Huber, Robert J.

Cytokinins (CKs) are adenine derivative molecules that are present in all kingdoms of life. CKs are known to have a role in cell growth and development in plants. However, the role of CKs in vertebrate systems is not well understood. Frog virus 3 (FV3) is a type species of the Iridioviridae family, genus Ranavirus. FV3 is a major contributor to the amphibian population decline in North America. In this study, we demonstrate that concurrent and pretreatment of 20 µM of either N6-isopentyldenine (iP), N6-ispoentyladenosine (iPR), N6-furfurladenine/kinetin, and N6-furfurladenosine/kinetin riboside (KR) decreased FV3 replication. To understand the mechanism of inhibition, we assessed morphological changes in host cell nuclei to assess the effect of CKs on infected nuclei. Our results show that infection with FV3 and 20µM treatment of iP or iPR reduced nuclei size. These results are the first to reveal insight into the potential mechanism in which FV3 replication is inhibited by iP and iPR.  

Author Keywords: cytokinins, frog virus 3

This thesis investigated the role of the neuronal ceroid lipofuscinosis protein, Cln5, during autophagy. This was accomplished by performing well-established assays in a Dictyostelium cln5 knockout model (cln5-). In this study, cln5- cells displayed a reduced maximum cell density during growth and impaired cell proliferation in autophagy-stimulating media. cln5- cells had an increased number of autophagic puncta (autophagosomes and lysosomes), suggesting that autophagy is induced when cln5 is absent. cln5- cells displayed increased amounts of ubiquitin-positive proteins but had no change in proteasome protein abundance. During the development of cln5- cells, fruiting bodies developed precociously and cln5- slug size was reduced. Lastly, when cln5- cells were developed on water agar containing ammonium chloride (NH4Cl), a lysosomotropic agent, the formation of multicellular structures was impaired, and the small slug phenotype was exaggerated. In summary, these results indicate that Cln5 plays a role in autophagy in Dictyostelium. The cellular processes that regulate autophagy in Dictyostelium are similar to those that regulate the process in mammalian cells. Thus, this research provides insight into the undefined pathological mechanism of CLN5 disease and could identify cellular pathways for targeted therapeutics.

Author Keywords: Autophagy, Batten disease, Cln5, Dictyostelium discoideum, NCL