published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Cell cycle-related kinase (CCRK) is a 42 KDa serine/threonine protein kinase homologous to Cdk1, 2 and 7. Previous work has shown that CCRK regulates cell cycle transition by phosphorylating Cdk2 and Rb. More importantly, it was found that CCRK was a candidate oncogene in both glioblastoma multiform (GBM) and human colorectal cancer. However, the mechanistic role of CCRK in tumorigenicity is still not completely understood. In the first part of this thesis, I found that casein kinas II beta (CKIIβ) was one of proteins that interact with CCRK using the high-throughput yeast-two-hybrid analysis. Then I confirmed their interaction by co-immunoprecipitation. CCRK phosphorylated CKIIβ at Ser-209 in a cell cycle-dependent manner. The phosphorylation of CKIIβ by CCRK enhanced the activity of CKII holoenzyme, protected CKIIβ against proteasome degradation, and facilitated CKIIβ translocation into the nucleus in U-87 MG and U-373 MG GBM cells. Importantly, CCRK de-sensitized GBM cells to the cytotoxic effect of three chemotherapy drugs, whereas knockdown of CCRK by siRNA reduced chemoresistance. Functionally, CKIIβ is responsible for CCRK-mediated inhibition of apoptosis, as suppression of CKIIβ by siRNA or CKIIβ inhibitor could re-sensitize cells to the cytotoxic effect of cisplatin in both wild type and CCRK-overexpressing U-87 MG cells. In vivo studies also showed that stable over-expression of CCRK increased tumor growth and decreased the anti-tumor efficacy of cisplatin in a nude mice GBM xenograft model. These results provide the first evidence that phosphorylation of CKIIβ is a new mechanism by which CCRK confers tumor growth and drug resistance to GBM cells. In the second part of this thesis I described a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600 and sebacoyl chloride. 1H-NMR, FT-IR and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. Cytotoxicity of the mPPS-FA/DNA nanoparticles was also evaluated on B16-F0, U87MG, CHO-1 and Ho-8910 cells. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910 and A549 cells was investigated in vitro as compared to the lipid-based transfection agent LipofectamineTM2000 and Linear PEI 22KDa (L-PEI 22KDa). I found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1 and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be significantly competed and blocked by the free folic acid molecules. All together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The notochord is a conserved structure in the phylum chordate, which includes all vertebrates and some closely related invertebrates. In mouse embryos, the notochord is a midline structure underneath the neural tube and it consists of a rod of cells constrained by a thick extracellular sheath, which is rich in fibronectin and other extracellular matrix molecule. During notochord formation, two key processes are involved: cell migration and convergent extension. Two molecules are essential for these processes: fibronectin and Protein Kinase C iota (prkci). For cell migration, fibronectin regulates this process by modulating cell protrusion via a signaling pathway in which atypical protein kinase C (aPKC) is an essential factor. For convergent extension, fibronectin has been shown to be important in this process by regulating both cell migration and cell adhesion. The role of aPKCin convergent extension is revealed as it is asymmetrically expressed in Ciona notochord during convergent extension, indicating possible function of aPKC in convergent extension process and in the morphogenesis of notochord. These studies raised the possibility that fibronectin and prkci are important in notochord formation, by regulating cell migration and convergent extension. In this thesis, Cre/loxP system was used to study the function of fibronectin and prkci in the development of notochord. I provided evidence that conditional deletion of fibronectin by Foxa2-Crein the notochord resulted in a notochord of smaller volume and fewer notochordal cells. The nucleus pulposus was also smaller in are and less in cell number. Fibronectin in the notochord was not affected at E9.5, but diminished in the core of the notochord at E12.5 and in nucleus pulposus at E15.5. The phenotypes of smaller notochord and the nucleus pulposus might be the results of reduced notochordal cell proliferation and increased cell death. However, more samples are needed to analyze to confirm this and perform statistical analysis. In addition, convergent extension of notochord seemed less effective. These results are consistent with previous study about fibronectin and α5β1 integrin. The results suggest that fibronectin is required for notochordal cell proliferation, survival, migration and efficient convergent extension, but not for notochordal cell fate determination. The results also demonstrated that prkci seemed not to be important for notochord development. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Background: Regulation of vascular tone is complex. Various complementary signaling pathways causing contraction and relaxation of vascular smooth muscle take place to ensure proper blood flow within the vasculature. Mitogen activated protein kinase (MAPK) signaling cascade is observed to be one of the many signaling pathways that regulate vascular tone. Aim: This study examines the role of the following MAPK: mitogen-activated extracellular-regulated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p38 MAPK in the regulation of relaxation in the endothelium and smooth muscle. Method: Isometric tension of isolated porcine coronary artery rings were measured with organ chamber setup. The effects of MEK inhibitor, PD98059 (30 μM), ERK inhibitor, U0126 (10 μM) and p38 MAPK inhibitor, SB203580 (10 μM), on relaxations induced by bradykinin (a vasodilating peptide), SKA-31 [an activator of small and intermediate conductance calcium-activated potassium channels (SKCa and IKCa,, respectively)], Deta NONOate (a nitric oxide donor) and forskolin (an adenylate cyclase activator) were examined in arteries with and without endothelium, contracted with an thromboxane A2 analog, U46619 (300 nM – 1 μM). In some experiments, rings were also incubated with the following pharmacological inhibitors, indomethacin (cyclooxygenase inhibitor, 10 μM), L-NAME (nitric oxide synthase inhibitor, 300 μM), TRAM34 (IKCa blocker, 1 μM), and UCL1684 (SKCa blocker, 1 μM), alone or in combination. Results: 1. Bradykinin-induced relaxation was potentiated by MEK and ERK inhibition but not by p38 MAPK inhibition. 2. SKA-31-induced relaxation was potentiated by MEK and p38 MAPK inhibition but not by ERK inhibition. 3. Deta NONOate-induced relaxation was potentiated by MEK, p38 MAPK inhibition, but not by ERK inhibition except in the presence of indomethacin, TRAM-34 plus UCL1684. 4. Forskolin-induced relaxation was potentiated by MEK and p38 MAPK inhibition, but not by ERK inhibition. Discussion: MAPK plays a role in regulating the vascular tone in both the endothelium and smooth muscle of porcine coronary arteries. MEK appears to have an inhibitory action on relaxation that is downstream of the generation of endothelium-derived nitric oxide, activation of IKCa and SKCa and activation of adenylate cyclase. ERK are unlikely to be the downstream target of MEK for inhibiting relaxation, in view of the lack of effects of its inhibitor on endothelium-derived hyperpolarizing factor (EDHF)-mediated and endothelium-independent relaxations. The involvement of ERK in relaxation pathways in the endothelium appears to be complicated, since U0126 caused opposing effects (inhibition and potentiation) on bradykinin-induced relaxation in the presence of indomethacin without and with L-NAME or TRAM-34 plus UCL1684. As inhibition of p38 MAPK results in potentiation of relaxations to all relaxing agents tested except bradykinin, this MAPK may have opposing action in the endothelium and smooth muscle; endothelial p38 MAPK may facilitate relaxation while smooth muscle p38 MAPK attenuates it. In conclusion, this study provided additional information on the influences of MEK, ERK and p38 MAPK on relaxation; this knowledge may contribute to the understanding of the mechanisms underlying the development of vascular disorders. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

The Aurora kinases are highly conserved serine/threonine kinases that play essential roles throughout mitosis. In metazoans, these functions are mediated by Aurora A and B at the spindle poles and the equatorial region respectively. I show here that Dictyostelium contains a single Aurora kinase, DdAurora that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box and localizes to the spindle poles during early mitosis. Like Aurora B, DdAurora localizes to centromeres in metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. In addition to these known features of Aurora A and B, I found that DdAurora remains associated with centromeres during anaphase and telophase which has not been shown in any other organisms. INCENP is known to be an important binding partner of Aurora B. In Dictyostelium the conserved C-terminal IN-box domain of DdINCENP is essential for its interaction with DdAurora and for the localization of DdAurora to the central spindle. In contrast, the centromeric and spindle pole localization of DdAurora does not require an interaction with DdINCENP. Surprisingly, a truncated DdINCENP protein lacking the IN-box domain can still localize on centromeres and the central spindle even though it does not bind to DdAurora. I also found that the localization of DdAurora to the central spindle requires Kif12, a protein similar to mitotic kinesin like protein 2 (MKLP2). However, this requirement is suppressed by the overexpression of GFP-DdINCENP. GFP-DdINCENP can localize to the central spindle in the absence of Kif12 and it probably recruits DdAurora to the same location through their strong interaction. Finally, I demonstrated that Myosin II heavy chain is important for the proper localization of the DdAurora/DdINCENP complex to the cleavage furrow during late cytokinesis. With the exception of DdINCENP, no other binding partner or substrate of DdAurora has been identified in Dictyostelium. By performing large-scale immunoprecipitation in wild-type cells, I identified several potential binding partners/substrates of DdAurora, including topoisomerase B and HspA. Future esearch on these proteins may help to elucidate DdAurora function in different stages of M phase.