Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

The historical game-changes in the philosophy of devotion and caste as used and misused by the Bhagavad-gita

This thesis considers that the purport of the Bhagavadgita is to prioritize the philosophy of loving devotion to God (bhakti), not the propagation of color-coded-caste (varna system). The distinction between bhakti and caste becomes clear when one sees their effect on human life and on the society. Jnana and karma, two of the other polarities with which the Gita contends, finally support bhakti towards betterment, not deterioration, if done selflessly and with balance. Caste, however, is a totally different tension, which is always detrimental to the well-being of the person and the society. In the Gita, the devotees' mystical or emotional love of, God apprehends their ~ oneness with the Supreme God and with all beings, and transcends the pitiless segregation of the caste system, and opens the path of salvation to all irrespective of race, color, caste, class or gender in life. In spite of much opposition from orthodoxy, the bhakti movement spread allover India, and bhakti itself rose to the level of orthodoxy and has become the faith of millions of people especially of the south, and surprisingly, of even of those of the so called highest caste. And yet, caste still remains as an indelible mark of every Hindu, even after they change their religion. Although caste is less venomous now, it is still openly present in all walks of Indian life and shows up its ugly head at important moments such as marriage, elections for public office, admission to school or employment. True, bhakti is the antidote for. caste; but only real bhakti can remove caste completely, not mere lip-service to it. This thesis claims that bhakti is the deliberate major thrust of the teaching of the Gita while caste seems to be a contradiction of this thrust.