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Addison, Christina Lynn. "Construction & characterization of adenoviral vectors expressing cytokines for cancer immunotherapy." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0003/NQ32564.pdf.
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Marr, Robert Anthony. "Tumour gene therapy using adenoviral vectors expressing tumour necrosis factor alpha." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0029/NQ66283.pdf.
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Addison, Christina Lynn. "Construction and characterization of adenoviral vectors expressing cytokines for cancer immunotherapy /." *McMaster only, 1997.
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Spurrell, Emma Louise. "The role of the innate immune system in oncolytic adenoviral therapy." Thesis, University of London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511359.
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Sridhar, Saranya. "Development of recombinant adenoviral vectors as a pre-erythrocytic malaria vaccine." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497101.
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Ralph, G. Scott. "Investigating AMPA-receptor mediated neurotoxicity using novel neurone-specific adenoviral vectors." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340079.
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Harding, Thomas C. "Development of adenoviral gene transfer systems : application to studies of neuroprotection." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297828.
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Vaillant, Remi. "The role of adenoviral capsid protein VI in cell cycle modulation." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0297/document.
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Les Adénovirus humains sont des virus non enveloppés se répliquant dans le noyau des cellules hôtes.Durant l’infection et après leur entrée par endocytose, les Adénovirus sont transportés au noyau pourinitier l’expression du génome viral. Dans l’endosome, les capsides virales subissent un désassemblagepartiel et libèrent le facteur viral lytique, la protéine VI (pVI). Au niveau de la membrane de l’endosome,cette protéine va alors induire sa rupture permettant ainsi le relargage des virions au sein du cytoplasmegrâce à son hélice amphipatique N-terminale. Par la suite, pVI est transportée vers des structuresnucléaires appelées PML nuclear bodies (PML-NB), associée une ubiquitine ligase cytoplasmique, laNedd4.2. Les PML-NB sont des complexes nucléaires multi-protéiques qui ont des propriétésantivirales. Celles-ci impliquent le recrutement de facteurs de transcription répressifs comme parexemple la protéine anti apoptotique Daxx ou encore le suppresseur de tumeur p53, impliqué dans larégulation du cycle cellulaire. Il a été montré que la protéine pVI en complexe avec Nedd4.2 induit larelocalisation de Daxx des PML-NB dans le cytoplasme, ce qui permet une expression efficace dugénome viral. Ainsi, l’inhibition fonctionnelle de Daxx par pVI suggère que cette protéine virale puisseaussi être impliquée dans la restriction de p53.Dans cette étude, nous avons montré que le nombre des modifications post-traductionnelles (PTM) dep53 augmentent en présence de pVI dans la cellule. De plus, les données obtenues montrent quel’expression de pVI affecte la transcription dépendante de p53 et que l’interaction avec Nedd4.2 n’estpas nécessaire pour inhiber les fonctions de p53. Pour étudier l’implication de pVI dans la modulationdu cycle cellulaire, nous avons créé une lignée cellulaire humaine exprimant cette protéine virale defaçon stable. La caractérisation de cette lignée a permis de mettre en évidence une prolifération cellulaireaccrue. Nos observations ont aussi montré une perte importante des PML-NB et une réduction desprotéines clés du cycle cellulaire p53 et pRb, un autre suppresseur de tumeur. Par des techniques demicro-injection et l’utilisation de l’inhibiteur MG132, nous avons observé que ces deux facteurscellulaires sont ciblés vers le protéasome et dégradés lors de la surexpression de pVI. L’étude desfonctions de cette protéine virale laisse penser que la protéine pVI présente un potentiel oncogéniquecar en effet, sa surexpression induit la dérégulation de l’homéostasie cellulaire et l’inhibition desuppresseurs de tumeur, comme p53 et pRbHuman Adenovirus are non-enveloped viruses which replicate inside the host cell nucleus. Uponinfection and after receptor-mediated entry, they are transported towards the nucleus to initiate the viralgene expression. Viral capsids deliver from the endosome into the cytoplasm by partial disassembly andrelease inside the endosome mediated by viral lytic factor protein VI (pVI). pVI is targeted to themembrane via an amphipathic helix structure in the N-terminus of the viral protein. After membranerupture and capsid release, pVI is transported to sub-nuclear structures, so-called PML nuclear bodies(PML-NBs), together with the cytoplasmic ubiquitin ligase Nedd4.2. PML-NBs represent multiproteinaggregates in the host-cell nucleus with an antiviral capacity, as to several PML-associated repressivetranscription factors, such as the anti-apoptotic Daxx protein and the tumor suppressor p53 were reportedto localize at these foci. In addition, pVI-mediated displacement of Daxx from PML-NBs was shown tooccur in dependency of Nedd4.2 to support efficient viral gene expression. Therefore, we postulate thatbesides Daxx functional inhibition, pVI might also be involved in p53 restriction.Here, we show that p53 posttranslational modification (PTM) is increased when pVI protein is presentin the host-cell. Moreover, we obtained data that pVI expression severely impacts p53 inducedtransactivation of cellular transcription. Biochemical approaches indicate that pVI binding of theubiquitin ligase Nedd4.2 is no prerequisite for the capacity to inhibit p53 functions. In a next step toelucidate the role of pVI on cell cycle regulation, we generated a human cell line stably expressing theviral pVI protein. Our characterization analyses show significantly that these cells benefit from thepresence of pVI as we proved increased cell proliferation rates. We also observed an intense loss ofPML-NBs and reduced protein concentrations of cycle key regulators p53 and pRb. Usingmicroinjection and the inhibitor MG132 we were able to show that both cellular restriction factors weresequestered into the proteasomal degradation pathway of the cell. Evaluation of pVI functions temptedus to speculate, whether pVI might execute oncogenic potential upon overexpression, due toderegulation of host-cell homeostasis and inhibition of tumor suppressive determinants
Humane Adenoviren (HAdV) sind unbehüllte Doppelstrang-DNA-Viren mit einem Proteinkapsid, diesich im Wirtzellkern replizieren. Der lytische Infektionsverlauf beginnt mit dem rezeptor-vermitteltenEintritt des Viruspartikels und dem gerichteten Transport des viralen Genoms zum Wirszellkern. Dasvirale Protein VI (pVI) ist nötig um den effizienten Austritt des bereits disassemblierten Viruspartikelsaus dem zellulären Endosom zu gewährleisten. Durch eine amphipathische Helix im N-terminalenProteinbereich interkaliert dieser lytische Faktor in die endosomale Membran und führt zum Aufbruchdes zellulären Organells. pVI wird anschließend an zelluläre Kernstrukturen, sogenannte PML nuclearbodies (PML-NBs) lokalisiert und komplexiert dort mit der zytoplasmatischen Ubiquitinligase Nedd4.2.PML-NBs stellen nukleäre Multiproteinkomplexe dar, die mittlerweile aufgrund ihrer antiviralenEigenschaften in den Mittelpunkt der virologischen Forschung gerückt sind. Diese zellulären Aggregatebestehen hauptsächlich aus repressiven Transkriptionsfaktoren, wie dem anti-apoptotischen DaxxProtein sowie dem Tumosupressor p53. In diesem Zusammenhang konnte bereits eine pVI-vermittelteRelokalisation des Daxx Proteins aus den PML-NBs gezeigt und als Vorraussetzung zur effizientenVirusgenexpression bestätigt werden. Es stellte sich im Rahmen der vorliegenden Arbeit die Frage, obneben der pVI-abhängigen Daxx Inhibition, auch p53 ein Zielprotein des viralen Capsidproteinsdarstellt.Unsere Arbeiten zeigen erstmals, dass nach der pVI Expression vermehrt posttranslationaleModifikationen am p53 Protein beobachtet werden. Weitere Befunde konnten außerdem einen Einflussvon pVI auf die p53-abhängige Transaktivierung zellulärer Promotoren beweisen. Mittelsbiochemischer Analysemethoden konnten wir zeigen, dass die Kooperation zwischen pVI und Nedd4.2keine Rolle bei der p53 Inhibition zu spielen scheint. Um im nächsten Schritt die Rolle von pVI imZellzyklus genau zu beleuchten, wurde zunächst ein zell-basiertes Modelsystem mit stabilerÜberexpression des viralen Faktors generiert, Anschließende phenotypische Analysen konnten zeigen,dass die Anwesenheit von pVI zur Steigerung der Zellproliferationsrate führt. Im Rahmen unsererUntersuchungen konnten wir auch einen signifikanten Verlust zellulärer PML-NBs beobachten sowieeine Reduktion der p53 und pRb Proteinkonzentration nachweisen. Mittels unter Verwendung vonMikroinjektion und dem Inhibitor MG132 war es uns möglich zu zeigen, dass pVI den proteasomalenProteinabbau der beiden Wirtszelldeterminaten p53 und pRb induziert. Deswegen kann man basierendauf den erhobenen Befunden zur pVI vermittelten Dysregulierung des zellulären Wachstums einonkogenens Potenzial des viralen Faktors annehmen
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Regardsoe, Emma Louise. "An investigation into the role of Fas ligand as a potential immunomodulatory molecule for CNS gene therapy." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365794.
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Duran, Amparo. "Investigation of a Trimeric Hemagglutinin Stem Domain from Influenza B for a Universal Vaccine." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38200.
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Influenza infection occurs in as much as 5–15% of the world population, resulting in 3–5 million cases of severe illness and up to 500,000 deaths annually. According to the CDC, on average 24% of all influenza positive respiratory samples during 2001 to 2011 tested positive for Influenza B. Influenza has two main surface glycoproteins, neuraminidase (NA) and hemagglutinin (HA), HA being responsible for the binding of the virus to the host cell. Currently, seasonal influenza vaccines are produced using two strains of Influenza A and one or two strains of Influenza B viruses recommended by the World Health Organization (WHO). These vaccines are mainly targeting the head domain of the HA protein, which mutates constantly, hence the need for annual vaccine updates. The goal of this research is to develop an experimental universal vaccine against influenza B and increase our knowledge to help pave the way for finding a one-time vaccination alternative, reducing the need for a yearly flu shot. To achieve the above, protection and toxicity studies were conducted in DBA/2 mice immunized with a designed HA2 adenoviral-vectored vaccine targeting the HA stem region of influenza B. Results showed that this designed vaccine was able to confer 100% survival protection, this was supported by lower viral titer in trachea and lung tissues. Additionally, we studied the influence of CD40L as a targeting adjuvant, by analyzing its effect on the humoral and cellular immune response, where results showed that it has a significant effect by inducing a higher TH1-bias response. This research is the first report that leads us to a better understanding of the potential use of a conserved consensus HA2 sequence to induce protection against influenza B virus.
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Mui, Melissa. "Role of PP2A in cell death induced by the adenoviral protein E4orf4." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110353.
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The E4orf4 protein of adenovirus induces cell death in human cancer cells and not normal primary cells when expressed alone. E4orf4 is also lethal to the yeast Saccharomyces cerevisiae, which we use as a model system. E4orf4-induced toxicity in both mammalian and yeast cells is largely dependent on its ability to associate with protein phosphatase 2A (PP2A), a highly conserved Ser/Thr phosphatase, more specifically, with the B class of regulatory subunits, of which Bα is best characterized in mammalian cells, as well as the yeast equivalent Cdc55. Binding to the B subunit appears to inhibit PP2A activity with some substrates. Consequently, E4orf4 is a useful agent in delineating specific functions of Bα/Cdc55-containing PP2A holoenzymes in vivo. E4orf4 induces mitotic arrest, suggesting that PP2ACdc55 plays a role in regulating mitotic exit. The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit complex involved in the timely degradation of cell cycle proteins to allow the mitotic exit of cells. Two forms of the APC, APCCdc20 and APCCdh1, control mitotic transitions and we demonstrate that E4orf4, through its interaction with Cdc55, induces unscheduled APCCdc20 activity in S-phase arrested cells, and results in the untimely degradation of both Pds1/securin and the cohesin Scc1. In contrast, E4orf4 prevents induction of APCCdh1. Thus, E4orf4 may uncouple PP2ACdc55 regulation of mitotic events. While the specific mechanism by which E4orf4 mediates cell death is unclear, its interaction with the B regulatory PP2A subunit plays a significant role. Cdc55 and Bα contain seven WD40 repeats organized into a seven-bladed propeller structure. In an attempt to functionally map the Cdc55 subunit, we identified regions in Cdc55 required for E4orf4 binding. A polypeptide composed only of blades 1 and 2 bound to E4orf4, and overexpression of this fragment blocked E4orf4 toxicity in yeast. Later on, with the knowledge of the Bα crystal structure, we delineated the precise regions of Bα/Cdc55 involved in E4orf4 binding. Mutational studies in both yeast Cdc55 and mammalian Bα suggest that E4orf4 binds across the substrate-binding groove of the B regulatory subunits, and binding interferes with the interaction of certain substrates, such as p107, with the PP2A holoenzyme. This deregulation of PP2A activity by E4orf4 may affect the functions of certain critical substrates, ultimately leading to cell death.La protéine adénovirale E4orf4 induit la mort cellulaire dans les cellules cancéreuses humaines et non dans les cellules normales primaires lorsqu'elle est exprimée seule. E4orf4 est également létale dans la levure Saccharomyces cerevisiae, que nous utilisons comme modèle. La toxicité induite par E4orf4 dans les cellules de mammifères et dans la levure dépend largement de son association avec la phosphatase protéique 2A (PP2A), une Ser/Thr phosphatase hautement conservée. Plus spécifiquement, E4orf4 s'associe avec les sous-unités régulatrices de classe B, parmi lesquelles Bα, la mieux caractérisée dans les cellules de mammifères, et Cdc55, son équivalent chez la levure. Il semble que la liaison avec la sous-unité B inhibe l'activité de PP2A pour certains substrats. En conséquence, E4orf4 est un outil très utile pour définir les fonctions spécifiques de l'holoenzyme PP2A contenant Bα/Cdc55 in vitro. E4orf4 induit un arrêt mitotique, ce qui suggère que PP2ACdc55 joue un rôle en régulant la sortie de mitose. Le complexe APC/C (Anaphase Promoting Complex/Cyclosome) est un complexe de plusieurs sous-unités impliqué dans la dégradation minutée des protéines du cycle cellulaire, permettant ainsi aux cellules de sortir de mitose. Deux formes de l'APC/C, APCCdc20 et APCCdh1, contrôlent les passages mitotiques et nous avons démontré qu'E4orf4, par son interaction avec Cdc55, induit une activité non programmée d'APCCdc20 dans les cellules arrêtées en phase S, ce qui résulte en une dégradation désynchronisée de Pds1/securin et de la cohésine Scc1, alors qu'au contraire, E4orf4 prévient l'induction d'APCCdh1. Il semble donc qu'E4orf4 soit responsable du découplage de la régulation, par PP2ACdc55, des évènements mitotiques. Bien que le mécanisme spécifique par lequel E4orf4 régule la mort cellulaire reste incertain, son interaction avec la sous-unité régulatrice B de PP2A joue un rôle important. Cdc55 et Bα contiennent sept répétitions WD40, organisées dans une structure en hélice à sept pales. Dans le but de cartographié fonctionnellement la sous-unité Cdc55, nous avons identifiés les régions de Cdc55 qui sont nécessaire pour la liaison avec E4orf4. Un polypeptide composé uniquement des pales 1 et 2 est toujours capable de se lier à E4orf4, et la surexpression de ce fragment bloque la toxicité d'E4orf4 dans la levure. Plus tard, avec la résolution de la structure cristalline de Bα, nous avons délimité les régions précises de Bα/Cdc55 impliquées dans la liaison avec E4orf4. Des études mutationnelles réalisées à la fois dans Cdc55 (levure) et dans Bα (mammifère) suggèrent qu'E4orf4 se lie aux sous-unités régulatrices B au travers de la zone de liaison du substrat, et que cette interaction interfère avec la liaison de certains substrats, tels que p107, avec l'holoenzyme. Cette dérégulation de l'activité de PP2A par E4orf4 pourrait affecter les fonctions de certains substrats vitaux, ouvrant ainsi la voie vers la mort cellulaire.
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Thirion, Christian. "Improving gene transfer into skeletal muscle through genetic retargeting of adenoviral vectors." Diss., [S.l.] : [s.n.], 2004. http://edoc.ub.uni-muenchen.de/archive/00006339.
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Coates, Patrick Toby Hewlett. "Gene therapy studies of Adenoviral IL-10 transduced Dendritic cells in allotransplantation." Title page, contents and abstract only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phc6525.pdf.
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Bibliography: leaves 151-186. Studies the capacity of dendritic cells transduced with the immunosuppressive gene construct adenoviral interleukin-10 to inhibit alloimmune responses in both small and large animal transplantation models. There is promising in vitro evidence for gene therapy to modify dendritic cell function, which in small animal models can modify skin graft rejection. In large animals, genetically modified dendritic cells alone were not capable of prolongation of allograft survival, suggesting that these cells may require adjuvant immunosuppressive therapy to be used in future protocols.
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Lopez, Gordo Estrella. "Investigation of adenoviral immune neutralization and tropism for improved targeted gene delivery." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8069/.
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Human adenovirus serotype 5 (HAdV-5)-based gene delivery vectors are an attractive option for gene therapy applications because they can deliver large transgenes to a broad range of tissues, allow high level transgene expression, have negligible risk of insertional mutagenesis and are easily produced at high titers. Unfortunately, their high immunogenicity and liver and spleen-associated toxicity when intravascularly administered and high prevalence of neutralizing antibodies in patients remain challenges to be overcome for the generation of safe HAdV-5-based vectors for systemic gene therapy. The discovery that coagulation factor X (FX), a zymogen of a vitamin K-dependent serine protease that circulates in the bloodstream, simultaneously binds HAdV-5 hexon and heparan sulphate proteoglycans (HSPGs) to mediate hepatic transduction enabled the generation of HAdV-5 vectors with substantially reduced liver transduction via the manipulation of key amino acid residues in the adenoviral hexon protein. However, FX was also recently shown to protect HAdV-5 from neutralization by preventing binding of natural IgM antibodies to HAdV-5 capsids and it was reported that, in the absence of FX-binding and neutralization, HAdV-5 vectors can use alternative FX-independent transduction pathways for hepatocyte transduction. These findings highlight the complex interactions between adenoviruses and host blood factors and cells as well as the need for further research on these processes. Here, the interactions that mediate hepatic and splenic tropism of HAdV-5-based vectors and activation of the anti-viral immune response following intravascular delivery were investigated and targeted HAdV-5 delivery to the kidney was assessed. Liver and spleen transduction was assessed in immunocompetent C57BL/6 and immunocompromised Rag 2-/- or NSG mice lacking different components of the immune response, following intravascular administration of wild type or FX-binding deficient HAdV-5 vectors. HAdV-5 virions were neutralized in C57BL/6 mice in the absence of FX-binding, confirming a role for FX in protecting HAdV-5 from in vivo neutralization. However, NSG mice, which lack both innate and adaptive immunity, failed to neutralize FX-binding deficient HAdV-5 virions. Interestingly, administration of FX-binding deficient HAdV-5 vectors to IgM antibody-deficient Rag 2-/- mice revealed that IgM antibodies might not be required for in vivo neutralization of HAdV-5, indicating that innate immunity alone might be sufficient. In agreement with previous reports, exposure of FX-binding deficient HAdV-5 vectors to C57BL/6 serum or wild type HAdV-5 vectors to C57BL/6 serum pre-incubated with the FX inhibitor X-bp led to HAdV-5 neutralization in vitro. In contrast, Rag 2-/- and NSG serum failed to neutralize HAdV-5 in vitro in the absence of FX-binding, indicating that IgM antibodies are essential for in vitro HAdV-5 neutralization. This suggests in vitro and in vivo adenovirus neutralization is mediated by different mechanisms. Importantly, administration of FX-binding deficient HAdV-5 vectors to NSG mice, which were unable to neutralize HAdV-5, confirmed the existence of alternative FX-independent pathways for liver and spleen transduction in the absence of neutralization. CAR and αvβ3,5 integrins were assessed as possible host cell receptors for HAdV-5 transduction of these tissues in immunocompetent and immunocompromised mice. The use of CAR or αvβ3,5 integrin-binding ablated HAdV-5 vectors revealed that CAR and αvβ3,5 integrins might serve as receptors for HAdV-5 liver transduction in immunocompetent C57BL/6 mice in contrast to immunocompromised Rag 2-/- mice. Neither CAR nor αvβ3,5 integrins mediated HAdV-5 spleen transduction in either mouse strain. Furthermore, administration of HAdV-5 vectors simultaneously ablated for FX and αvβ3,5 integrin-binding to NSG mice showed that αvβ3,5 integrins play no role in liver or spleen transduction in the absence of neutralization. With the aim to define novel FX-independent pathways of HAdV-5 transduction in vitro that might be relevant in vivo, cell transduction was assessed for wild type or FX-binding deficient HAdV-5 vectors in the presence of immunocompromised Rag 2-/- serum or serum that had been pre-incubated with X-bp. These studies confirmed the existence of FX-independent mechanisms able to enhance HAdV-5 cell transduction in vitro in the presence of mouse serum and absence of neutralization. To identify the receptor(s) involved in in vitro HAdV-5 transduction in the presence of mouse serum, soluble recombinant HAdV-5 fiber knob was used to block access of virions to CAR and wild type or FX-binding deficient HAdV-5 cell transduction was assessed in high and low CAR-expressing cell lines in the presence of C57BL/6 or Rag 2-/- serum with or without X-bp. HAdV-5 predominantly used a FX-independent pathway for cell transduction of high CAR-expressing cell lines in the presence of Rag 2-/- serum and soluble fiber knob substantially reduced both C57BL/6 and Rag 2-/- serum-enhanced transduction in such cell lines, suggesting a role for CAR, Conversely, HAdV-5 used the FX-mediated pathway or other FX and CAR-independent pathways for low CAR-expressing cell line transduction. Importantly, the use of CAR-binding deficient HAdV-5 vectors demonstrated that CAR usage in this setting does not rely on direct interactions of HAdV-5 with CAR, thus implicating a role for a mouse serum protein(s) in this process. To investigate these different pathways further, virions were fluorescently labelled with Alexa Fluor 488 dye and HAdV-5 cell binding, uptake and endosomal membrane penetration were characterized in the presence of FX by microscopic assessment of individual virions at the single cell level. FX substantially enhanced virion cell binding but had minimal effect on virion uptake and it was suggested to decrease efficiency of endosomal membrane penetration, limiting escape of virions from endosomes into the cytosol. Finally, FX and CAR-binding deficient HAdV-5 vectors were engineered to incorporate renal-targeting peptides found by in vitro phage display into the HI loop of the fiber knob domain for kidney-specific gene therapy. The resultant mutant HAdV-5 vectors were tested for their specificity in mediating gene delivery to ligand-expressing cells in vitro but failed to achieve specific targeting. Together, these findings contribute to a deeper understanding of the interactions between HAdV-5 vectors and host blood components and cell receptors, and their implications for liver and spleen transduction and neutralization of virions. The studies presented in this thesis highlight the limitations of current re-targeting strategies and the need for further research to successfully develop efficient HAdV-5-based vectors for systemic gene therapy.
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JUILLARD, VERONIQUE. "Impact de l'utilisation d'un vecteur adenoviral sur la reponse immunitaire de l'hote." Paris 6, 1996. http://www.theses.fr/1996PA066205.
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Notre objectif a ete d'etudier les differents parametres des reponses immunitaires, cellulaire et humorale, induites par un vecteur adenoviral recombinant, defectif pour la replication. Nous avons mis en evidence, apres une seule immunisation par le vecteur adenoviral exprimant un gene codant la -galactosidase, une forte reponse cellulaire cytotoxique dirigee contre cette proteine, par les lymphocytes t (lt) cd8#+. Nous avons montre que cette reponse etait capable de proteger les animaux immunises, contre une infection par voie intracranienne par virus de la vaccine recombinant exprimant le gene codant la -galactosidase. Par ailleurs, nous avons observe une reponse proliferative et la production d'il-2 par les lt cd4#+ et l'induction d'une reponse humorale, specifiques de la -galactosidase. Une reponse memoire est obtenue pour l'ensemble des parametres analyses. L'intensite des reponses immunitaires, apres plusieurs immunisations par le vecteur, n'est pas significativement augmentee. L'apparition d'anticorps neutralisants diriges contre le vecteur lui-meme, des la deuxieme immunisation, pourrait constituer le facteur limitant l'efficacite des epreuves de rappel. A l'instar de la reponse humorale dirigee contre la particule adenovirale recombinante, nous observons une reponse dirigee contre la -galactosidase dominee par les isotypes igg2. L'etude comparative de la reponse humorale, dirigee contre la -galactosidase, apres immunisation par le vecteur, la proteine soluble ou par un plasmide codant le gene lac-z fait apparaitre des differences qualitatives importantes pour l'ensemble des criteres analyses. Ces resultats sont discutes dans le cadre de la modulation de la reponse immunitaire dirigee contre le produit du transgene, la -galactosidase, par le vecteur adenoviral. Enfin, nous avons mis en place un modele d'etude de l'induction d'une reponse immunitaire au niveau des tissus muqueux chez la souris. Nous avons, pour cela, realise un vecteur adenoviral exprimant un antigene de toxoplasma gondii (gra4)
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Anchim, Aleksandra. "Vaccination Potential Of Adenoviral Vectors Displaying Heterologous Epitopes In Their Capsid Proteins." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS070/document.
Full textAbstract:
Mes travaux de thèse ont pour but d’évaluer l’approche « épitope display » pour sa capacité d'induire les réponses cellulaires. Ad à capside modifiée par insertion de différents épitopes T issus de la ovalbumine ont étés produit. Après administration à des souris C57BL/6, j'ai mis en évidence l'induction de la réponse cellulaire dirigée contre les épitope insérés (grâce aux techniques ELISPOT, tétramères, ou bien en quantifiant la production d’IFNg par les splénocytes restimulés in vitro). J’ai démontré que ces réponses sont limités chez des souris préalablement immunisées avec Ad. Des manipulations en course ont pour but de confirmer ces résultats et d'évaluer la cinétique de ces réponses. Mon 2ème objectif est de comprendre les paramètres qui contrôlent l’immunogénicité des Ad présentant des épitopes. Ainsi, j’ai montré que l’ablation des interactions des Ad porteurs d’un épitope issu de l’ovalbumine avec les récepteurs/facteurs (intégrines, facteur X de la coagulation) impliqués dans l’entrée de l’Ad dans les cellules ne modifiait pas leur capacité à induire une réponse humorale contre l’ovalbumine. Ces résultats suggèrent que le processus d’infection virale n’est pas requis pour l’induction d’une réponse humorale par les Ad porteurs d’épitopesRecombinant adenoviruses (Ad) have recently been employed for a wide range of vaccination strategies. Unfortunately, highly prevalent pre-existing neutralizing antibodies (Abs), reduce their ability to trigger transgene expression. To avoid the step of gene transfer a new vaccination strategy has been proposed based on the use of Ad displaying epitopes inserted into their capsid proteins. Using an ovalbumin-derived B cell epitope, our group demonstrated that vaccination efficiency depends on both the site of peptide insertion and the host immune status towards Ad (Lanzi et al; 2011). The present work aims at (1) evaluating the potency of Ad displaying T-cell epitopes from ovalbumin to elicit cellular responses and (2) understanding the molecular bases controlling the efficacy of this vaccination strategy. 1) Ad displaying T-cell epitopes from ovalbumin were constructed, produced and characterized in vitro. First in vivo experiments in naive mice showed induction of cellular responses, assessed with techniques like ELISPOT, tetramer staining and in vitro splenocyte restimulation. Subsequent experiments showed that pre-exisitng anti-vector immunity is hampering the potent induction of anti-epitope cellular responses. Current work is aiming at confirming the obtained results as well as at evaluating the kinetics of cellular responses induced upon "epitope display" vaccination. 2)First, the influence of interactions of Ad (displaying OVA peptide) with their natural receptors was investigated. Different detargeted Ads were produced and characterized in vitro. Upon mice immunization these vectors led to unmodified anti-epitope humoral responses, suggesting that their efficacy does not depend on the ability to transduce cells. In parallel we sought to evaluate the impact of innate immunity on the outcome of anti-epitope adaptive immune responses. Upon immunization of WT and MyD88-/- mice with Ad displaying OVA epitope we observed that cellular responses induced in MyD88-/- mice are significantly diminished while humoral responses were not altered. These results remain to be confirmed but question the role of other innate immunity sensors in the immunogenicity of Ad-based vaccines. Altogether, our work is expected to provide the foundations for the development of Ad-based vaccines with minimized side effects and unaltered adjuvant properties
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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.
Full textAbstract:
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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Backström, Ellenor. "Regulation of Adenoviral Gene Expression by the L4-33K and L4-22K Proteins." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-101324.
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The splicing pattern during an adenovirus infection is shifted at the late phase towards using weaker splice sites, splicing out larger introns. Splicing of weak 3´ splice sites usually requires recognition of the 3´AG dinucleotide before the first catalytic step of splicing. Such splicing events are said to be AG-dependent and requires an interaction of both subunits of the cellular splicing factor U2AF with the 3´ splice site. We show that splicing of transcripts that are AG-dependent in uninfected nuclear extracts (NE) becomes AG-independent in nuclear extracts prepared form adenovirus late-infected HeLa cells (Ad-NE). Further we demonstrate that the first step in splicing of a model transcript, IgM, becomes completely U2AF-independent in Ad-NE. This finding supports our working model that 3´ splice site recognition in Ad-NE is altered, and in fact might be U2AF-independent. We further show that the adenovirus late protein L4-33K acts as a virus encoded alternative splicing factor. L4-33K activates splicing of both cellular and viral transcripts containing weak 3´ splice sites. This supports the hypothesis that adenovirus alter splicing during the infection to favour usage of weak, suboptimal 3´ splice sites. However, we were unable to find an alternative U2AF-related factor that could stimulate L4-33K splicing enhancer activity. Furthermore, we demonstrate that the serine residues in the C-terminal part of L4-33K are important for the splicing enhancer activity but also for its nuclear localisation. The adenovirus major late promoter is highly activated after the onset of viral genome replication. Protein complexes binding to downstream elements of the promoter are required for full enhancement of this promoter. We show that an L4-33K-related protein, L4-22K, stimulates transcription from the major late promoter. This stimulation is mainly via the downstream elements and does not require the viral IVa2 protein, which is a transcription factor of the major late promoter.
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Quinlan, Jonathan Mark. "In vitro culture of embryonic mouse intestinal epithelium and adenoviral-mediated gene delivery." Thesis, University of Bath, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501614.
Full textAbstract:
Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes.