Thesis (M.S.)--State University of New York at Binghamton, Thomas J. Watson School of Engineering and Applied Science, Department of Bioengineering, Biomedical Engineering, 2009. / Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.

Thesis (Ph.D.)--Baylor University, 2009. / Includes bibliographical references (p. 172-194).

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are potent toxins produced by a wide range of evolutionarily diverse plants. These toxins cause cell death by physically dismantling ribosomal RNA and shutting down protein synthesis. They also have a strong antiviral activity. Some believe that the antiviral property of RIPs is a function of ribosomal inactivation, others believe that the two properties are unrelated. RIPs are non-specific in their antiviral activity. Transgenic RIPexpressing plants are resistant to a wide spectrum of viruses. Many different viruses threaten grapevine. It is not practical to design individual remedies for each of these viruses. In this study, we screen the grapevine genome for the presence of a RIP gene using degenerate PCR primers. If a RIP gene does exist in grapevine, it is not being expressed in a useful way. We also clone several well-documented RIP genes from various plants into pGEM-T Easy: dianthin from Dianthus caryophyllus; p-Iuffin from Luffa octandra and mirabilis antiviral protein (MAP) from Mirabilis jalapa. These isolated genes are then subcloned into a selection of expression vectors: dianthin into pKK223-3, a bacterial expression vector; p-Iuffin into pCambia3301, a plant expression vector; and MAP into pFLAG, a yeast expression vector. The constructs prepared in this project may be used for the synthesis of RIP molecules. The exogenous application of RIPs has been shown to protect plants from viruses. Transformation of grapevine with the RIP-containing plant expression vector may result in a variety of vine that is resistant to a wide range viruses. This thesis describes preliminary work in an attempt to impart broad-spectrum antiviral resistance to grapevine. / AFRIKAANSE OPSOMMING: Ribosomale-inaktiverende proteïne (RIPs) is kragtige toksienes wat deur 'n wye verskeidenheid evolusionêr diverse plante verskaf word. Hierdie toksienes veroorsaak die dood van die selle deur fisies die ribosomale RNA af te breek en proteïensintese stop te sit. Hulle toon ook 'n sterk antivirale aktiwiteit. Sommige voel dat die antivirale eienskap van RIPs 'n funksie van ribosomale inaktivering is, terwyl ander glo dat die twee eienskappe onafhanklik optree. RIPs is in hul antivirale aktiwiteit onspesifiek. Transgeniese RIP-weergewende plante toon weerstand teen 'n wye spektrum virusse. Wingerd word deur baie verskillende virusse aangeval. Dit is onprakties om spesifieke teenmiddels vir elk van die virusse te ontwerp. In hierdie studie word die wingerdgenoom vir die voorkoms van 'n RIP-geen ondersoek, deur die gebruik van degeneratiewe PKR primers. As daar wel 'n RIP-geen in wingerd voorkom, word dit nie in 'n nuttige manier uitgedruk nie. Ons het ook 'n groep goedgedokumentêre RIP-gene vanuit verskeie plante in pGEM- T Easy gekloneer: dianthin vanuit Dianthus caryophyllus; p-Iuffin vanuit Luffa octandra; en mirabilis antivirale proteïen (MAP) vanuit Mirabilis jalapa. Hierdie geïsoleerde gene is toe in verskeie uitdrukkingsvektore gesubkloneer: dianthin in pKK223-3, 'n bakterïele uitdrukkingsvektor; p-Iuffin in pCambia3301, 'n plant uitdrukkingsvektor; en MAP in pFLAG, 'n gis uitdrukkingsvektor. Die constructs wat in hierdie projek voorberei is, kan gebruik word vir die sintese van RIP molekules. Dit is gevind dat die eksogeniese toepassing van RIPs plante teen virus-infeksie beskerm. Die transformasie van wingerd met die RIP-bevattende plant ekspressievektor kan 'n wingerd wat teen 'n wye verskeidenheid virusse bestand is tot stand bring. Hierdie tesis beskryf die voorlopige werk in 'n poging om breë-spektrum antivirale weerstand in wingerd deelagtig te maak.

Please read the abstract in the 00front part of this document / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Orientador: Erich Vinícius de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:53:53Z (GMT). No. of bitstreams: 1 Faiotto_VanessaBoury_M.pdf: 2777980 bytes, checksum: 26068f6f5813665c531b744d7a8c8f60 (MD5) Previous issue date: 2015 / Resumo: A sepse representa uma condição potencialmente fatal em que a resposta do organismo a uma infecção resulta em lesão em seus próprios tecidos. A terapia gênica (TG) consiste na modificação do repertório de células somáticas com fins terapêuticos, substituindo genes defeituosos que causam doenças. Na sepse, vetores não virais podem representar uma estratégia excelente para a transferência do gene terapêutico, uma vez que não provoca resposta imune significativa e são expressos apenas transitoriamente. Além disso, a sua produção é mais simples. Métodos: O estudo foi dividido em três etapas. Em primeiro lugar, dois genes repórter (lacZ e F9) foram testados em dois modelos experimentais de sepse (endotoxemia e ligadura e punção cecal, CLP) para confirmar a viabilidade da transferência gênica no contexto da sepse. A expressão foi avaliada por métodos qualitativos (histoquímica) e quantitativos (avaliação funcional; métodos coagulométricos). Em seguida avaliou-se a eficácia terapêutica da transferência do gene de sFlt-1, um antagonista de VEGF natural, no modelo de endotoxemia. A expressão foi avaliada por ELISA, e a eficácia foi avaliada através de uma curva de sobrevida. Os camundongos foram tratados com o plasmídeo DNA (pDNA) contendo o cDNA do gene Flt1 (tratamento) lacZ (controle), 6 horas após o desafio com LPS. A propósito, sFlt-1 foi capaz de proteger da sepse experimental murina em outros estudos, devido a sua propriedade estabilizadora da barreira endotelial (BE). Na última etapa, avaliamos os níveis séricos de quatro proteínas envolvidas na modulação da integridade BE, utilizando amostras de soro de indivíduos diagnosticados com sepse grave e choque séptico (n = 53), através de um kit Multiplex comercial. Resultados: No primeiro passo, a transferência gênica por vetores não virais foi demonstrada no modelo de endotoxemia, pois tanto a expressão de ?-galactosidase quanto de F IX mostraram-se aumentadas nos animais tratados neste modelo. A expressão não foi confirmada no modelo CLP, embora o número reduzido de animais por grupo não permita uma conclusão definitiva sobre o assunto. Em relação à segunda etapa, apenas 4/14 animais tratados apresentaram níveis detectáveis de sFlt-1 por Elisa. Além disso, não houve diferença na sobrevida entre os animais tratados e controles. Juntos, estes resultados confirmam que a transferência gênica com vetores não virais é possível no contexto de uma inflamação grave, mas apresenta baixa eficiência e previsibilidade. Por fim, observamos diferenças significativas nos níveis séricos de endoglina (maior expressão) e HB-EGF (menor expressão) em pacientes com choque séptico, em comparação ao grupo controle. Os níveis de BMP-9 e FGF-2 não foram significativamente diferentes no choque séptico. Conclusões: Nosso estudo nos permite concluir que, apesar de factível, a utilização de vetores não-virais não parece representar uma estratégia terapêutica eficaz na sepse experimental. Maiores estudos são necessários para validar o uso de endoglina e HB-EGF como biomarcadores de sepse grave / Abstract: Sepsis represents a potentially fatal condition that occurs when the body's response to infection results in injury to its own tissues. Gene therapy (GT) consists in modification of the repertory of somatic cells with therapeutic purposes, replacing defective genes that cause diseases. In sepsis, non-viral vectors, could represent an excellent strategy for therapeutic gene transfer, since they do not elicit intense immune responses in the individual and are expressed only transiently. Besides, their production is easy and inexpensive. Methods: The study was divided in 3 steps. First, two reporter genes (lacZ and F9) were tested in two experimental models of sepsis (endotoxemia and cecal ligation and puncture, CLP) to confirm the feasibility of gene transfer in the context of sepsis. We assessed expression by qualitative (histochemistry) and quantitative methods (functional evaluation, through coagulometric methods). Next we evaluated the efficacy of the therapeutic gene transfer of sFlt-1, a natural VEGF antagonist, in the endotoxemia model. Expression was evaluated by ELISA, and efficacy was evaluated by a survival curve. Mice were treated with pDNA containing the Flt1 (treatment) or lacZ (control) cDNA, 6 hours after challenge with LPS. Of note, sFlt-1 has been previously shown to protect from experimental sepsis due to its endothelial barrier (EB) stabilizing properties. In the last step, we evaluated serum levels of 4 proteins involved in the modulation of EB integrity, using serum samples of subjects diagnosed with severe sepsis and septic shock (n=53), through a commercial Multiplex kit. Results: In the first step, we could confirm the expression of both reporter genes by non-viral vectors in the endotoxemia model. Of note, expression of ?-galactosidase showed a notable increase when compared with control group. Similarly, we noted increased expression of factor IX in the mice which were submitted to gene transfer with pDNA, when compared with the controls that received the lacZ pDNA (111,4 ± 16,10 vs 64,73 ± 12,34; p<0,001) in the endotoxemia model, and similar result in the group without sepsis induction by endotoxemia (163,4± 73,46 vs 79,88 ± 9,39; p=0,0006). Expression was not confirmed in the CLP model, although the limited number of animals per group does not allow a definite conclusion on this matter. In relation to the second step, only 4/14 treated animals presented detectable levels of sFlt-1 by Elisa. In addition, no difference could be observed in the survival between treated and control animals. Together, these results confirme that gene transfer with non-viral vectors is feasible in the context of severe inflammation, but with a low efficiency and predictability. Finally, we demonstrated significant differences in serum levels of endoglin (higher expression) and HB-EGF (lower expression) in patients with septic shock, compared to controls. BMP-9 and FGF-2 levels were not significantly different in septic shock. Conclusions: Our study allows us to conclude that although feasible, the use of non-viral vectors does not seem to represent an effective therapeutic strategy in experimental sepsis. Larger studies are needed to validate the use of endoglin and HB-EGF as biomarkers of severe sepsis / Mestrado / Clinica Medica / Mestra em Ciências

A integridade do DNA é ameaçada pelos efeitos lesivos de inúmeros agentes físicos e químicos que podem vir a comprometer sua função. Um dos mais versáteis e estudados mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (NER). Este mecanismo remove lesões que causam distorções na dupla fita de DNA, incluindo dímeros de pirimidina ciclobutano (CPDs) e 6-4 fotoprodutos (6-4 PPs), provocados pela radiação de luz ultravioleta (UV). Em humanos, a síndrome genética xeroderma pigmentosum (XP) apresenta uma alta sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e degeneração neurológica progressiva. O gene xpa parece estar envolvido diretamente no reconhecimento de lesões produzidas pela luz UV, atuando tanto no reparo global (GGR) como no reparo acoplado à transcrição (TCR). A modulação da expressão deste gene deve alterar as taxas de reparo no genoma celular, fornecendo valiosa contribuição para o estudo do reparo de DNA no NER e em outras vias distintas. No entanto, não foi possível a atenuação ou inativação total do transcrito XPA, provavelmente devido ao baixo número de moléculas de mRNA nas células e da relativa estabilidade da proteína XPA. A expressão controlada do cDNA xpa em células deficientes XP12RO foi conseguida através da transfecção do vetor indutível por muristerona A, pINXA. O clone INXA15M mostrou-se eficiente na indução da proteína XPA, complementando células XP12RO. Quantidades reduzidas de XPA foram suficientes para a complementação total de células XP12RO na sobrevivência frente à luz UV, ou para a atividade de reparo de DNA no genoma global. Entretanto, observou-se uma maior incidência de células apoptóticas em períodos de tempo curtos após a UV, quando comparamos com células proficientes para o reparo de DNA (HeLa). O vetor adenoviral portando o cDNA xpa (AdyXPA) mostrou-se eficiente na complementação de fibroblastos derivados de pacientes XPA. Apesar do curto período de expressão do transgene e da conhecida reação imunológica produzida pelos adenovirus, este vetor representa uma potencial ferramenta para testes de complementação, identificação de mutações e busca de sistemas de correção gênica de pacientes XP. / The DNA integrity is always threatened by the damage effects of physical and chemical agents that could jeopardy its function. The nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove DNA double-helix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproduct, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during the UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). This gene modulation may modify the DNA repair rate in the cell genome, providing valuable contribution to the NER understanding and other DNA repair pathways. However, the complete inactivation or even the attenuation of the XPA transcript was not possible, mainly because of the low abundance mRNA per cell and the high stability of the XPA protein. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15M clone shows good induction of the XPA protein and total complementation of XP12RO cells deficiency. Small quantities of the XPA protein do not interfere in the cellular UV sensitivity and the DNA repair activity in the global genome. Nevertheless, a higher number of cells in the apoptotic process were detected in short periods of time after UV light when compared to normal cells (HeLa). The adenovirus vector carrying the cDNA xpa (AdyXPA) can efficiently complement XPA patients’ fibroblast cells. In spite of the short period of the transgene expression and the known imunological reaction caused by adenovirus, this vector represents a potential tool for gene complementation diagnostic and gene correction in XP patients.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 124-147).

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies. / AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.