Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 138-139

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Chau Wing-Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 92-101). / Abstracts in English and Chinese. / Abstract --- p.I / Abstract (Chinese version) --- p.III / Acknowledgement --- p.IV / List of Abbreviations --- p.VIII / List of Tables --- p.IX / List of Figures --- p.X / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Neurodegenerative diseases --- p.2 / Chapter 1.2 --- Tauopathies --- p.5 / Chapter 1.3 --- Function and structure of Tau --- p.9 / Chapter 1.4 --- Post-translational modifications of Tau --- p.13 / Chapter 1.5 --- Tau protein kinases --- p.17 / Chapter 1.6 --- Tau protein kinase inhibitors --- p.19 / Chapter 1.7 --- Drosophila model of Tauopathies --- p.20 / Chapter 1.8 --- Aims of study --- p.24 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Drosophila manipulation / Chapter 2.1.1 --- Drosophila genetics --- p.26 / Chapter 2.1.2 --- External fly eye and adult wing morphology examination --- p.27 / Chapter 2.1.3 --- Study of fly wings deformation on Tau kinase overexpression --- p.27 / Chapter 2.2 --- RNA extraction / Chapter 2.2.1 --- Method --- p.28 / Chapter 2.2.2 --- Buffers and reagents --- p.29 / Chapter 2.3 --- Reverse transcription-PCR / Chapter 2.3.1 --- Method --- p.30 / Chapter 2.3.2 --- Buffers and reagents --- p.31 / Chapter 2.4 --- SDS-Polyacrylamide gel electrophoresis / Chapter 2.4.1 --- Method --- p.31 / Chapter 2.4.2 --- Buffers and reagents --- p.32 / Chapter 2.5 --- Western blotting / Chapter 2.5.1 --- Method --- p.32 / Chapter 2.5.2 --- Buffers and reagents --- p.33 / Chapter 2.6 --- Phosphatase treatment of proteins / Chapter 2.6.1 --- Method --- p.34 / Chapter 2.6.2 --- Buffers and reagents --- p.34 / Chapter 2.7 --- Sequential extraction of proteins / Chapter 2.7.1 --- Methods --- p.35 / Chapter 2.7.2 --- Buffers and reagents --- p.36 / Chapter 2.8 --- Sarkosyl extraction of proteins / Chapter 2.8.1 --- Method --- p.37 / Chapter 2.8.2 --- Buffers and reagents --- p.37 / Chapter 2.9 --- Immunostaining / Chapter 2.9.1 --- Method --- p.38 / Chapter 2.9.2 --- Buffers and reagents --- p.38 / Chapter 2.10 --- Lithium treatment of flies / Chapter 2.10.1 --- Method --- p.39 / Chapter 2.10.2 --- Buffers and reagents --- p.40 / Chapter 2.11 --- Quantitation of Lithium ion by atomic absorption spectrometry / Chapter 2.11.1 --- Method --- p.40 / Chapter 2.12 --- Statistical analysis --- p.41 / Chapter Chapter 3 --- Results / Chapter 3.1 --- GAL4/UAS gene expression system in transgenic fly / Chapter 3.1.1 --- Introduction --- p.43 / Chapter 3.1.2 --- Results --- p.47 / Chapter 3.1.3 --- Discussion --- p.52 / Chapter 3.2 --- Tau phosphorylation and Tau-induced toxicity in transgenic fly / Chapter 3.2.1 --- Introduction --- p.52 / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Overexpressed Tau is phosphorylated and toxic in fly --- p.53 / Chapter 3.2.2.2 --- Coexpression of GSK3β/Shaggy or Cdk5 enhance Tau phosphorylation and Tau-induced toxicity --- p.57 / Chapter 3.2.2.3 --- Lithium suppresses Tau phosphorylation and Tau-induced toxicity --- p.64 / Chapter 3.2.3 --- Discussion --- p.68 / Chapter 3.3 --- Tau solubility properties in transgenic fly / Chapter 3.3.1 --- Introduction --- p.69 / Chapter 3.3.2 --- Results / Chapter 3.3.2.1 --- Coexpression of GSKlβ/Shaggy does not alter the sarkosyl solubility of Tau --- p.70 / Chapter 3.3.2.2 --- Coexpression of GSK3β/Shaggy causes a minor alteration of Tau solubility properties --- p.73 / Chapter 3.3.3 --- Discussion --- p.78 / Chapter 3.4 --- Tau aggregate formation in transgenic fly / Chapter 3.4.1 --- Introduction --- p.79 / Chapter 3.4.2 --- Results / Chapter 3.4.2.1 --- Tau aggregates are detected in aged transgenic flies --- p.80 / Chapter 3.4.3 --- Discussion --- p.82 / Chapter 3.5 --- Effect of Lithium on GSK3p/Shaggy-induced wing deformation / Chapter 3.5.1 --- Introduction --- p.83 / Chapter 3.5.2 --- Results / Chapter 3.5.2.1 --- Lithium rescues GSK3β/Shaggy-induced wing deformation --- p.84 / Chapter 3.5.3 --- Discussion --- p.86 / Chapter Chapter 4 --- General discussion --- p.87 / References --- p.92

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related deaths. Systemic therapies are the main treatment options for HCC patients with advanced disease (∼ 80% of all cases). However, only very moderate clinical responses are achieved with most of the conventional therapies. Thus, more effective therapeutic strategies are much needed. The PI3K/Akt/mTOR signaling pathway, which plays a critical role in controlling cell proliferation and survival, is aberrantly activated in ∼ 45% HCC, suggesting it to be a potential target for HCC treatment. Moreover, emerging evidences indicate that activation of the PI3K/Akt/mTOR pathway may be associated with resistance to many cytotoxic chemotherapies, including microtubule targeting agents. In this study, by gene expression profiling and gene ontology analysis, "microtubule-related cellular assembly" was identified to be the major biological/functional process involved in HCC development, suggesting that microtubule is also an important therapeutic target for HCC. With these understandings, it is hypothesize in this thesis that combined targeting of a key component ofthe PI3K/Akt/mTOR pathway, namely the mammalian target of rapamycin (mTOR) and the microtubule would be an effective therapeutic strategy for HCC. The objectives of the thesis are to examine the therapeutic potential of microtubule targeting, mTOR targeting, and combined targeting of the microtubule and mTOR in both in vitro and in vivo models of HCC. / In summary, the PI3K/Akt/mTOR pathway and the microtubule represent promising therapeutic targets for HCC treatment. The findings from this thesis offer a rationale for combining mTOR inhibitors with microtubule targeting agents for effective HCC treatment. / In the second part, the effect of mTOR inhibition, either alone or in combination with an additional microtubule targeting agent (vinblastine) was investigated in HCC. Temsirolimus, an mTOR inhibitor, suppressed HCC cell proliferation in as early as 24 hrs with an IC50 of 1.27+/-0.06muM (Huh7), 8.77+/-0.76muM (HepG2), and 52.95+/-17.14muM (Hep3B). Vinblastine (1nM) alone caused 30--50% growth inhibition in 3 HCC cell lines. In these HCC cell lines, it was found that temsirolimus/vinblastine combination resulted in an additive to synergistic effect (when compared to single agents alone) with maximum growth inhibition of 80--90% as early as 24 hrs upon treatment. This marked growth inhibition was accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage (a hallmark for apoptosis). Moreover, the combination specifically caused concerted down-regulation of several important anti-apoptotic and survival proteins (survivin, Bcl-2 and Mcl-1), which was not observed in single agent treatments. It was hypothesized that inhibition of these key anti-apoptotic/survival proteins may represent a novel mechanistic action of this highly effective combination approach of dual targeting of mTOR and microtubule by temsirolimus/vinblastine in HCC cells. Indeed, transient over-expression of each of these genes (survivin, Bcl-2 or Mcl-1) in HCC cells did partially rescue the growth inhibitory effect of the temsirolimus/vinblastine combination. More importantly, this novel combination significantly suppressed the growth of HCC xenografts in nude mice (when compared with single agents alone). / In the third part, the anti-tumor effect of another mTOR inhibitor everolimus in combination with microtubule targeting agents, vinblastine and patupilone (a microtubule-stabilizing agent), was investigated in HCC cells. Everolimus/vinblastine combination resulted in an additive to synergistic effect accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage. The combination also caused concerted down-regulation of anti-apoptotic and survival proteins (survivin, Bel-2 and Mel-1) as observed with the temsirolimus/vinblastine combination. However, everolimus only moderately enhanced the sensitivity of patupilone for reasons unknown. / Taxanes are the major chemotherapeutic agents that target the microtubule. In the first part of the thesis, the anti-tumor activity of two taxanes, paclitaxel and docetaxel (which are known to stabilize microtubules) was examined and compared with doxorubicin (a DNA intercalating agent). Across all three HCC cell lines tested, it was found that the microtubule targeting agents, taxanes, were more efficacious than doxorubicin. This supports the initial finding that microtubule assembly process is functionally important in HCC. Recent studies demonstrated that using nanoparticles for drug delivery can greatly enhance therapeutic efficacy and reduce side-effects. Therefore, the nanoparticle albumin-bound (nab)-paclitaxel was employed to further evaluate the therapeutic efficacy of such a delivery strategy in HCC models. In all three HCC cell lines tested, nab-paclitaxel was found to be the most effective agent, with an average IC50 value of 0.16--10.42nM, when compared to non-conjugated taxanes (paclitaxel, docetaxel) and doxorubicin. In vitro analysis showed that nab-paclitaxel was able to induce cell cycle arrest at G2/M phase and apoptosis in HCC cells. In vivo study demonstrated that nab-paclitaxel readily inhibited the growth of HCC xenografts with lower toxicity when compared to paclitaxel, docetaxel and doxorubicin. Moreover, specific silencing of a key regulatory protein for microtubule dynamics, Stathmin 1, by siRNA significantly enhanced the effect of nab-paclitaxel in HCC cells, resulting in synergistic growth inhibition in vitro. / Zhou, Qian. / Advisers: Winnie Yeo; Vivian Lui; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 148-164). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 107-130).

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

The intracellular human pathogen Salmonella enterica serovar Typhi (S. typhi) causes the systemic disease known as typhoid fever. This disease afflicts approximately 17,000,000 people every year, of which over 600,000 cases are fatal. / Sequencing of the S. typhi genome has allowed a better understanding of the pathogenesis caused by this bacterium. In silico research on the genome sequence identified three open reading frames, termed prpZ gene cluster, present in the Ty2 and multi-drug resistant CT18 strains of S. typhi but absent in all other sequenced serovars of S. enterica. Further analysis of this gene cluster revealed that the three genes are transcribed as an operon that encodes two eukaryotic-like Ser/Thr kinases (PrkX and PrkY) and a protein phosphatase 2C (PP2C) (PrpZ). / A previous study has shown that the recombinant His-PrpZ protein has all the hallmarks of a PP2C. Typically, PP2Cs hydrolyze phosphoserine and phosphothreonine residues. In addition, His-PrpZ was found to hydrolyze phosphotyrosine residues, making it a dual specificity phosphatase. A subsequent investigation implicates the prpZ gene cluster in S. typhi virulence as the survival of a prpZ operon deletion mutant is compromised after 48 hours of macrophage infection when compared to wild type bacteria. / It is clear from these results that the prpZ operon plays a role in the pathogenesis of S. typhi. To determine the role of these three genes in virulence, an in vitro characterization of PrkY was carried out as well as an examination of the possible physiological roles of PrpZ. / We have demonstrated that PrkY is an active protein kinase capable of phosphorylating artificial substrates in the presence of Mg2+ and/or Mn2+. Optimal phosphorylation of substrates is achieved in the presence of 5mM Mg2+ at pH 8.0. In addition, we have identified a putative interaction between PrkY and PrpZ, leading to an inhibition of the kinase activity of PrkY. While exploring the possible physiological functions of PrpZ, we have found that this protein is secreted by Ty2 S. typhi in both LB and in the low pH, low phosphate and low Mg 2+ LPM medium. / These findings suggest that PrkY and PrpZ may have antagonistic effects in a S. typhi specific virulence pathway involved in the modulation of host cell signaling by secreted bacterial virulence factors.

Macrophages are active participants in many important biological processes, including antimicrobial activity, tumour surveillance, apoptotic cell clearance, homeostasis and wound healing. The activity of all cells is under the direct influence of their genetic makeup and macrophages are no exception. Natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1) is a macrophage-restricted gene that confers resistance to intracellular pathogens in mice. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to stressors, including the Toll-like receptor (TLR)-4 ligand LPS. Like NRAMP1, MK2 has also been shown to influence the efficiency of the antibacterial response. The present study evaluates the role of NRAMP1 and MK2 in TLR-mediated cytokine induction and their role in cutaneous wound healing. Mice lacking NRAMP1 are severely impaired in their rate of cutaneous wound healing. Nramp1 gene ablation has been associated with lower levels of SLPI, a protein previously demonstrated to influence the rate of wound healing in a non-redundant fashion. Macrophages derived from Nramp1-null mice are less efficient in activating p38 MAPK signaling, which results in lower levels of MK2 phosphorylation. The reduced level of p38 MAPK and MK2 activation in Nramp1-null macrophages also correlates with decreased cytokine induction in response to TLR7 ligand stimulation of these cells. Using p38 MAPK inhibitor and MK2-deficient macrophages, we demonstrate that TLR7- and TLR9-mediated cytokine induction is directly under the control of this signaling pathway. Furthermore, cytokine induction is regulated by MK2 at the post-transcriptional level. Macrophage-induced cytokines play an important role in cutaneous wound healing. Since MK2-deficient macrophages are severely impaired in their ability to induce cytokines following activation, we next evaluated the role of MK2 in cutaneous wound healing. Our results demonstrate that the rate of wound healing is significantly delayed in the absence of MK2. The level of cytokine expression in the wounds is impaired and macrophages are major players in cutaneous wound healing. Our data also show that intradermal transfer of macrophages with intact MK2 significantly improved wound healing kinetics. Overall, the studies presented in this dissertation demonstrate the importance of NRAMP1 and MK2 in the modulation of macrophage gene expression, and their important role in the control of cutaneous wound healing.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.