The Hippo pathway plays a key role in controlling organ growth and size. In mammals, core pathway components include the Lats1/2 and Mst1/2 kinases, which phosphorylate the transcriptional regulators, Taz and Yap. To identify novel upstream pathway regulators high throughput protein-protein interaction screens, called LUMIER (LUminescence-based Mammalian IntERactome) were performed together with a functional screen using a luciferase reporter that examines Hippo pathway responses. The screens revealed 1103 protein-protein interactions and 227 transcriptional regulators, which were particularly enriched in cytoskeletal regulators. A subset of these hits including BTK, Dvl1, Dvl2, Dvl3, Ing2, Magi2, Mark4 and Trip6 were validated by manual LUMIER assays and co-immunoprecipitation (Co-IP). Of particular interest was the microtubule dynamics regulatory protein MARK4. Loss of Mark4 prevents Taz activity demonstrating its role as a potential negative regulator of the Hippo pathway. Further studies could help decipher mechanisms of how Mark4 and the other cytoskeletal hits act to modulate the Hippo pathway.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by an excess production of a specific, fully functional blood cell type. Many cases involve deregulation of a protein tyrosine kinase. JAK2 is one such kinase, involved in a subset of MPNs. JAK2-selective inhibitors are currently being evaluated in clinical trials. In order to identify inhibitor-resistant JAK2 mutations before they appear in the clinic, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signalling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles. Chronic myeloid leukemia is an MPN characterized by the presence of the BCR-ABL fusion gene. We determined that a specific cohort bearing deletions near the ABL gene, which is associated with poor prognosis, do not suffer from genomic instability. We also examined the role of a putative tumour suppressor gene EXOSC2 as an explanation for the reduced survival time, and suggest it may have a role in disease progression.

The Hippo pathway plays a key role in controlling organ growth and size. In mammals, core pathway components include the Lats1/2 and Mst1/2 kinases, which phosphorylate the transcriptional regulators, Taz and Yap. To identify novel upstream pathway regulators high throughput protein-protein interaction screens, called LUMIER (LUminescence-based Mammalian IntERactome) were performed together with a functional screen using a luciferase reporter that examines Hippo pathway responses. The screens revealed 1103 protein-protein interactions and 227 transcriptional regulators, which were particularly enriched in cytoskeletal regulators. A subset of these hits including BTK, Dvl1, Dvl2, Dvl3, Ing2, Magi2, Mark4 and Trip6 were validated by manual LUMIER assays and co-immunoprecipitation (Co-IP). Of particular interest was the microtubule dynamics regulatory protein MARK4. Loss of Mark4 prevents Taz activity demonstrating its role as a potential negative regulator of the Hippo pathway. Further studies could help decipher mechanisms of how Mark4 and the other cytoskeletal hits act to modulate the Hippo pathway.

<p>Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells, but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a CDK mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive feedback loops active at the G2/M transition.</p> / Dissertation

Typewritten / 117 leaves, [28] leaves of plates : ill. ; 27 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physical & Inorganic Chemistry,1960

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007. / Source: Dissertation Abstracts International, Volume: 68-05, Section: B, page: 2790. Adviser: Susan Strome. "Title from dissertation home page (viewed Jan. 24, 2008)."

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007. / Source: Dissertation Abstracts International, Volume: 68-05, Section: B, page: 2766. Adviser: Claire E. Walczak. "Title from dissertation home page (viewed Jan. 24, 2008)."

Thesis (Ph. D.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.