Dissertations / Theses on the topic 'Gene transfer, viral genome'
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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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Monjezi, Razieh [Verfasser], and Michael [Gutachter] Hudecek. "Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing / Razieh Monjezi ; Gutachter: Michael Hudecek." Würzburg : Universität Würzburg, 2018. http://d-nb.info/1162062231/34.
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Ibraheim, Raed R. "Genome Engineering Goes Viral: Repurposing of Adeno-associated Viral Vectors for CRISPR-mediated in Vivo Genome Engineering." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1114.
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One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.
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Mills, Ryan Edward. "New AB initio methods of small genome sequence interpretation." Diss., Available online, Georgia Institute of Technology, 2006, 2006. http://etd.gatech.edu/theses/available/etd-04062006-182528/.
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Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.Tannenbaum, Allen, Committee Member ; Choi, Jung, Committee Member ; Borodovsky, Mark, Committee Chair ; Voit, Eberhard, Committee Member ; Lee, Eva, Committee Member.
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Bremner, K. Helen. "Application of nuclear localization sequences to non-viral gene delivery systems." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273725.
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Bowden, Jonathan Kirk. "Development of a viral and a non-viral based gene transfer systems using the yeast Saccharomyces cerevisiae." Thesis, Brunel University, 2016. http://bura.brunel.ac.uk/handle/2438/14538.
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VSV-G has been used for several years to pseudotype reteroviral and lentiviral vectors to increase the range of cell types that these vectors can be targeted to as well as increasing transfection efficiency and serum resistance. It has previously been shown that purified VSV-G protein can be added to several types of non-viral complexes to produce these same advantages. VSV-G therefore holds great potential in gene therapy for both viral and non-viral vectors. Due to the cellular toxicity of VSV-G in mammalian cells VSV-G pseudotyped viral vectors are generally produced from transiently transfected cells which greatly limit the scale of viral production. VSV-G for non-viral vectors is also limited in the same manner but also suffer from expensive and time consuming methods to purify the VSV-G from the expression media. To address these problems with production we attempted to generate strains of the yeast Saccharomyces cerevisiae that can produce VSV-G pseudotyped lentivirus and VSV-G protein from inducible integrated vectors. We theorised that the cell wall of Saccharomyces cerevisiae would prevent syncytia and cellular toxicity of VSV-G during production, allowing the continuous production of virus or protein. In this report we show that this new production method allows us to produce and purify VSV-G from yeast using simple and scalable methods and that this produces a greater enhancement of transfection efficiency than mammalian derived VSV-G. However we were not able to demonstrate the production of VSV-G pseudotyped virus, seemingly due to the genotoxic effects of viral integrase.
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Xenariou, Stefania. "Magnetofection and sonoporation to enhance non-viral gene transfer to airway epithelium." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433595.
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Collins, Louise. "A non-viral vector system for efficient gene transfer via membrane integrins." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321961.
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Odom, Mary Rebecca. "Poxvirus evolution the role of horizontal gene transfer /." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2010. https://www.mhsl.uab.edu/dt/2010p/odom.pdf.
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Zhou, Chen, and 周辰. "Genome-informed studies on Penicillium marneffei: horizontal gene transfer survey and differentialsecretomics." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41633672.
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Zhao, Yu-Guang. "Progress towards genome manipulation in the mosquito : gene transfer in cultured cells." Thesis, University of Liverpool, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338455.
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McKechnie, Victoria Margaret. "Variation in the NS5A gene of Hepatitis C Virus in response to interferon alpha therapy." Thesis, University of Glasgow, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301364.
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Huentelman, Matthew J. "HIV-1 based viral vector development for gene transfer to the cardiovascular system." [Gainesville, Fla.] : University of Florida, 2003. http://purl.fcla.edu/fcla/etd/UFE0000974.
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Chilton, Jamie Meredith. "Investigation of the limitations of viral gene transfer to murine embryonic stem cells." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/29745.
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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008.Committee Chair: Joseph Le Doux; Committee Member: Anthanassios Sambanis; Committee Member: David Archer; Committee Member: Michelle LaPlaca; Committee Member: Steve Stice; Committee Member: Todd McDevitt. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Soeda, Emiko. "Developing polyoma viral pseudocapsids for gene transfer : delivery into cells with poly-L-lysine." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298352.
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Zhou, Chen. "Genome-informed studies on Penicillium marneffei horizontal gene transfer survey and differential secretomics /." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41633672.
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Eubanks, Aleida C. (Aleida Christine). "Nucleotide Sequence of a Bovine Arginine Transfer RNA Gene." Thesis, University of North Texas, 1996. https://digital.library.unt.edu/ark:/67531/metadc278753/.
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A single plaque-pure lambda clone designated λBA84 that hybridized to a ˆ32P-labeled bovine arginine tRNA was isolated from a bovine genomic library harbored in a lambda bacteriophage vector. A 2.3-kilobase segment of this clone was found to contain an arginine transfer RNAccg gene by Southern blot hybridization analysis and dideoxyribonucleotide DNA sequencing. This gene contains the characteristic RNA polymerase III split promoter sequence found in all eukaryotic tRNAs and a potential RNA polymerase III termination site, consisting of four consecutive thymine residues, in the 3'-flanking region. Several possible cis-acting promoter elements were found within the 5'-flanking region of the sequenced gene. The function of these elements, if any, is unknown.
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Cooney, Ashley L. "Integrating viral vectors as a gene therapy approach for cystic fibrosis." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6083.
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Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasian populations. CF affects multiple organ systems including pancreas, liver, intestines, sweat glands, and male reproductive organs, however the leading cause of morbidity and mortality in CF patients is chronic lung disease. CF is caused by a mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene which leads to chloride (Cl-) and bicarbonate (HCO3-) anion dysregulation at the airway surface. Without adequate anion exchange, thick, viscous mucus accumulates at the airway surface allowing bacterial colonization to occur. Complementing CFTR in the appropriate airway cells restores the anion channel activity in CFTR-deficient cells. The ultimate goal for CF gene therapy is to design an integrating vector that would lead to persistent and efficient expression of CFTR in the airways.
Performing gene therapy experiments is dependent upon a relevant animal model. The CF pig is a large animal model similar in size, anatomy, and physiology to humans. Importantly, the CF pig recapitulates human lung disease. From the CF pig, we have learned much about CF lung disease and have developed relevant assays to measure anion channel correction. We have learned that loss of CFTR leads to a decreased airway surface ASL pH, bacterial killing ability, and increased mucus viscosity. Standardized assays have been developed to evaluate the change in current by Ussing chambers, ASL pH, bacterial killing in vivo and ASL pH and viscosity on primary airway cultures in vitro. Ultimately, these metrics allow us to make conclusions about the efficiency of CFTR restoration.
Viral vectors are promising candidates for CF gene therapy. Viral vectors such as adenovirus (Ad), adeno-associated virus (AAV), and pseudotyped lentiviral vectors such as feline immunodeficiency virus (FIV) or human immunodeficiency virus (HIV) can efficiently transduce airway cells and express CFTR. Ad and AAV have both been tested in CF clinical trials, but CFTR expression was transient, if detected at all. Understanding vector biology and overcoming barriers in the lung have allowed us to improve vector delivery to the airways. However, the next major hurdle was achieving persistent expression. Ad and AAV are both transiently expressing vectors, and vector readministration is implausible due to the presence of neutralizing antibodies that develop against the vector. Creating a hybrid nonviral/viral vector in which the integrating nonviral piggyBac transposon system is delivered by an Ad or AAV vector has allowed us to achieve persistent expression in mice. In a third integrating vector system, lentiviral vectors have historically been challenging to work with due to low titer levels. However, improvement in vector purification methods have allowed us to validate a lentiviral vector as a viable gene therapy option. In total, we have validated three integrating vector systems by restoring CFTR to CF pigs to correct the phenotypic defect.
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Queen, Jillian May. "Viral mediated gene transfer for modification of cell signalling and remodelling in the ischaemic myocardium." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301367.
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Zinselmeyer, Bernd H. "Evaluation of polypropylenimine dendrimers and their quaternary amino derivates as non-viral gene transfer agents." Thesis, University of Strathclyde, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432089.
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Chu, C. "Modulation of the intraocular immune environment by viral gene transfer in mouse models of uveitis." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1461402/.
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Treatment options for severe uveitis are predominantly limited to systemic immunosuppressants, which are often accompanied by significant side effects. Ocular gene transfer, though, may serve as a sustained local immunomodulating therapy. New adeno-associated virus (AAV) serotypes were used to investigate gene therapy for uveitis in mouse models. Experimental autoimmune uveoretinitis (EAU) was used to model human disease; whilst endotoxin induced uveitis (EIU) was employed to dissect factors affecting immune suppression by AAV. Refinement of AAV production processes improved the quality of vectors including AAV8, AAV9 and ShH10. Technical issues were assessed, confirming apical transgene secretion from RPE and examining the effects of endotoxin contamination. New methods for the evaluation and quantitative scoring of disease severity in both animal models were developed, based on flow cytometry and optical coherence tomography (OCT) for EIU and EAU in C57BL/6J mice, respectively. The complexity of the role of IL-10 in EAU became evident, and pronounced species differences between murine and human IL-10 were identified in EIU. Despite generating intraocular levels comparable to those of recombinant protein known to be effective, AAV-derived human IL-10 did not suppress EIU. Gene delivery of soluble monomeric TNF receptors did not suppress EAU or EIU, and in vitro exhibited less TNF neutralising effects than an equivalent dimeric Fc-fusion protein. Local expression of this dimeric protein from different vectors however was also unable to attenuate EAU. An NFκB-motif based promoter to regulate the expression of AAV-delivered transgenes in uveitis was developed in parallel. It demonstrated activation only in the presence of, and in proportion to the degree of inflammation in EAU. Despite these advances, neither IL-10 nor TNF-inhibitors proved optimal targets for local suppression of intraocular inflammation by AAV. Further work is needed to assess the response of the eye to viral gene transfer in the context of uveitis.
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Tanabe, Soshi. "Developing novel techniques for primate neural network analyses by retrograde gene transfer with viral vectors." Kyoto University, 2020. http://hdl.handle.net/2433/253133.
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Shariat, Parvaneh. "Use of tRNA Gene Probes to Identify Polymorphic Loci in the Bovine Genome." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc278338/.
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A 30-mer oligonucleotide probe encoding the "A box" and anticodon loop regions of a human glycine tRNA gene was used to isolate a 581bp DNA fragment from a bovine genomic DNA library. Although the cross-hybridizing segment of DNA was found not to encode any tRNA gene or pseudogene, a region with homology to the "C-element" of the "BOV-tA" type Alulike artiodactyl retroposons was identified. This cross-hybridization was determined to be the result of conserved RNA polymerase III promoter elements in the probe portion of the tRNA gene and these repetitive elements. A microsatellite repeat (TC) was also found associated with this element. Future screening for bovine tRNA genes will require the use of a) longer probes and higher stringency hybridization conditions or b) the simultaneous screening with probes from the 5' and 3' ends of the gene which avoid the conserved Pol III promoter boxes.
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INGUSCI, SELENE. "Herpetic vectors for safe, long-term gene transfer in the central nervous system." Doctoral thesis, Università degli studi di Ferrara, 2019. http://hdl.handle.net/11392/2487861.
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After a dramatic stop following the initial clinical trials in 1990s, gene therapy has had an encouraging new start in recent years. Significant improvements in vector engineering, delivery, and safety have placed viral vector-based therapy at the forefront of modern medicine for the treatment of many metabolic, cardiovascular, muscular and oncologic diseases, with nearly 3000 clinical trials completed by 2017. However, one of the major challenges of gene therapy remain the development of therapeutic strategies for central nervous system (CNS) disorders. No cure is available for the vast majority of CNS diseases, and gene therapy offers a promising new approach to compensate for lacking genes, to substitute abnormal genes or to produce therapeutic proteins. Unfortunately, many different hurdles are not yet overcome, including stability and regulation of transgene expression, and safety of both the vector and the expressed transgene(s). In addition, the brain adds several challenges that do not pertain to peripheral gene therapy paradigms, such as post-mitotic target cells (neurons), heterogeneity of cell types and circuits, and limited access to the tissue due to the presence of the blood-brain barrier (BBB). Among a variety of possible gene delivery systems, the Herpes Simplex virus type 1 (HSV-1) appears to be a promising viral vector for CNS gene therapy. Advantageous properties include natural neurotropism, high transduction efficiency, large payload capacity, and the ability of enter a latent state in neurons.
The primary aim of this PhD project was to develop new gene transfer tools based on herpetic vectors for application in CNS diseases.
The challenge has been to genetically engineer the HSV genome to prevent expression of toxic viral genes while retaining the ability to infect multiple cell types and express multiple therapeutic transgenes under independent transcriptional control. Specifically, this thesis focuses on the in vitro and in vivo characterization of a new family of replication-defective viral vectors (JΔNI) and the in vivo characterization of amplicon vectors based on HSV. Attention has been mainly given to safety and expression kinetics. in addition, we evaluated mechanisms to regulate the strength of transgene expression. The principal finding was that this new generation of HSV-1 based vectors exhibit a robust and long-lasting expression of reporter genes, essentially limited to neurons, together with the absence of signs of toxicity or infiltration of inflammatory cells. Therefore, these vectors represent a highly promising tool for gene deliver in many CNS pathologies.Dopo una battuta d'arresto seguita ai primi, problematici trial clinici negli anni ‘90, la terapia genica ha recentemente trovato un incoraggiante nuovo inizio. Significativi miglioramenti nell'ingegnerizzazione, nel delivery e nella sicurezza hanno posto la terapia genica in una posizione di avanguardia nella medicina moderna per il trattamento di varie malattie metaboliche, cardiovascolari, muscolari e oncologiche, con quasi 3000 studi clinici completati al 2017. Tuttavia, una delle principali sfide della terapia genica rimane lo sviluppo di strategie terapeutiche per i disturbi del sistema nervoso centrale (SNC). Per la stragrande maggioranza delle malattie del SNC non esistono ancora terapie curative, e la terapia genica offre un approccio nuovo che promette di compensare la mancanza di geni, di sostituire geni mutati o di produrre proteine terapeutiche. Rimangono molti ostacoli da superare, come la stabilità e la regolazione dell'espressione del transgene e la sicurezza del vettore e del transgene. Inoltre, l’approccio al cervello pone numerosi problemi che non si presentano nella terapia genica periferica, come la presenza di cellule-bersaglio post-mitotiche (i neuroni), l'eterogeneità dei tipi e dei circuiti cellulari e l'accesso limitato dovuto alla presenza della barriera emato-encefalica. Tra i tanti possibili sistemi di delivery di geni, il virus dell'Herpes Simplex di tipo 1 (HSV-1) sembra particolarmente promettente per la terapia genica del SNC. Le proprietà vantaggiose comprendono il neurotropismo naturale, l'elevata efficienza di trasduzione, la capacità di ospitare geni di elevate dimensioni e la capacità di entrare in uno stato di latenza nei neuroni. Lo scopo principale di questo progetto di dottorato è stato sviluppare nuovi strumenti di trasferimento genico basati su vettori erpetici per applicazioni terapeutiche nel SNC. La sfida è stata quella di ingegnerizzare il genoma di HSV in modo da prevenire l'espressione di geni virali tossici conservando la capacità di infettare più tipi cellulari e di esprimere più transgeni terapeutici in modo indipendente e controllato. Nello specifico, la tesi tratta la caratterizzazione in vitro e in vivo di una nuova famiglia di vettori virali difettivi per la replicazione (JΔNI), e la caratterizzazione in vivo di vettori ampliconi basati su HSV. L'attenzione è stata posta prima di tutto sulla sicurezza e sulla cinetica di espressione. In aggiunta, sono stati studiati meccanismi atti a regolare l’intensità di espressione del transgene. La scoperta principale è stata che questa nuova generazione di vettori HSV-1 esibisce una robusta e duratura espressione dei geni reporter, essenzialmente limitata ai neuroni, in assenza di segni di tossicità o di infiltrazione da parte di cellule infiammatorie. Questi nuovi vettori rappresentano quindi uno strumento promettente per la terapia genica in molte patologie del SNC.
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Berglund, Eva Caroline. "Genome Evolution and Host Adaptation in Bartonella." Doctoral thesis, Uppsala universitet, Molekylär evolution, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-108376.
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Bacteria of the genus Bartonella infect the red blood cells of a wide range of wild and domestic mammals and are transmitted between hosts by blood-sucking insects. Although most Bartonella infections are asymptomatic, the genus contains several human pathogens. In this work, host adaptation and host switches in Bartonella have been studied from a genomic perspective, with special focus on the acquisition and evolution of genes involved in host interactions. As part of this study, the complete genome of B. grahamii isolated from a Swedish wood mouse was sequenced. A genus-wide comparison revealed that rodent-associated Bartonella species, which have rarely been associated with human disease, have the largest genomes and the largest number of host-adaptability genes. Analysis of known and putative genes for host interactions identified several families of autotransporters as horizontally transferred to the Bartonella ancestor, with a possible role both during early host adaptation and subsequent host shifts. In B. grahamii, the association of a gene transfer agent (GTA) and phage-derived run-off replication of a large genomic segment was demonstrated for the first time. Among all acquisitions to the Bartonella ancestor, the only well conserved gene clusters are those that encode the GTA and contain the origin of the run-off replication. This conservation, along with a high density of host-adaptability genes in the amplified region suggest that the GTA provides a strong selective advantage, possibly by increasing recombination frequencies of host-adaptability genes, thereby facilitating evasion of the host immune system and colonization of new hosts. B. grahamii displays stronger geographic pattern and higher recombination frequencies than the cat-associated B. henselae, probably caused by different lifestyles and/or population sizes of the hosts. The genomic diversity of B. grahamii is markedly lower in Europe and North America than in Asia, possibly an effect of reduced host variability in these areas following the latest ice age.
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Farraha, Melad. "Recombinant Adeno-associated Viral Vector Mediated Gene Transfer of hTBX18: Advancing the Development of a Biological Pacemaker." Thesis, The University of Sydney, 2019. http://hdl.handle.net/2123/20886.
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Recombinant adeno-associated viral (rAAV) vectors, have been developed and are being clinically trialled for treatment of cardiovascular diseases. With an improved understanding of the molecular mechanisms involved in pacemaker dysfunction, gene therapy is being used to advance viable treatment options that can potentially replace the need for electronic devices. Currently, the only viable management option for pacemaker dysfunction is the insertion of an electronic pacemaker. We however proposed an alternative gene therapy approach using rAAV vectors to deliver the gene human T-box 18 (hTBX18), to advance the creation of a biological pacemaker. The work in this thesis aimed to address the limitations with current approaches to biological pacemaker development, to set up a relevant animal model to assess this gene transfer approach and to assess rAAV vector biosafety in this novel model. In the first phase of work, we successfully generated an original rAAV construct expressing hTBX18 and showed that rAAV6-hTBX18 gene transfer to ventricular cardiomyocytes resulted in molecular, physiological, morphological and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. In the second body of work, we successfully developed, characterised and validated a large animal model of atrioventricular block that is stable and technically feasible in adult sheep. In the third body of work, we successfully performed rAAV cardiac infusions in sheep and analysed vector shedding in excreta samples from urine, nasal mucus, saliva and faeces. We concluded that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles following vector delivery. This thesis therefore lays the groundwork for the next phase of pre-clinical development of biological pacemakers using clinically relevant rAAV vectors in a previously non-existent sheep animal model.
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Power, Jeffrey John [Verfasser], Berenike [Gutachter] Maier, and Tobias [Gutachter] Bollenbach. "Horizontal Gene Transfer Between Subspecies Affects Bacterial Genome Dynamics / Jeffrey John Power ; Gutachter: Berenike Maier, Tobias Bollenbach." Köln : Universitäts- und Stadtbibliothek Köln, 2018. http://d-nb.info/1206334975/34.
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Rohwedder, Carolin [Verfasser], and Martin [Akademischer Betreuer] Müller. "Generation of a shut-off system for Adeno-associated viral gene transfer vectors / Carolin Rohwedder ; Betreuer: Martin Müller." Heidelberg : Universitätsbibliothek Heidelberg, 2017. http://d-nb.info/118098546X/34.
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Håfström, Therese. "Genome closure and bioinformatic analysis of the parallel sequenced bacterium Brachyspira intermedia PWS/AT." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-164335.
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Brachyspira species are bacteria that colonize the intestines of some mammalian and avian species with different degrees of pathogenicity. Brachyspira intermedia is a mild pig and bird pathogen with an unknown genomic sequence. In this project, we completed the genome of Brachyspira intermedia PWS/AT and did a comparative genomic analysis between B. intermedia PWS/AT and the already completed genomes of B. hyodysenteriae WA1, B. murdochii 56-150T and B. pilosicoli 95/1000. A table containing 15 classes of unique and shared genes was developed and analyzed in order to gain a better understanding of species-specific traits and clues behind the different degree of pathogenicity. Our result shows that genes are overall poorly annotated and further studies are of great importance for understanding different and shared properties. The largest number of unique features was found in B. intermedia and B. murdochii. B. hyodysenteriae and B. pilosicoli has most likely developed independently towards different biological niches and B. pilosicoli has undergone a major reductive evolution. One plasmid and six prophages were found in B. intermedia, where two of the phages appear to be capable of horizontal gene transfer. Further genome sequencing of more strains will probably increase the understanding of species-specific traits even more.
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Gluck, Thaler Emile. "Computational, Evolutionary and Functional Genetic Characterization of Fungal Gene Clusters Adapted to Degrade Plant Defense Chemicals." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555406081422532.
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Xie, Gang. "Evolution of aromatic metabolism a genomic perspective on the complexity contributed by genome expansion, reductive evolution, and lateral gene transfer /." [Gainesville, Fla.] : University of Florida, 2002. http://purl.fcla.edu/fcla/etd/UFE1001196.
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Cheeseman, Kevin. "Aspects of Penicillium genomics : Molecular combing genome assembly, genetic exchange in food and potential for secondary metabolite production." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112280/document.
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Les Penicilliums sont des champignons filamenteux appartenant au genre Ascomycota. Ces champignons ont été utilisés par l’homme pour la production de nourriture depuis des siècles. Plus récemment, ils ont aussi été utilisés dans l’industrie biotechnologique pour la production de composés chimiques d’intérêts pharmaceutiques. Certaines espèces de Penicillium sont par ailleurs des moisissures contaminants certains aliments, d’autres sont des pathogènes de plantes, y compris de certains fruits. Leur génomique est globalement peut connue. Dans cette étude, nous avons analysé les génomes de deux espèces nouvellement séquencées, Penicillium roqueforti et Penicillium camemberti. Nous reportons ici le développement d’une nouvelle méthodologie pour l’amélioration et la validation d’assemblage de génomes en utilisant une technologie permettant l’observation de molécules d’ADN unique, le Peignage Moléculaire. En utilisant cette méthode, nous avons amélioré l’assemblage de Penicillium roqueforti. Ce manuscrit décrit aussi de multiples occurrences d’un transfert horizontal d’un ilot génomique de plus de cinq cent kilobases entre plusieurs Penicillium. Ce cas de transfert horizontal indique une fréquence d’échange latéral de matériel génétique plus forte qu’attendue. Enfin nous présentons un inventaire préliminaire du potentiel génomique pour la production de métabolites secondaires dans ces importants Penicillium alimentairesPenicillium are filamentous fungi belonging to the Ascomycota genus. Penicillium species have been used by Man for centuries in food making processes. More recently they have also been used in the biotechnology industry for the production of compounds of pharmaceutical interest. Some Penicillium species are food spoilage agents, pathogens of plants including fruits. Aspects of their genomics are largely unknown. In this study, we analysed the genomes of two newly sequenced species, Penicillium roqueforti and Penicillium camemberti. Here we report the development of a new methodology for improving and validating genome assembly using an original single DNA molecule technology, Molecular Combing. Using this methodology we were able to produce a high quality genome assembly of Penicillium roqueforti. This work also reports the multiple and recurrent horizontal transfer of a large genomic island of over half a megabase between several Penicillium species. This horizontal transfer indicates a higher frequency of lateral genetic exchange between cheesemaking fungi than previously expected. Finally, we present an early assessment of the genomic potential for secondary metabolite production in these important food associated penicilliums
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Sokal, Nadia. "Defining the role of autographa californica multiple nucleopolyhedrovirus immediate early-0 and immediate early-1 proteins in viral genome replication and early gene transactivation." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42940.
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The immediate early-0 (IE0) and IE1 proteins of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are key transregulators in the viral replication cycle. If both proteins are absent, the virus is inactive. Either protein can support viral replication but both are required to achieve wildtype infection. Both IE0 and IE1 are involved in viral DNA replication and transcriptional transactivation of early genes. In this study, to analyze IE0 and IE1’s function, ie0, ie0MtoA or ie1 were placed under the control of identical promoters (ie1 or gp64) to achieve comparable levels of protein expression. The ie1 promoter produced higher levels of IE0, IE0MtoA and IE1 compared to the gp64 promoter. Time course assays of infected Spodoptera frugiperda 9 (Sf9) cells allowed examination of viral DNA replication and budded virus (BV) production. The results showed that when IE0 and IE1 protein levels were high, either IE0, IE1 or IE0 and IE1 together maintained DNA replication and BV production similar to wildtype levels. However, when IE0 and IE1 protein levels were low, only when IE0 and IE1 were present together was DNA replication and BV production similar to wildtype. These results suggest that during the virus replication cycle when cellular levels of IE0 or IE1 are low, for example at the beginning of infection, the presence of both proteins results in more efficient DNA replication. Transient transactivation studies were also performed to examine IE0 and IE1’s ability to activate nineteen viral early gene promoters. At low levels of IE0 and IE1 expression, a group of viral early gene promoters were found to be differentially transactivated by IE0 alone or when both IE0 and small amounts of IE1 were together. These results suggest that during early times post-infection, when cellular levels of viral proteins are low, IE0 in the presence of a small amount of IE1 results in rapid onset of viral DNA replication and efficient transactivation of a specific set of viral early gene promoters. In context of virus infection, rapid viral replication by IE0 and IE1 and transactivation of early genes by IE0 may counter the insect’s defense mechanisms like sloughing and apoptosis.
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Mwinyi, Adina, Achim Meyer, Christoph Bleidorn, Bernhard Lieb, Thomas Bartolomaeus, and Lars Podsiadlowski. "Mitochondrial genome sequence and gene order of Sipunculus nudus give additional support for an inclusion of Sipuncula into Annelida." Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2010/4491/.
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Background:
Mitochondrial genomes are a valuable source of data for analysing phylogenetic
relationships. Besides sequence information, mitochondrial gene order may add phylogenetically useful information, too. Sipuncula are unsegmented marine worms, traditionally placed in their own phylum. Recent molecular and morphological findings suggest a close affinity to the segmented Annelida.
Results:
The first complete mitochondrial genome of a member of Sipuncula, Sipunculus nudus, is presented. All 37 genes characteristic for metazoan mtDNA were detected and are encoded on the same strand. The mitochondrial gene order (protein-coding and ribosomal RNA genes) resembles that of annelids, but shows several derivations so far found only in Sipuncula. Sequence based phylogenetic analysis of mitochondrial protein-coding genes results in significant bootstrap
support for Annelida sensu lato, combining Annelida together with Sipuncula, Echiura, Pogonophora and Myzostomida.
Conclusion:
The mitochondrial sequence data support a close relationship of Annelida and
Sipuncula. Also the most parsimonious explanation of changes in gene order favours a derivation from the annelid gene order. These results complement findings from recent phylogenetic analyses of nuclear encoded genes as well as a report of a segmental neural patterning in Sipuncula.
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McKenna, Duane D., Erin D. Scully, Yannick Pauchet, Kelli Hoover, Roy Kirsch, Scott M. Geib, Robert F. Mitchell, et al. "Genome of the Asian longhorned beetle (Anoplophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle–plant interface." BIOMED CENTRAL LTD, 2016. http://hdl.handle.net/10150/622572.
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Background: Relatively little is known about the genomic basis and evolution of wood- feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.
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Nair, Amrithraj Muraleedharan. "Studies of retroviral vectors for in utero gene transfer and investigation of calcium-mediated gene regulation by Human T-lymphotropic virus type-1." The Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1088785797.
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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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Carrera, Sandra Garcés. "Virulence of Mayetiola destructor (Say) field populations in the Great Plains and levanase/inulase-like genes in the Hessian fly genome." Diss., Kansas State University, 2013. http://hdl.handle.net/2097/16873.
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Doctor of PhilosophyDepartment of Entomology
Ming-Shun Chen
C. Michael Smith
The Hessian fly, Mayetiola destructor (Say), is a major pest of wheat, and is controlled mainly through deploying fly-resistant wheat cultivars. This study investigated five M. destructor populations collected from Texas, Louisiana, and Oklahoma, where infestation by Hessian fly has been high in recent years. Eight resistance genes including H12, H13, H17, H18, H22, H25, H26, and Hdic, were found to be highly effective against all tested M. destructor populations in this region, conferring resistance to 80% or more of plants containing one of these resistant genes. The frequency of biotypes virulent to resistant genes ranged from 0 to 45%. A logistic regression model was established to predict biotype frequencies based on the correlation between the percentages of susceptible plants obtained in a virulence test. In addition to the virulence test, the log-odds of virulent biotype frequencies were determined by a traditional approach to predict the logistic regression model. Characterization of a bacterial artificial chromosome (BAC) clone identified a gene encoding a protein with sequence similarity to bacterial levanases. Blast searching with the levanase-like protein identified 14 levanase/inulase-like genes or gene fragments. In this study, we determined the expression levels of these genes in different developmental stages and different tissues of 3-d old larvae of M. destructor. Sequence analysis revealed that six genes encode full length proteins, three were truncated at the 5’ end, and five truncated at the 3’ end. Sequences of putative proteins showed approximately 42% similarities to bacterial levanases or inulases, and 36% similarity to fungal levanases or inulases. No sequence similarities were found with any known animal or plant proteins. Comparative analysis of sequences among 14 levanase/inulase-like genes revealed that positions for intron/exon boundaries are conserved among different genes even though the length of each intron and exon varied among different genes. The expression patterns of the levanase/inulase-like genes were different among developmental stages and larval tissues of M. destructor. Interestingly, three genes presented alternative splicing bands in different developmental stages, and two genes exhibited splicing bands in different tissues of 3 d old M. destructor. This study would be useful for future studies of the characterization and function of levanase/inulase-like genes of these enzymes in plant-insect interactions.
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Frank, Anna Carolin. "Lifestyle and Genome Evolution in Vector-Borne Bacteria : A Comparison of Three Bartonella Species." Doctoral thesis, Uppsala universitet, Molekylär evolution, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5913.
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Bacterial genomes provide records of the molecular processes associated with emergence and evolution of different bacterial lifestyles. This thesis is based on whole-genome comparisons within the genus Bartonella, an excellent model system for studies of host- and vector-specificity and infection outcome in animal-associated bacteria. The louse-borne human specialist and trench fever agent Bartonella quintana was contrasted to the flea-borne generalist relatives Bartonella henselae and Bartonella grahamii, which cause asymptomatic infection in cat and mouse respectively. While B. henselae is commonly isolated from humans, and causes cat scratch disease, there is only one reported case of B. grahamii human infection. The gene complements of the three species are nested like Russian dolls with the smaller genome (B. quintana) being entirely contained in the medium sized (B. henselae), which in turned is contained in the largest (B. grahamii). Size differences reflect differences in the horizontally and vertically acquired gene content, and in the number of genus- and species- specific genes, owing to differential impact of bacteriophages and plasmids, and to different degrees of genome decay. These processes can be attributed to the three distinct lifestyles. Comparisons with other alpha-proteobacteria suggest that the Bartonella genus as a whole evolved from plant-associated species, and that horizontal transfer, in particular of genes involved in interaction with the host, played a key role in the transition to animal intracellular lifestyle. The long-term genome decay associated with this lifestyle is most advanced in the host-restricted B. quintana. The broad host-range species B. grahamii has the largest genome and the largest proportion of auxiliary DNA of the three, probably because it has access to a larger gene pool. In encodes all the known pathogenicity determinants found in the genomes of B. henselae and B. quintana, suggesting that these genes primarily evolved to facilitate colonization in the reservoir host.
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Driscoll, Timothy. "Host-Microbe Relations: A Phylogenomics-Driven Bioinformatic Approach to the Characterization of Microbial DNA from Heterogeneous Sequence Data." Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/50921.
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Plants and animals are characterized by intimate, enduring, often indispensable, and always complex associations with microbes. Therefore, it should come as no surprise that when the genome of a eukaryote is sequenced, a medley of bacterial sequences are produced as well. These sequences can be highly informative about the interactions between the eukaryote and its bacterial cohorts; unfortunately, they often comprise a vanishingly small constituent within a heterogeneous mixture of microbial and host sequences. Genomic analyses typically avoid the bacterial sequences in order to obtain a genome sequence for the host. Metagenomic analysis typically avoid the host sequences in order to analyze community composition and functional diversity of the bacterial component. This dissertation describes the development of a novel approach at the intersection of genomics and metagenomics, aimed at the extraction and characterization of bacterial sequences from heterogeneous sequence data using phylogenomic and bioinformatic tools.
To achieve this objective, three interoperable workflows were constructed as modular computational pipelines, with built-in checkpoints for periodic interpretation and refinement. The MetaMiner workflow uses 16S small subunit rDNA analysis to enable the systematic discovery and classification of bacteria associated with a host genome sequencing project. Using this information, the ReadMiner workflow comprehensively extracts, assembles, and characterizes sequences that belong to a target microbe. Finally, AssemblySifter examines the genes and scaffolds of the eukaryotic genome for sequences associated with the target microbe. The combined information from these three workflows is used to systemically characterize a bacterial target of interest, including robust estimation of its phylogeny, assessment of its signature profile, and determination of its relationship to the associated eukaryote.
This dissertation presents the development of the described methodology and its application to three eukaryotic genome projects. In the first study, the genomic sequences of a single, known endosymbiont was extracted from the genome sequencing data of its host. In the second study, a highly divergent endosymbiont was characterized from the assembled genome of its host. In the third study, genome sequences from a novel bacterium were extracted from both the raw sequencing data and assembled genome of a eukaryote that contained significant amounts of sequence from multiple competing bacteria. Taken together, these results demonstrate the usefulness of the described approach in singularly disparate situations, and strongly argue for a sophisticated, multifaceted, supervised approach to the characterization of host-associated microbes and their interactions.Ph. D.
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Henning, Karen [Verfasser]. "Adeno-associated viral gene transfer to prevent the cellular phenotype of cortical organotypic brain-slice cultures derived from Gaucher’s disease type II mice. / Karen Henning." Berlin : Freie Universität Berlin, 2014. http://d-nb.info/1050978242/34.
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Sallach, Jessica [Verfasser], Dagmar [Akademischer Betreuer] Knebel-Mörsdorf, and Bent [Akademischer Betreuer] Brachvogel. "Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes / Jessica Sallach. Gutachter: Dagmar Knebel-Mörsdorf ; Bent Brachvogel." Köln : Universitäts- und Stadtbibliothek Köln, 2013. http://d-nb.info/1050577019/34.
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Higuti, Eliza. "Correção fenotípica do nanismo avaliada por diferentes parâmetros de crescimento após administração de DNA plasmidial em modelo animal de deficiência isolada do hormônio do crescimento." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-07032016-091035/.
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A deficiência de hormônio de crescimento (DGH) é a deficiência mais comum entre os hormônios pituitários. A terapia utilizada atualmente consiste de injeções diárias de hormônio de crescimento humano recombinante (r-hGH), entretanto esta terapia apresenta alguns inconvenientes, como a necessidade de frequentes injeções de r-hGH durante um longo período de vida, dependendo da severidade da deficiência, e o alto custo do hormônio, em razão dos dispendiosos processos de purificação. Uma alternativa ao tratamento padrão seria aquele no qual fossem evitados estes tipos de inconvenientes e o processo de liberação da proteína fosse sustentável, por um longo período e promovesse níveis normais e sustentáveis do fator de crescimento semelhante à insulina I (IGF-I), o principal mediador dos efeitos do GH. Uma alternativa é a terapia gênica in vivo, baseada na administração de DNA plasmidial em diversos órgãos/tecidos, seguida de eletroporação. É considerada uma metodologia bastante promissora e que tem sido alvo de vários estudos para diversos tipos de deficiências sistêmicas. Neste trabalho foram realizadas diversas administrações de um plasmídeo contendo o gene do hormônio de crescimento humano, nos músculos quadríceps exposto ou tibial anterior sem exposição, seguidas de eletroporação, em camundongos anões e imunodeficientes (lit/scid) com 40-80 dias de idade, na tentativa de obter uma correção fenotípica do nanismo, mediante a avaliação de parâmetros de crescimento. A administração deste plasmídeo no músculo tibial anterior, em camundongos com a idade inicial de 40 dias, foi capaz de proporcionar uma normalização dos níveis de mIGF-I, quando comparados aos dos camundongos não-deficientes de GH. Além disso, foram obtidos valores de catch-up dos parâmetros de crescimento longitudinal de 36-77%. Visando uma maior eficiência na expressão de GH, foram construídos plasmídeos parentais, e a partir destes, foram produzidos minicírculos de DNA com os promotores do CMV e Ubiquitina C e com os cDNAs de hGH e mGH. Estes minicírculos de DNA foram transfectados em células HEK 293 e foram até 2 vezes mais eficientes em relação aos plasmídeos convencionais com o promotor do CMV. Estes dados são bastantes promissores e abrem caminho para ensaios mais eficientes, utilizando este tipo de protocolo de terapia gênica para a DGH, visando uma normalização de todos os parâmetros de crescimento.The human growth hormone deficiency (GHD) is the most common deficiency related to pituitary hormones. The current therapy is based on daily injections of recombinant human growth hormone (r-hGH). This therapy, however, presents some disadvantages, as the need for frequent injections of r-hGH during a long life time, depending on the deficiency severity and the high cost of this hormone, due to the expensive purification processes. An alternative to the standard treatment should be to avoid these inconveniences via a sustainable hormone release, acting for a long time and providing normal and sustainable levels of insulin-like growth factor-I (IGF-I). A possible alternative is in vivo gene therapy, based on the administration of plasmid DNA in several organs/tissues, followed by electroporation. This methodology is considered very promising and has been the target of many different studies for several types of systemic deficiencies. In the present work several administrations of a plasmid containing the human growth hormone gene were carried out, in the exposed quadriceps or non-exposed tibialis cranialis muscle, followed by electroporation, using immunodeficient dwarf mice 40-80 days old. The goal was to obtain a phenotypic correction of dwarfism, through the evaluation of different growth parameters. The administration of this plasmid, in the tibialis cranialis muscle of 40 day old mice, was able to provide a normalization of mIGF-I levels, when compared to non GHD mice. Furthermore, catch-up increases of longitudinal growth parameters of 36-77% were obtained. Aiming a high efficiency on GH expression, parental plasmids were constructed and from these DNA minicircles were generated with CMV and Ubiquitin C promoter and hGH or mGH cDNA sequences. These DNA minicircles were transfected into HEK 293 cells and were even 2 times moren efficient than conventional plasmids with CMV promoter. This data are very promising and pave the way for more efficient assays utilizing this type of gene therapy protocol for GHD, aiming at a normalization of all growth parameters.
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Reul, Johanna [Verfasser], Beatrix [Akademischer Betreuer] Süß, Gerhard [Akademischer Betreuer] Thiel, and Christian [Akademischer Betreuer] Buchholz. "Viral gene transfer systems for cancer immunotherapy: semireplication-competent VSV and receptor-targeted AAV for the delivery of immunomodulatory proteins / Johanna Reul ; Beatrix Süß, Gerhard Thiel, Christian Buchholz." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2019. http://d-nb.info/1176107585/34.
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Valdor, Markus Verfasser], Hermann [Akademischer Betreuer] [Wagner, and Jens [Akademischer Betreuer] Kurreck. "Functional characterization of the Kv7.2/Kv7.3 ion channel in rat dorsal root ganglion neurons following RNA interference-based knockdown by viral gene transfer / Markus Valdor ; Hermann Wagner, Jens Kurreck." Aachen : Universitätsbibliothek der RWTH Aachen, 2018. http://d-nb.info/1171818645/34.
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Valdor, Markus [Verfasser], Hermann [Akademischer Betreuer] Wagner, and Jens [Akademischer Betreuer] Kurreck. "Functional characterization of the Kv7.2/Kv7.3 ion channel in rat dorsal root ganglion neurons following RNA interference-based knockdown by viral gene transfer / Markus Valdor ; Hermann Wagner, Jens Kurreck." Aachen : Universitätsbibliothek der RWTH Aachen, 2018. http://d-nb.info/1171818645/34.
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Tamarit, Daniel. "Evolution of symbiotic lineages and the origin of new traits." Doctoral thesis, Uppsala universitet, Molekylär evolution, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-301939.
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This thesis focuses on the genomic study of symbionts of two different groups of hymenopterans: bees and ants. Both groups of insects have major ecological impact, and investigating their microbiomes increases our understanding of their health, diversity and evolution. The study of the bee gut microbiome, including members of Lactobacillus and Bifidobacterium, revealed genomic processes related to the adaptation to the gut environment, such as the expansion of genes for carbohydrate metabolism and the acquisition of genes for interaction with the host. A broader genomic study of these genera demonstrated that some lineages evolve under strong and opposite substitution biases, leading to extreme GC content values. A comparison of codon usage patterns in these groups revealed ongoing shifts of optimal codons. In a separate study we analysed the genomes of several strains of Lactobacillus kunkeei, which inhabits the honey stomach of bees but is not found in their gut. We observed signatures of genome reduction and suggested candidate genes for host-interaction processes. We discovered a novel type of genome architecture where genes for metabolic functions are located in one half of the genome, whereas genes for information processes are located in the other half. This genome organization was also found in other Lactobacillus species, indicating that it was an ancestral feature that has since been retained. We suggest mechanisms and selective forces that may cause the observed organization, and describe processes leading to its loss in several lineages independently. We also studied the genome of a species of Rhizobiales bacteria found in ants. We discuss its metabolic capabilities and suggest scenarios for how it may affect the ants’ lifestyle. This genome contained a region with homology to the Bartonella gene transfer agent (GTA), which is a domesticated bacteriophage used to transfer bacterial DNA between cells. We propose that its unique behaviour as a specialist GTA, preferentially transferring host-interaction factors, originated from a generalist GTA that transferred random segments of chromosomal DNA. These bioinformatic analyses of previously uncharacterized bacterial lineages have increased our understanding of their physiology and evolution and provided answers to old and new questions in fundamental microbiology.
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Weigl, Julia [Verfasser]. "Development of protocols and workflows for a fast gene synthesis and de novo synthesis of viral genomes : Entwicklung von Protokollen und Arbeitsabläufen für eine schnelle Gensynthese und de novo Synthese vitaler Genome / Julia Weigl." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2018. http://d-nb.info/1223620972/34.
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Filha, Eliana Rosa Lima. "Otimização de parâmetros de transferência in vivo do gene do hormônio de crescimento visando a correção fenotípica de camundongos anões." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-29072016-145236/.
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A deficiência de hormônio de crescimento (DGH) é tratada convencionalmente com repetidas injeções do hormônio recombinante. Este trabalho teve como objetivo estabelecer uma alternativa de tratamento baseada na transferência dos genes do hormônio de crescimento humano (hGH) ou de camundongo (mGH), em camundongos anões lit/lit ou lit/scid, mediante administração de DNA plasmidial associada à eletrotransferência, com a finalidade de atingir a máxima recuperação de crescimento em comparação ao camundongo normal (catch-up growth). Inicialmente foi realizada a administração do plasmídeo contendo o gene do mGH no músculo quadríceps exposto ou tibial anterior (TA) não exposto. Utilizando diferentes condições de eletrotransferência, baseadas em pulsos alternados de baixa (100 V/cm) e alta (1000 V/cm) voltagem (HV/LV, HV/8LV) ou em pulsos seguidos de baixa voltagem (8 pulsos de 150 V/cm), o músculo TA na condição HV/LV apresentou os maiores níveis de expressão de mGH: 6,7 ± 2,5 ng/mL. O tempo de exposição e a quantidade da enzima hialuronidase (HI) necessária para a eletrotransferência foram também analisados. O tempo de 30 minutos e a dose de 20 U de HI proporcionaram os melhores resultados de expressão. Diferentes quantidades de DNA foram também testadas, mas a administração de 50 μg DNA/animal foi confirmada como a melhor. Na padronização do volume de solução do plasmídeo administrado no TA, foi observado que a injeção de 20 μL de DNA apresentou expressão significativamente maior da proteína em comparação a de 10 μL. Buscando uma maior expressão de GH, foi realizado experimento adicionando poli-L-glutamato ao diluente do DNA, comparando também diferentes condições de eletrotransferência (HV/LV e 375 V/cm). A condição de 375 V/cm, sem a adição do polímero, proporcionou as maiores concentrações, tanto de hGH como de mGH, no soro de camundongos lit/scid e lit/lit, respectivamente. Quando utilizados 3 pulsos de 375 V/cm e a administração do plasmídeo com o gene do mGH em dois locais de cada músculo TA, foram obtidos os mais altos níveis de expressão atingindo 14,7 ± 3,7 ng mGH/mL. Estes foram os parâmetros utilizados em um bioensaio, no qual foi também determinada a medida do comprimento inicial e final do fêmur por radiografia. Neste bioensaio de 36 dias, a curva de crescimento dos camundongos lit/lit tratados foi similar a de camundongos heterozigotos não tratados e os níveis de mGH do grupo DNA foram significativamente maiores (P<0,0002) em relação ao grupo controle. Os camundongos tratados também apresentarem concentração de mIGF-I no soro superior a do grupo controle. Considerando os parâmetros de crescimento avaliados, o grupo tratado com DNA apresentou percentuais de incremento altamente significativos em relação ao grupo controle, com P<0,001 para o peso corpóreo e P<0,002 para o comprimento do corpo, da cauda e para ambos os fêmures, com valores de catch-up da ordem de 79% para o comprimento dos fêmures. Podemos concluir que foi estabelecida uma metodologia eficiente de transferência gênica não viral, que poderá levar a uma completa normalização de crescimento de camundongos anões mediante utilização de animais mais jovens, como mencionado na literatura e em trabalho recente do nosso grupo.Growth hormone deficiency (GHD) is conventionally treated with repeated injections of the recombinant hormone. This work aimed at establishing an alternative treatment based on the transfer of the human (hGH) or mouse growth hormone (mGH) genes into lit/lit or lit/scid dwarf mice, using plasmid DNA administration associated with electrotransfer, in order to achieve the maximum growth recovery compared to normal mice (catch-up growth). Administration of the plasmid containing the mGH gene was first carried out in the exposed quadriceps or non-exposed anterior tibialis (TA) muscle. Using different electrotransfer conditions, based on alternate pulses of high (1000 V/cm) and low (100 V/cm) voltage (HV/LV, HV/8LV) or consecutive pulses of low voltage (8 pulses of 150 V/cm), the TA muscle in the HV/LV condition showed the highest levels of mGH expression: 6.7 ± 2.5 ng/mL. Exposure time and amount of the enzyme hyaluronidase (HI) required for electrotransfer were also analyzed. The time of 30 minutes and the dose of 20 U HI provided the best results of expression. Different amounts of DNA were also tested, but the administration of 50 μg DNA/animal was confirmed as the best. In the optimization of the volume of plasmid solution administered to TA, it was observed that injection of 20 μL of DNA showed a significantly higher expression of the protein compared with 10 μL. Aiming at a higher GH expression, an experiment was carried out by adding poly-L-glutamate to the DNA diluent, comparing also different electrotransfer conditions (HV/LV and 375 V/cm). The condition of 375 V/cm, without the polymer addition, provided the higher concentrations of both hGH and mGH in the serum of lit/scid or lit/lit mice, respectively. Using 3 pulses of 375 V/cm and administration of mGH-DNA in two locations on each TA muscle, the highest expression levels of up to 14.7 ± 3.7 ng mGH/mL were obtained. These were the parameters utilized in a bioassay, which was also carried out by measurement of the initial and final femur length by radiography. In this 36-day bioassay, the growth curve of treated lit/lit mice was similar to that of heterozygous untreated mice and the mGH levels of DNA group were significantly higher (P<0.0002) than the control group. Treated mice also showed a higher mIGF-I concentration in the serum compared to the control group. Concerning growth parameters, DNA-treated group showed percentages of increase highly significant compared to the control group, with P<0.001 for body weight and P<0.002 for body, tail and both femurs lengths, with catch-up values of the order of 79% for femur lengths. We can conclude that an efficient non-viral gene transfer methodology has been established, which lead to a complete growth normalization of the dwarf mice through the use of younger animals, as reported in the literature and in a recent paper of our group.
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Walther, Wolfgang. "In vitro- und in vivo Untersuchungen für eine nicht-virale und Therapie-regulierbare Tumorgentherapie." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2004. http://dx.doi.org/10.18452/13932.
Full textAbstract:
Die Gentherapie hat in den letzten Jahren wesentliche Entwicklungen im Vektordesign, der kontrollierte Expression sowie der Sicherheit ihrer Anwendung durchgemacht. Die Erkenntnis, dass die Tumorgentherapie allein nur in begrenztem Maße zum erhofften therapeutischen Benefit für den Patienten beitragen kann, führte zum Konzept der lokalen Gentherapie als Teil anderer, etablierter Tumortherapien. In diesem Zusammenhang wird die Gentherapie als eine moderne Option zur Steigerung der Effizienz von Chemotherapie, Strahlentherapie oder Hyperthermie verstanden. Zum Erreichen dieses Zieles ist die Etablierung Therapie-regulierbarer Vektorsysteme von besonderer Attraktivität. Im Rahmen der Strategie des lokalen Transfers therapeutischer Gene bietet inzwischen die Anwendung nicht-viraler Transfersysteme, wie z.B. in vivo-Elektrotransfer, Gene-Gun oder Jet-Injection eine klinisch applikable Technologie. Die Etablierung einer effizienten, auf der Jet-Injection basierenden nicht-viralen Transfertechnologie und die Analyse ihres Potentials für eine klinische Anwendung in einem multimodalen Therapiekonzept war ein wesentliches Ziel der Arbeit. Es wurde gezeigt, dass die Jet-Injection in tierexperimentellen Tumormodellen zur effizienten Expression der Transgene führt, dass sowohl Eindringtiefen, als auch Verteilung der Jet-Injection optimal für einen effizienten Gentransfer sind und die Höhe der Genexpression mit etablierten Gentransfer-Technologien, wie z.B. der in vivo-Lipofektion, vergleichbar ist. Basierend auf der Strategie des Einsatzes der Gentherapie in Kombination mit anderen Therapien, bestand ein weiteres Ziel der Arbeit in der Charakterisierung und Anwendung konditioneller Vektorsysteme, mit denen die Expression therapeutischer Gene durch Chemotherapie oder Hyperthermie kontrollierbar ist. Derartige Vektoren, in denen der humane Multidrug Resistenzgen 1- (mdr1) Promotor genutzt wurde, exprimierten vor allem Zytokingene, die die therapeutische Effizienz von Zytostatika oder der Hyperthermie verbessern. Die Zytostatika-und auch Hitze-Induzierbarkeit der mdr1-Promotor gesteuerten Genexpression konnte in verschiedenen Tumormodellen in vitro und in vivo erfolgreich demonstriert werden Diese Untersuchungen zeigten, dass eine Zytostatika-induzierte Gentherapie zu einer besseren Tumortherapie beiträgt. Die Kombinations-Experimente der konditionellen Gentherapie im Kontext einer Hyperthermie geben erste Hinweise, dass auch hier die therapeutische Effektivität in vitro und in vivo gesteigert werden kann. Im Rahmen des Konzepts der kombinierten Gen- und Chemotherapie von Tumoren ist in der Arbeit vor allem auf das chemosensitivierende Potential von Zytokinen gesetzt worden. Besonders für TNF-a, IL-2 sowie IFN-g konnte gezeigt werden, dass diese Zytokine zu einer Modulation der Expression MDR-assoziierter Gene, wie dem mdr1, MVP/LRP und auch MRP1 in der Lage sind und dadurch zur Chemosensitivierung in verschiedenen Tumormodellen führt. Diese Befunde bildeten eine wichtige Rationale für den Einsatz von Zytokingenen im Rahmen der Tumorgentherapie zur Überwindung der MDR. Gentransferexperimente mit TNF-a- und IL-2-exprimierenden Vektoren konnten analog zur Applikation rekombinanter Zytokine die Modulation der Gene mdr1 und MVP/LRP zeigen, die mit der Erhöhung der Sensitivität gegenüber Zytostatika wie Vincristin oder Adriamycin assoziiert ist.Gene therapy has made great achievements in vector design, controlled gene expression and in safety. The fact, that gene therapy as single therapy has only limited potential for the benefit in the therapy for cancer patients, has led to the concept of local gene therapy as part of other, established therapies. In this context, gene therapy serves as a modern option to improve the efficiency of chemotherapy, radiotherapy or hyperthermia. To achieve this goal, the establishment of therapy-regulatable vectors is of particular attractiveness. For the concept of local transfer of therapeutic genes non-viral transfer systems, such as in vivo electrotransfer, gene gun or jet-injection represent clinically applicable transfer technologies. One major issue of this work was the establishment of an efficient, jet-injection based non-viral transfer technology and the analysis of its potential for clinical application in a concept of multimodal therapy. It has been shown in vivo, that efficient transgene expression can be achieved by jet-injection, that penetration and distribution of the transgene are optimal for an efficient gene transfer and that the level of gene expression is comparable to established gene transfer technologies, sch as in vivo lipofection. Based on the strategy of combination of gene therapy with other therapies, another goal of this work aimed at the characterization and utilization of conditional vector systems, by which expression of therapeutic genes is controllable by chemotherapy or hyperthermia. By such vectors, in which the human multidrug resistance gene 1 (mdr1) promoter was employed, cytokine genes were expressed, which are capable to improve the therapeutic efficacy of cytostatic drugs or of hyperthermia. The drug- and heat-inducibility of mdr1 promoter-driven gene expression has successfully been demonstrated in in vitro and n vivo tumor models. The studies have also shown, that drug-induced gene therapy leads to improved tumor treatment. Combination experiments of conditional gene therapy in the context with hyperthermia give first indication of an increased therapeutic efficiency in vitro and in vivo. For the concept of combined gene- and chemotherapy the chemosensitizing potential of cytokines was exploited. It has been shown, particularly for TNF-a, IL-2 and IFN-g, that these cytokines are capable to modulate the expression of MDR-associated genes, such as mdr1, MVP/LRP or MRP1 leading to chemosensitization in different tumor models. These observations represent an important rationale for the use of cytokine genes in gene therapy for MDR-overcoming. Gene transfer experiments with TNF- or IL-2 expressing vectors showed the modulation of mdr1 or MVP/LRP expression, associated with increased sensitivity towards cytostatic drugs, such as vincristine or adriamycin.