Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts. Despite intensive chemotherapy and allogeneic bone marrow transplantation, the treatment outcome of AML remains unsatisfactory, with a cure rate of only about 30%. Therefore, novel therapeutic strategies targeting the pathogenetic pathways of leukemia initiation and progression are needed. Using intracellular phospho-flow analysis with normal bone marrow as reference, we detected an increase in phosphorylated-STAT5 (pSTAT5) in three leukemic cell lines (K562, KG-1 and ML-2) and 15 primary AML samples. Treatment with specific JAK2 inhibitor TG101209 and JAK2/3 inhibitor AG490 significantly reduced pSTAT5 level and leukemia cell growth associated with an increase in apoptosis and decrease in cellular proliferation. The clonogenic activities of these leukemia cell lines were also significantly reduced. Furthermore, treatment with these inhibitors in K562 and KG-1 also significantly reduced the WNT signaling activity, as enumerated by the TOP/FLASH luciferase assay. In addition, genes associated with oncogenic potential and anti-apoptosis were significantly reduced, consistent with the pathogenetic role of JAK-STAT pathway. In summary, the present study highlighted the importance of the JAK2-STAT5 signaling pathway in sustaining AML. The results may open up a new avenue whereby new therapeutic strategies targeting AML can be designed. / published_or_final_version / Medicine / Master / Master of Philosophy

Liver kinase B1 (LKB1), a serine/threonine kinase, is responsible for the activation of AMP-activated protein kinase (AMPK), the master regulator of energy metabolism. LKB1/AMPK signaling pathway possesses a wide range of biological functions in regulating cell cycle progression, cell polarity, senescence and inflammation. In cultured endothelial cells, the pro-senescence function of LKB1/AMPK signaling pathway has been observed. However, the mechanisms by which LKB1 is regulated in endothelial cells remain largely uncharacterized. Furthermore, little is known about the effects of activated endothelial LKB1/AMPK signaling pathway on vascular and energy homeostasis. The present study aimed to investigate the upstream molecular mechanisms regulating LKB1 protein stability during endothelial senescence and the downstream pathophysiological effects of hyperactivated AMPK signaling in endothelial cells. In cultured model of cellular senescence, the lysine (K) 64 residue of LKB1 was found to be crucial for mediating its pro-senescence activities. The protein stability and intracellular localization of LKB1 mutant with K64 replaced by arginine (R) was different from the wild type protein. K64R exhibited enhanced effects on promoting endothelial senescence. Moreover, mutation of this residue attenuated the binding to HERC2, a newly identified E3 ubiquitin ligase for LKB1, in turn preventing its ubiquitination and degradation. Using a transgenic mouse model that selectively over-expresses a constitutively active AMPK α1 subunit (EC-AMPK) in endothelial cells, the influence of hyperactivated AMPK signaling on metabolic and vascular functions was investigated. Under standard chow condition, the metabolic phenotypes were similar between wild type and EC-AMPK mice; under high fat diet condition, EC-AMPK mice showed more severe obesity-induced fatty liver injury. Selective activation of AMPK in endothelial cells caused vascular and hepatic inflammation. Cyclooxygenase-2 (COX-2) was found to be the mediator for the pro-inflammatory functions of AMPK in vascular endothelial cells and facilitated to the development of obesity-induced fatty liver injury in EC-AMPK mice. Evaluation using isolated arteries revealed an increased systolic blood pressure and abnormal endothelial function in EC-AMPK miceunder high fat diet. AMPK activation in endothelium of the blood vessel could not block vascular remodeling associated with dietary obesity. Taken in conjunction, the above findings suggest that continuous activation of LKB1/AMPK signaling elicits adverse effects on both energy and vascular homeostasis. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Esophageal cancer is the third most common digestive tract malignancy. Along with surgery, genotoxic drugs (e.g. cisplatin) and radiotherapy are the mainstays of treatment for this disease. Environmental factors and environmental stress-induced responses contribute to esophageal tumorigenesis and chemoresistance. Studying key molecules in stress-induced signal pathway can help unravel the underlying mechanisms and discover rational therapeutic targets. Cyclin D1 is DNA damage response protein. Genotoxic stress induces rapid cyclin D1 degradation and the molecules mediating this response are cell-type dependent. The first part of this study investigated the changes of cyclin D1 expression in response to genotoxic stress in immortalized esophageal epithelial cells, which are experimental models commonly used to study the early events of cancer development. The results showed that cyclin D1 underwent rapid proteasomal degradation before p53-induced p21 accumulation, which substantiates that cyclin D1 plays a role in eliciting cell cycle arrest very early in the DNA damage response. FBXO31 and FBX4, two F-box proteins previously reported to mediate cyclin D1 degradation, were found to be accumulated and unchanged, respectively, after ionizing irradiation in immortalized esophageal epithelial cells and esophageal squamous cell carcinoma (ESCC) cell lines. Yet, knockdown of FBXO31 did not rescue rapid cyclin D1 degradation upon UV or ionizing irradiation. This led to the hypothesis that accumulation of FBXO31 may have novel functions beyond mediating cyclin D1 degradation in cells responding to genotoxic stress. The second part of this study explored the function of FBXO31 in genotoxic stress response. The accumulation of FBXO31 in cancer cells after exposure to various genotoxic stresses was found to coincide with p38 deactivation, giving the clue that FBXO31 may negatively regulate this important pathway. Further studies revealed that knockdown of FBXO31 resulted in sustained activation of stress-activated MAPKs (SAPKs) p38 and JNK, as well as increase in UV-induced cell apoptosis, whereas overexpression of FBXO31 had opposite effects. The inhibitory role of FBXO31 on SAPK activation and apoptosis was confirmed by shRNA rescue experiments. Consistent with the observed anti-apoptotic effect, soft agar, colony formation and in vivo xenograft experiments showed that FBXO31 had oncogenic function in ESCC. Moreover, in vitro and in vivo results showed that knockdown of FBXO31 could sensitize ESCC cells to cisplatin treatment. The mechanism underlying the inhibition of SAPKs by FBXO31 was investigated in the third part of this study. Co-immunoprecipitation results showed that FBXO31 could interact with MKK6 (a p38 activator), but not p38, JNK1, or other MAP2Ks. FBXO31 was found to be co-localized with MKK6 in the cytoplasm. Mapping of interaction domains of FBXO31 revealed that aa 115-240 and aa 351-475 were responsible for binding to MKK6. Further study found that binding of FBXO31 to MKK6 could facilitate the K48-linked polyubiquitination and degradation of MKK6. Taken together, the results of this study showed that FBXO31 accumulation upon genotoxic stress can promote the degradation of MKK6 via K48-linked ubiquitination, thereby inhibiting SAPK activation and protecting cancer cells from genotoxic stress-induced apoptosis. FBXO31 may be a potentially useful therapeutic target to overcome chemoresistance in cancer therapy. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Presenilin (PS) is the catalytic subunit of the gamma-secretase which is responsible for the cleavage of amyloid precursor protein to form beta amyloid (Aβ). Mutations in PS cause familial Alzheimer’s disease (FAD) by increasing the Aβ plaques formation in the brain and thereby induce neurodegeneration. Apart from this, FAD-linked PS mutations have been demonstrated to disrupt cellular calcium (Ca2+) homeostasis. Ca2+is a vital secondary messenger that involved in various neurophysiological functions, including memory, learning, and neuroplasticity and mounting evidence suggesting that Ca2+dysregulation associated with PS mutations may play a proximal role in the AD pathogenesis. Yet, the molecular mechanism for Ca2+dysregulation in AD remains debatable. It has been reported that cellular Ca2+homeostatsis can be disrupted in various ways. On the one hand, mutant PS has been demonstrated to exaggerate Ca2+release from the endoplasmic reticulum (ER) through different pathways. On the other hand, attenuatedCa2+influx from the extracellular medium through the capacitative Ca2+entry (CCE) pathway has also been reported to bring about cellular Ca2+disruption. However, the molecular mechanism for the PS mutation-mediated CCE deficits is largely unknown. For this reason, the objective of the current study is to elucidate the underlying molecular mechanism for attenuated CCE in AD. In this study, human neuronal cell line SH-SY5Y is employed as a cellular model to investigate the effect of wild-type or FAD-linked PS1 mutation on CCE pathway. Using single cell Ca2+imaging technique, significant CCE deficits was observed in SH-SY5Y stably expressing FAD-linked PS1mutation, PS1M146L. Interestingly, this CCE attenuation in PS1 mutant expressing cells was not mediated by the down-regulation of STIM1 and Orai1 expression, the known essential molecular players in the CCE pathway. Instead, co-immunoprecipitation and proximity ligation assay have suggested a physical interaction between PS1 and STIM1 proteins. Moreover, a putative gamma-secretase mediated STIM1 cleavage was discovered by western blotting. In addition, confocal imaging showed that PS1M146L significantlyreduceSTIM1 puncta formation and ER translocation followed by the activation of CCE pathway by ER Ca2+store depletion with thapsigargin. This indicated that mutant PS1 attenuates CCE by affecting STIM1 oligomerization or its recruitment with Orai1. Taken together, our results suggested the negative regulatory role of PS on CCE pathway and hypothesized the molecular mechanism of CCE where FAD-linked PS mutation is perceived as a gain-of-function mutation and enhanced the negative impact on STIM1 to inhibit Ca2+entry.This hypothetic model provides new insights into the molecular regulation for CCE pathway and the identification of the interacting domains between PS1 and STIM1 may suggest novel targets for the development of therapeutic agents that help to treat the disease. / published_or_final_version / Physiology / Master / Master of Philosophy

Human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs) for disease modeling, cardiotoxicity screening, drug discovery and cell‐based therapies. However, as shown in previous studies conducted by our lab (Poon, Kong et al. 2011), human embryonic stem cells (hESCs)‐derived CMs display immature〖Ca〗^(2+)–handing properties with smaller transient amplitudes, slower rise and decay kinetics than those of adult CMs. Although the cytosolic 〖Ca〗^(2+) signaling of hESC‐CMs has only recently been understood, there is no investigation on the nuclear 〖Ca〗^(2+) signal in hESC‐CMs, despite its importance. In this dissertation, delayed kinetics of nuclear 〖Ca〗^(2+), as compared to that of cytosol during 〖Ca〗^(2+)waves or 〖Ca〗^(2+) transients, was found in hESC‐derived ventricular (V) CMs, indicating that nuclear 〖Ca〗^(2+) was initiated by 〖Ca〗^(2+) diffusion from cytosol. Besides global 〖Ca〗^(2+) signals, local nuclear 〖Ca〗^(2+) signals were observed and identified as Ca2+ release from ryanodine receptors (RyRs), and nucleoplasmic reticulum (NR) served as their structural basis. In addition, targeted expression of 〖Ca〗^(2+) buffering protein parvalbumin (PV) in cytosol or nucleus altered 〖Ca〗^(2+) transient and stimuli‐induced apoptosis of hESC‐VCMs. For cytosolic 〖Ca〗^(2+) signaling in hESC‐VCMs, the mechanistic basis of excitation‐contraction coupling of hESC‐VCMs was studied by using 〖Ca〗^(2+) sparks, which are the unitary 〖Ca〗^(2+) ‐events. The results indicated that RyRs could be sensitized by 〖Ca〗^(2+) in permeabilized hESC‐VCMs. Increasing external 〖Ca〗^(2+) dramatically escalated the basal 〖Ca〗^(2+) and spark frequency. Furthermore, RyR‐mediated Ca2+ release sensitized nearby RyRs, leading to compound 〖Ca〗^(2+) sparks, whereas inhibition of mitochondrial 〖Ca〗^(2+) + uptake promoted Ca2+ waves. The aforementioned immature 〖Ca〗^(2+)–handing properties of hESC‐CMs can be attributed to their differential expression of crucial Ca2+–handling proteins. During diastole, SERCA and NCX sequester and extrude 〖Ca〗^(2+) ions, respectively, to return cytosolic 〖Ca〗^(2+) to the resting level. As previously published in our lab, NCX, robustly expressed in hESC‐CMs but much less so in the adult counterparts, is a functional determinant of immature 〖Ca〗^(2+) homeostasis. Unlike NCX, SERCA is expressed less in hESC‐CMs than in adult‐CMs. The present study first demonstrated the effects of lentivirus‐based genetic manipulation of SERCA2a and NCX1 in hESC‐VCMs, and the results indicated that SERCA2a overexpression shortened the decay phase of low‐frequency (0.5 Hz) electrical stimulation‐elicited Ca2+ transient. Increasing pacing frequency from 0.5 Hz to 2 Hz led to a decrease of relative transient amplitude, showing that hESC‐VCMs harbored a negative‐frequency response. At a high‐stimulation frequency of 2 Hz, it was revealed that SERCA overexpression, but not NCX1 suppression, increased the amplitude of 〖Ca〗^(2+) transient by accelerating 〖Ca〗^(2+) sequestration to sarcoplasmic reticulum (SR), indicating partial rescue of the negative‐frequency response. Taken collectively, the findings provide 1) novel information on nuclear 〖Ca〗^(2+) signaling in hESC‐VCMs, 2) the first lines of direct evidence that hESC‐VCMs have functional 〖Ca〗^(2+)‐induced‐〖Ca〗^(2+)+‐release (CICR), and 3) evidence of driving hESC‐VCMs maturation by SERCA2a overexpression, which may facilitate clinical and other applications of hESC‐VCMs. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

abstract / Biochemistry / Doctoral / Doctor of Philosophy

abstract / Biochemistry / Master / Master of Philosophy

Not available / text

Prolonged, heavy consumption of alcohol produces marked neuroadaptations in excitatory neurotransmission. These effects are accelerated following patterns of intermittent heavy drinking wherein periods of heavy consumption are followed by periods of abstinence. Studies have shown that neuroadaptive changes in the glutamatergic N-methyl-D-aspartate (NMDA) receptor produces excitotoxicity during periods of withdrawal; however, upstream targets were not adequately characterized. The present studies sought to identify these targets by assessing the role of group 1 metabotropic glutamate receptors (mGluR) and intracellular calcium in promoting cytotoxicity of hippocampal cell layers in vitro. It was hypothesized that ethanol-induced activity of mGluR1-and-5 contributes to hippocampal cytotoxicity and promotes the behavioral effects of withdrawal in vivo. In order to identify and test this theory, rat hippocampal explants were co-exposed to chronic intermittent ethanol exposure with or without the addition of a group 1 mGluR antagonist to assess cytotoxicity in neuronal cell types. In a second study, adult male rodents were co-exposed to chronic intermittent ethanol exposure with or without the addition of an mGluR5 antagonist to assess the role of these receptors in the development of dependence as reflected in withdrawal behaviors. Together, these studies help to identify and screen toxicity of putative pharmacotherapies for the treatment of ethanol dependence in the clinical population.

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy