Typescript (Photocopy) / Bibliography: leaves 170-198. / xx, 198 leaves, 1 leaf of col. plates : ill. ; 30 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 Medicine, 1991

The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common clinical problem. Tanshinone IIA is a compound purified from the Chinese herb Danshen ( Radix Salviae Miltiorrhiza Bge). Thrombopoietin (TPO) and Erythropoietin (Epo) are hematopoietic growth factors. The effects of tanshinone IIA, EPO and TPO on hypoxia-ischemia brain injury were investigated in this study, using in vitro model of neural cell culture and an in vivo model of hypoxic-ischemic brain damage. / Our observation provided the first evidence showing the expression of functional TPO receptor c-mpl in central nervous system. It revealed that novel agents TPO, EPO and tanshinone IIA have neuroprotection effects against brain injury induced by hypoxia-ischemia in neonatal rats, and these agents could be developed for clinical applications. / To investigate the effect of TPO, EPO and tanshinone IIA on in-vivo neural protection, a neonatal rat model of hypoxic-ischemic brain damage was established. Our results demonstrated significant and sustained brain injury in the hypoxic-ischemic and vehicle-treated group, measured by the reduction in relative weights of the ipsilateral (right) to the contralateral (left) brain at 1 and 3 weeks post-surgery, compared with those of sham-operated animals. At 3 weeks post-surgery, the hypoxic-ischemic animals had decreased cortical neuron density quantified by neuron-specific enolase (NSE) staining, and compromised sensorimotor functions in response to the postural reflex test. Treatment with TPO, EPO and tanshinone IIA significantly reduced the severity of brain injury, as indicated by the significantly increased ipsilateral brain weight and neuron density. Recoveries of sensorimotor functions (p < 0.05) and histopathology were also observed in animals that received TPO, EPO and tanshinone IIA. The plasma of tanshinone IIA-treated animals exhibited higher antioxidant activities (oxygen radical absorbance capacity assay) than those from vehicle-treated rats. / TPO and TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres, cerebellum, mouse neural progenitor cell line C17.2 and four neuroblastoma cell lines (SK-N-MC, MHH-NB-11, SK-N-AS and SH-SY-5Y) using RT-PCR methods. TPO proteins were detected in human cerebrospinal fluid (CSF) and plasma by ELISA. Furthermore, TPO receptor c-mpl was confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunohistostaining. TPO had a stimulating effect on the growth of neural progenitor cell C17.2 in culture via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway as demonstrated by Western blot. The anti-apoptotic effects of TPO, EPO on C17.2 cells were demonstrated by staining with Annexin-V and PI. EPO exerted a protective effect against SHSY-5Y cell damage induced by NMDA (N-methyl-d-aspartate), as demonstrated by the MTT and LDH assay. The anti-oxidative property of tanshinone IIA was studied in the C17.2 cell line. Tanshinone IIA increased the viability of these cells subjected to 2,2'-azobis (2-amidino propane hydrochloride) (AAPH)-induced oxidative stress. / by Xia Wen-Jie. / "May 2005." / Advisers: Kwok-Pui Fung, Tai-Fai Fok. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0126. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 126-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Indiana University-Purdue University Indianapolis (IUPUI) / The development and function of hematopoietic stem cells (HSCs) is regulated by numerous signaling pathways including Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway. Dysregulation of this pathway results in impaired HSC function and contributes to the development of hematologic malignancies. Activated mTORC1 phosphorylates and subsequently activates ribosomal protein S6 kinase 1 (S6K1). To study the role of S6K1 in hematopoiesis as well as leukemogenesis, we used a genetic model of S6K1 deficient mice (S6K1-/-). We found that loss of S6K1 expression in HSCs results in reduction of absolute HSC number in bone marrow (BM). Following chemotherapy, cycling HSCs undergo apoptosis and quiescent HSCs are required to cycle to regenerate the hematopoietic system. S6K1 regulates the quiescence of HSCs and in the absence of S6K1, mice are more susceptible to repeated myeloablative stress. We also observed that loss of expression as well as gain of expression of S6K1 affects the self-renewal ability of HSCs. Interestingly, when we overexpressed S6K1, it also resulted in reduced self-renewal of HSCs. Next, we assessed the role of S6K1 in the propagation of acute myeloid leukemia (AML). The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult HSCs. Translocations in MLL are detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and performed serial transplantation. Upon secondary transplantation, recipients of S6K1 deficient AML cells survived significantly longer compared to controls. In vitro, pharmacological inhibition of S6K1 activity resulted in reduced growth of primary human cells expressing MLL-AF9. Both human and murine HSC/Ps expressing MLL-AF9 showed reduced mTORC1 activity upon inhibition of S6K1 suggesting that loss of S6K1 activity results in reduced Akt-mTORC1 activation both upstream and downstream of mTORC1. Overall, our studies establish a critical role of S6K1 activity in the maintenance of HSC function and in the propagation of leukemia.

Indiana University-Purdue University Indianapolis (IUPUI) / Adult hematopoietic stem cells (HSC) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment or self-renewal. We have demonstrated that mouse and human HSC express prostaglandin E2 (PGE2) receptors, and that short-term ex vivo exposure of HSC to PGE2 enhances their homing, survival and proliferation, resulting in increased long-term repopulating cell and competitive repopulating unit (CRU) frequency. HSC pulsed with PGE2 are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon multiple serial transplantations, with full multi-lineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression and enhances their migration to SDF-1α in vitro and homing to bone marrow in vivo and stimulates HSC entry into and progression through cell cycle. In addition, PGE2 enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. While PGE2 pulse of HSC promotes HSC self-renewal, blockade of PGE2 biosynthesis with non-steroidal anti-inflammatory drugs (NSAIDs) results in expansion of bone marrow hematopoietic progenitor cells (HPC). We co-administered NSAIDs along with the mobilizing agent granulocyte-colony stimulating factor (G-CSF) and evaluations of limiting dilution transplants, assays monitoring neutrophil and platelet recoveries, and secondary transplantations, clearly indicate that NSAIDs facilitate mobilization of a hematopoietic graft with superior functional activity compared to the graft mobilized by G-CSF alone. Enhanced mobilization has also been confirmed in baboons mobilized with G-CSF and a NSAID. Increases in mobilization are the result of a reduction of signaling through the PGE2 receptor EP4, which results in marrow expansion and reduction in the osteoblastic HSC niche. We also identify a new role for cannabinoids, an eicosanoid with opposing functions to PGE2, in hematopoietic mobilization. Additionally, we demonstrate increased survival in lethally irradiated mice treated with PGE2, NSAIDs, or the hypoxia mimetic cobalt chloride. Our results define novel mechanisms of action whereby eicosanoids regulate HSC and HPC function, and characterize novel translational strategies for hematopoietic therapies.

Indiana University-Purdue University Indianapolis (IUPUI) / Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.