Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 94-101). Also available in electronic version.

No description available.

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "May, 2008." Includes bibliographical references (leaves 117-133). Online version available on the World Wide Web.

Τα stem κύτταρα είναι ένας υποπληθυσμός κυττάρων με δύο κύριες ιδιότητες: αυτοανανέωση και διαφοροποίηση. Τα stem κύτταρα διαμένουν σε ένα εξειδικευμένο μικροπεριβάλλον, την φωλεά, η οποία παίζει σημαντικό ρόλο στη διατήρηση της ισορροπίας μεταξύ της αυτοανανέωσης και της διαφοροποίησης. Τελευταία δεδομένα εισηγούνται ότι ο καρκίνος αναπτύσσεται από ένα υποσύνολο κυττάρων με ιδιότητες ανάλογες αυτών των φυσιολογικών stem κυττάρων. Τα κύτταρα αυτά αποκαλούνται καρκινικά stem κύτταρα. Η θεωρία των καρκίνικών stem κυττάρων υποστηρίζει ότι τα καρκινικά stem κύτταρα εγκαινιάζουν και συντηρούν την ανάπτυξη και εξέλιξη του όγκου, ευθύνονται για την κυτταρική ετερογένεια των καρκίνικών κυττάρων του όγκου, είναι υπεύθυνα για τις μεταστάσεις και παραμένουν στους ασθενείς παρά τη χρήση των συμβατικών χημειοθεραπευτικών παραγόντων. Πρόσφατα δεδομένα πιστοποιούν την ύπαρξη καρκινικών stem κυττάρων στην ωοθήκη. / Stem cells are a subpopulation of cells with two key properties: self-renewal and differentiation. Stem cells reside in a specialized microenvironment, i.e. niche, which plays an important role in the balance between self-renewal and differentiation. Recent data suggest that cancer develops from a subset of cells with properties similar to those of normal stem cells. These cells are called cancer stem cells. Cancer stem cell hypothesis suggest that cancer stem cells initiate and preserve the growth of tumor, they are responsible for cellular heterogeneity and metastasis of tumor and they are, finally, drug-resistant.Latest data suggest the presence of cancer stem cells in the ovary.

Prostate cancer is one of the most common cancers affecting one of every six men in United States. It is increasingly appreciated that tumor or cancer stem cells are the cells responsible for initiating tumor formation and therefore should be targeted for eradication in cancer treatment. But the mechanism involved in the acquisition of unlimited self-renewal and tumor initiation by cancer stem cells is unknown. Nanog, along with Oct3/4 and Sox-2, constitute the core transcriptional circuitry for the maintenance of stemness in embryonic stem cells. Herein we report that Nanog expression was detected at mRNA and protein levels in prostate cancer cells. The Nanog-expressing LNCaP-T and DU145 cells were enriched by infection with lentiviruses expressing GFP under the control of Nanog promoter. The Nanog-enriched prostate cancer cells had stronger expressions of stem and progenitor cell surface markers, including CD44 and CD133, when compared with those in the control group. Colony formation assay found that the Nanog-enriched LNCaP-T and DU145 cells formed more holoclones and prosta-spheres, which contained more self-renewing cells, than the control cells did. On the other hand, knockdown of Nanog in DU145 or LNCaP-T cells, via shRNAs, reduced their ability to form holoclones. Instead, most clones derived were meroclone and paraclones as result of increased differentiation and senescence due to knockdown of Nanog. When injected into mice, Nanog-enriched DU145 cells were found to possess increased tumorigenic potentials when compared to the vector controls. On the other hand, LNCaP-T cells with Nanog knocked down did not form tumors, while the vector controls readily formed tumors. Taken together, our data suggest an essential role for Nanog in the self-renewal and tumor initiation of prostate cancer cells. Chemotherapy is the major salvage therapeutic modality available for the patients with advanced cancers. However, drug resistance by some prostate cancer cells is a major barrier to efficacious chemotherapy. It has been increasingly appreciated that cancer stem cells are responsible for resistance to chemo- or radio-therapy, in addition to tumor initiation. However, the mechanisms involved remain unknown. In this study, we examined whether Nanog plays an essential role of Nanog in resistance to chemotherapy. In the surviving fractions of prostate cancer cells, we found increased levels of Nanog protein when compared to the cells treated with solvent control. To determine the role of Nanog in resistance of prostate cancer cells, we marked and enriched Nanog-expressing prostate cancer DU145 and LNCaP-T cells using a reporter gene under control of 2.5 kb hNanog1 promoter. When compared to the control, the prospectively enriched Nanog-expressing cells presented increased resistance to Taxol, vinblastine, and doxorubicin. Profiling of genes in drug resistance and metabolism revealed a marked increase in the mRNA level of ATP-binding cassette (ABC) efflux transporters B1 and G2 in tumor cells enriched with endogenous Nanog expression. The increased expression of ABCB1 and ABCG2 at protein levels in Nanog expressing cells was confirmed by Western blot and immunocytochemistry. Inhibition of ABCB1 activities sensitized Nanog expressing cells toward Taxol and vinblastine, and to less extent, doxorubicin. Blocking of ABCG2 activity sensitized Nanog expressing cells toward doxorubicin, but not Taxol and vinblastine. In addition, the tumor cells enriched with Nanog expression showed reduced apoptosis in response to Taxol treatment. Interestingly, Nanog-enriched prostate carcinoma cells displayed aberrantly activated â-catenin signaling, which is potentially associated with their increased chemo-resistant ability as well as the increased acquisition of epithelial to mesenchymal transition. In summary, Nanog is expressed in prostate cancer cells, especially in those positive for stem/progenitor markers. Enrichment of Nanog expressing cells led to enrichment of tumor cells with increased tumor initiating ability and increased resistance toward chemotherapy. Knockdown of Nanog reduces tumor initiating ability of prostate cancer cells and further sensitizes them toward chemotherapy. The gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to initiate tumor formation and resist chemotherapy.

Bioactive lipids have emerged as prominent regulators of neural stem and progenitor cell (NPC) function under both physiological and pathological conditions. However, how lipid metabolism is regulated, and its role in modulation of NPC function remains unknown. In this regard, my study defines a novel epigenetic pathway that regulates lipid metabolism to determine NPC proliferation versus differentiation. Specifically, I show that activation of an atypical protein kinase C (aPKC)-mediated Ser436 phosphorylation of CREB binding protein (CBP) by aging, metformin stimulation and continued passaging in vitro, represses expression of monoacylglycerol lipase (Mgll) to promote neuronal differentiation of adult NPCs. Mgll, a lipase that hydrolyzes the endocannabinoid 2-arachidonoyl glycerol (2-AG) to produce arachidonic acid (ARA), is thus a key regulator of two critical bioactive lipid signaling pathways in the brain and a potential modulator of NPC function. I observed elevated Mgll levels, concomitant with neuronal differentiation deficits in both the lateral ventricle sub-ventricular zone (SVZ) and the hippocampal subgranular zone (SGZ) NPCs of phospho-null CBPS436A mice, that lack a functional aPKC-CBP pathway. Genetic knockdown of Mgll or inhibition of Mgll activity rescued these neuronal differentiation deficits. In addition, I found that CBPS436A SVZ NPCs exhibit enhanced proliferation at the expense of differentiation as an outcome of increased Mgll levels in culture. Interestingly, I also observed that SVZ NPCs from an Alzheimer’s disease (AD) model, the 3xTg mice, closely resemble CBPS436A NPC behaviour in culture. 3xTg NPCs exhibit attenuation of the aPKC-CBP pathway, which is associated with elevated Mgll expression and increased NPC proliferation at the expense of neuronal differentiation. Reactivation of the aPKC-CBP mediated-Mgll repression in 3xTg AD NPCs mitigates their differentiation deficits. These findings implicate Mgll as a critical switch that regulates NPC function by altering bioactive lipid signaling (2-AG versus ARA). They demonstrate that the aPKC-CBP mediated Mgll repression is essential for normal NPC function, and that when perturbed in AD, it causes impaired NPC function to generate fewer neurons, contributing to AD predisposition.

Thesis (Ed.D.)--Boston University

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases, in part by modulating adaptive and innate immune responses. Since metabolism governs the phenotype and function of immune cells, the aim of this thesis was to investigate whether NSCs have the ability to regulate the immunometabolic components underpinning neuroinflammation. Herein I have identified a new mechanism by which transplanted somatic and directly-induced NSCs counteract CNS-compartmentalised chronic inflammation in mice. NSC transplantation reduces the immunometabolite succinate in the cerebrospinal fluid, while decreasing the burden of mononuclear phagocyte (MP) infiltration and secondary CNS damage. Mechanistically, the anti-inflammatory activity of NSCs arises in response to succinate released by inflammatory MPs, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, thus initiating prostaglandin E2 secretion and extracellular succinate scavenging. This work uncovers a succinate-SUCNR1 axis in NSCs that clarifies how stem cells respond to inflammatory metabolic signals to inhibit the activation of pro-inflammatory MPs in the chronically inflamed brain.

Polydopamine (PDA), a unique bioinspired polymer, has been a subject of interest in fields including orthopedic biomaterials and antibacterial surfaces. Its osteogenic effects and ability to control surface traits through precise variables (e.g., pH, temperature) have led to its use as a coating in the enhancement of a wide range of materials, including metals and ceramics. In this Thesis, two studies were carried out to better understand the capability and mechanism of PDA-mediated bioactivity. In the first study, we investigated whether PDA coatings can further enhance the bioactivity of nanoporous Titanium (NPTi). While physicochemical traits were in line with literature, PDA was effective in enhancing cell proliferation, beyond NPTi, as early as 8 hours with enhancement in cell spreading and focal adhesion prevalence as early as 1 hour. No changes in adsorptive capacity were found, suggesting a serum-independent component (SIC) of the surface. The second study was focused on (1) determining how treatment parameters influence the physiochemical makeup of PDA surfaces, (2) assessing how PDA surfaces influence stem cell behavior and (3) confirming and investigating the SIC of PDA effect. Results confirm that there is indeed a SIC of PDA coatings with enhancement in cell spreading that improves with the increased size and density of PDA particles. Our findings show that the SIC works in concert with circulating sera to elicit the bioactive effects of PDA. The novel rPDA surface, obtained by adding rotation during the coating deposition, is also shown to elevate bioactivity during normal culturing, beyond classical coatings, with ongoing work suggesting enhancement in the osteogenic differentiation of hMSCs. Taken together, this work has demonstrated novel aspects underlying the potential and mechanism of action for the bioactivity of PDA, ultimately providing new evidence supporting the use of PDA as a biomedical material.

Mesoderm is one of the three primary germ layers from which the cardiovascular system, muscle and bone originate and derivatives of the mesoderm lineage are affected in a number of pathologies. Therefore, understanding the mechanisms regulating formation of mesoderm is interesting for a diversity of diseases and clinical application. In vivo study of human development beyond gastrulation is technically challenging and the mechanisms controlling mesoderm specification are difficult to study since the maximum number of days allowed to grow human embryos is 14 days. Thus, in this dissertation I use human pluripotent stem cells (hPSCs) as a simplified model of human development. Studies have shown that the cell cycle machinery plays a direct role in the differentiation of endoderm and ectoderm lineages but its role in guiding mesoderm subtype formation remains elusive. In this dissertation, I provide new insights of the importance of the cell cycle regulators in mesoderm specification. I first developed tools such as the FUCCI2A reporter line to isolate cells in the different cell cycle phases and to investigate propensity of mesoderm differentiation. I have shown that the propensity of differentiation into the three mesoderm subtypes lateral plate mesoderm, cardiac mesoderm and presomitic mesoderm varies during the cell cycle phases, with differentiation being more efficient in the G1 and to a lesser extend in G2/M phase. Furthermore, I developed a protocol where cells can be efficiently synchronised in the different cell cycle phases using the G2/M inhibitor nocodazole. Using this tool, I showed that developmental signalling pathways such as BMP and WNT are active in all cell cycle phases indicating that alternative mechanisms are involved in the differentiation process. In order to further explore these mechanisms, I investigated the role of cell cycle regulators controlling the G1 and G2 checkpoint. I have shown that the cell cycle regulators CDK4/6, CDK2, Retinoblastoma phosphorylation and CDK1 are essential for mesoderm subtype formation. Furthermore, I have shown that CDK1 regulates the activity of ERK1/2 signalling, an important pathway for the differentiation process confirming the existence of complex interplays between cell cycle machinery, signalling pathways and transcription factors in mesoderm subtype formation. This knowledge will be useful to further improve protocols for generating mesoderm subtypes from hPSCs for clinical applications such as drug screening, disease modelling and cell based therapy.