Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Brent Iverson. Vita. Includes bibliographical references.

Th. 3e cycle--Électronique, électrotech., autom.--Toulouse 3, 1978. N°: 2111.

The last decade has seen widespread application of recombinant DNA technology to the study of picornaviruses. Comparative sequence analysis has revealed that the most highly conserved region amongst many members of this family of viruses is the 5' non-coding region. Using recombinant type 3 polioviruses it has been shown that a single point mutation located in this region dramatically reduces neurovirulence and inhibits the intracellular life-cycle of the virus. Mutation at this nucleotide contributes to the observed reversion to neurovirulence of the Sabin attenuated poliovirus type 3 vaccine strain currently used in vaccination programmes throughout the world. Knowledge concerning the function of the 5' non-coding region remains scant, and as a result, the mechanism whereby a single point mutation within this region results in alteration of the expressed phenotype of the virus remains unknown. Clearly, an understanding of the molecular mechanism(s) involved requires greater knowledge of the function of the 5' non-coding region. This thesis describes the design and construction of vectors that allow analysis of the role of the 5' non-coding region in the control of viral translation, replication, and encapsidation of viral RNA. In the plasmid pRSV-5'polio-CATm2 (N+), the 5' non-coding region of poliovirus was fused to the coding region of the bacterial chloramphenicol acetyltransferase reporter gene. The presence of the 5' non-coding region resulted in the inhibition of CAT expression when this plasmid was introduced into eukaryotic cells in culture. Deletion analysis of the 5' non-coding region in this vector identified two regions that were responsible for the marked inhibition of expression of the reporter gene. It would appear from the results of these experiments that the poliovirus/CAT chimaeric message is translatsed as a normal eukaryotic mRNA and is subject to the rules of the "scanning model". This observation suggests that the 5' non-coding region of poliovirus on its own does not possess features which enable a message containing it to be translated efficiently. It is concluded that a second factor, present in infected cells, is required for the efficient translation of poliovirus. A second plasmid was designed and constructed to investigate the role of the 5' non-coding region in replication and encapsidation of viral RNA. Preliminary data suggest that the product of this vector does undergo replication while its ability to be encapsidated has still to be tested.

La littérature décrit des différences fondamentales entre l’AAV2 sauvage et ses pendants recombinants. La forme sauvage serait plus efficiente en terme de production, d’encapsidation et d’infectieusité, allant de facteurs de deux à cents en fonction de la propriété étudiée. A cause de ces différences, la quantité de rAAV nécessaire pour traiter un patient atteint d’une maladie implique une injection systémique estimée à 1.1015 particules par kilogramme de tissue à traiter. C’est dans cette optique que s’inscrivent mes travaux de thèse. Essayer de comprendre qu’elles sont les différences entre l’AAV sauvageet les rAAV qui peuvent engendrer tant de différences en terme d’efficacité. L’étude du comportement du génome de l’AAV2 sauvage dans le système baculovirus/cellules Sf9, a permis de découvrir que la régulation de l’AAV2 sauvage était similaire en baculovirus. Nous avons aussi découvert, grâce à notre analyse transcriptomique que les promoteurs naturels del’AAV2 étaient actifs dans notre système, ce qui nous a permis d’imaginer de nouvelles constructions génétiques afin d’améliorer la quantité et la qualité des particules rAAV. Nous avons aussi réaliser des études structurales sur différentesparticules AAV afin d’améliorer notre connaissance de ces particules. / The literature describes several fundamental differences between WT AAV and rAAP properties. WT form obtains an one hundred higher production yield compared to rAAV, with the possibility to obtain only full particles, and most importantly, all the WT AAV particles are infectious, compared to only 1% for the rAAV. These lower values for rAAV, implied to inject up to 1.1015 particles per kilogram of tissue. These quantities induce a non-negligible cost for rAAV based gene therapies, even with the productions techniques improvements or the development of baculoviruses based techniques. It is with these ideas in mind that my PhD works were developed. Trying to understand differences between WT and rAAV, trying to produce WT AAV in baculovirus has bring important knowledges about AAV comportment in baculovirus. Our RNA-Seq results have demonstrated than the WT AAV natural promoters were all active, allowing us to design to genetic constructs in order to improve rAAV quantity and quality. We have also tried to solve the AAV crystal structure, to improve our knowledges about these particles.

No description available.

Infection with Human papillomaviruses has been suggested to play an important role in the etiology of a number of human malignancies. The most investigated of HPVs is HPV-16. Infection with HPV-16, in association with other unknown factors, is implicated in the development of cervical cancer. Genetic analysis of HPVs has been hampered by the lack of an in vitro system in which the complete replicative cycle and the transcription of the various genes is possible. Replication and transcription of HPVs seem to be in part regulated by cellular factors expressed at different stages in the maturation of epithelial cells which constitute the normal hosts of those viruses. This study was primarily designed to address this difficulty. HPV-16 genome was introduced into a viral system, HSV-1, hoping that virally encoded factors can substitute for cellular factors required for the expression of HPV genes. This hypothesis was tested by analyzing the activity of the HPV-16 early promoter P₉₇ , in the constructed recombinant, in cells which are unsusceptible to infection with HPV-16. In Vero cells infected with the recombinant, P₉₇ was shown to be active. This suggests that HSV-1 encoded factors can influence transcription from endogenous papilloma promoters. / Thesis / Master of Science (MS)

A cost effective approach to the delivery of therapeutic gene products in vivo is to immunoprotect genetically-engineered, universal, non-autologous cells in biocompatible microcapsules before implantation. Myoblasts may be an ideal cell type for encapsulation due to their inherent ability to differentiate into myotubes, thereby eliminating the problem of cell overgrowth within the capsular space. To evaluate the interaction between the differentiation program and the secretory activity of the myoblasts within the microcapsule environment, we transfected C2C12 myoblasts to express human growth hormone and followed their expression of muscle differentiation markers, such as creatine phosphate kinase (CPK) protein and up-regulation of muscle-specific genes (ie. myosin light chains 2 & 1/3, Troponin I slow, Troponin T, myogenin and MyoD1). As the transfected myoblasts were induced to differentiate for up to two weeks, their myogenic index (i.e. the percentage of multinucleate myoblasts) increased from 0 to ~50%. Concomitantly, up-regulation of differentiation marker RNA levels, and as much as a 23-fold increase in CPK activity, were observed. After encapsulation and the induction of differentiation, the myoblasts showed a lag phase of ~3 days before an increase in CPK was observed, although the level of CPK activity increased by as much as 63-fold. The myogenic index of the encapsulated cells remained at zero. The rate of human growth hormone secretion was relatively constant throughout the two-week differentiation period, at an average of 7.78 x 10^-2 ng hGH per hour per (mu)g protein, however, human growth hormone secretion was slightly decreased by about twofold during the differentiation of encapsulated myoblasts. In conclusion, the differentiation of myoblasts into myotubes is retarded after encapsulation while the secretion of a recombinant product is slightly reduced. Further studies are necessary to elucidate the cause of this atypical differentiation pattern such that the proliferation and differentiation of the encapsulated myoblasts may be optimized to provide a stable vehicle for gene delivery. / Thesis / Master of Science (MS)

The production of recombinant glycoproteins from mammalian cell cultures requires robust processes that can achieve high protein yield while ensuring the efficacy of these proteins as human therapeutics. We describe two approaches currently being developed in our group to genetically engineer cell lines with desirable characteristics for recombinant protein production. To enhance the degree of sialylation in the glycoprotein product, we propose to increase intracellular sialic acid availability by overexpressing the CMP-sialic acid transporters. We are also interested in engineering mammalian cells that can proliferate at reduced cultivation temperatures. Low temperature cultivation of mammalian cells has been shown to enhance glycoprotein production but reduces cell growth. It is hypothesized that a mutant cell line that can proliferate at low temperatures may be coupled with low temperature cultivation to improve recombinant protein production. / Singapore-MIT Alliance (SMA)

No description available.