In the field of integrative oncology, polyphenols have gained attention for their ability to modulate key signaling pathways involved in breast cancer prevention. One noteworthy product, the Polyphenol-Enriched Blueberry Preparation (PEBP), produced by the fermentation of blueberries by the bacterium Rouxiella badensis subsp. acadiensis has demonstrated various beneficial properties, including anti-inflammatory effects and the ability to control cancer stem cells. Cancer Stem Cells (CSCs) are highly tumorigenic cells involved in carcinogenesis and can cause relapses. MicroRNAs (miRNAs) can act as regulators of CSCs, by controlling stemness and invasion. We postulate that PEBP or its polyphenolic components induce specific epigenetic changes by modulating miRNA networks, reducing CSCs, and preventing breast carcinoma. Thus, the overarching aim of this thesis is to better understand the mechanisms underlying the protective effects of the polyphenol-enriched preparation in mitigating breast cancer. The objectives are: 1. To investigate the chemopreventive effects of PEBP on breast cancer stem cell development in cell models and in vivo, as well as to study the involvement of STAT3 and MAPKs signaling pathways 2. Assess the impact of the polyphenol-enriched blueberry preparation on breast cancer by regulating the expression signatures of miRNA involved with cell proliferation, survival, and CSC self-renewal pathways in in vitro experiments. 3. Characterize PEBP and investigate the effect of a subset of its components on miRNA expression. Furthermore, validate the role of those components in regulating the functional behavior of breast cancer stem cells through experiments using a 4T1 animal model. The results have shown a decrease in the formation of CSCs by delaying the development of tumors in vivo, decreasing metastasis to the lungs, and controlling the PTEN/PI3K/AKT axis, a central node in CSC signaling and homeostasis. In addition, several miRNAs associated with different clinical-pathological characteristics of breast cancer were shown to be differentially expressed in CSCs after exposure to PEBP. Notably, the expression of hypoxamir miR-210, associated with a poor prognosis in breast cancer patients, was downregulated, while tumor suppressor miR-145, which prevents metastasis through FOXO1 was over-expressed. The chemopreventive potential of a polyphenolic mixture containing PCA, gallic acid, and catechin, found in PEBP, was also shown to successfully reduce tumour growth and metastasis in our animal model and decrease the presence of stem-like tumour cells by favouring the upregulation of tumour-suppressor miR-145. These findings provide novel evidence in translational medicine, highlighting the effectiveness of a natural epigenetic modulator in chemoprevention by specifically targeting CSCs.

The recent discovery of adult neural precursor cells (NPCs) in the dentate gyrus and the subventricular zone of the lateral ventricles of most mammals holds much hope for the potential regeneration of damaged brain tissue. However, their use has been limited by their low numbers and relatively quiescent state, particularly in the aging brain. Previous studies from our laboratory have demonstrated a crucial role for the Rb/E2F pathway in the regulation and proliferation of NPCs, and the direct mechanistic involvement of E2F3 in regulating the pluripotency factor, Sox2. More recently, our investigations into the roles of E2F1 and E2F3 in during adult neurogenesis have revealed that loss of both these genes results in a dramatic loss of adult NPCs. Here, we have employed the Emx1-Cre and Nestin-CreERT2 transgenic models, to specifically delete E2F1 and E2F3 in the cerebral cortex and in NPCs in order to investigate the role of both these genes in embryonic neurogenesis. Our results suggest a switch in the requirement for both E2Fs 1 and 3 between embryonic and adult NPCs, demonstrated by a decrease in NPC proliferation and numbers starting only during late embryonic development and persisting through postnatal neurogenesis. These findings suggest that E2Fs 1 and 3 are essential for the maintenance of stem cells and neurogenesis in the adult brain. Moreover, their deletion results in defects in learning and memory. These studies reveal a crucial role for activating E2Fs in the long-term maintenance and proliferation of neural stem cells.

Many countries in Latin America, for cultural, religious and regulatory reasons, have struggled and failed to appear as competent players in the global bio-economy of emerging technologies in the biosciences field. This investigation takes Mexico as a country case study to map out the factors hampering the development of the governance of emergent biomedical biotechnologies in this context, particularly that applied to stem cell science. This research aims to contextualise and portray prevailing ethical, legal, political and religious concerns regarding stem cell research in this context. Exploring the debates in these arenas, it seeks to elucidate the perceptions of key stakeholders and to appraise critically the divergences and convergences among the actors who currently shape the debate and who may have significant influence on the creation of any legislative framework in the area. It explores whether it is feasible to draw on the approach taken to stem cell science and tissue regulation in the United Kingdom, in order to illuminate the way forward for governing stem cell research and its clinical applications in Mexico. It also aims to evaluate the risks posed by the persistent lack of regulation in this scientific field, since Mexico appears to be an ideal destination for stem cell tourism among Latin American countries. Drawing on empirical data gathered from prominent Mexican stakeholders in the stem cell issue, this research elucidates the key themes influencing the debate which need to be addressed in detail in order to prepare the ground for the effective governance of stem cell science and its clinical applications. By detailing the emergent themes and providing reflexive explanations of the elements influencing the views of all the actors in this arena, this thesis aims to provide ethical, empirical and normative proposals to be translated by policymakers into purposive regulation of biomedical innovations. Thus, it delineates two main features of the debate over stem cell science regulation in Mexico and shows the urgent need to create a legal framework to deal with problematic situations provoked by the legal vacuum in this area: a) the legal inertia preponderant in the Federal Congress, which is mainly caused by the constant lobbying of politicians by the Roman Catholic hierarchy to endorse prohibitive policies in sensitive areas, such as sexual matters, reproduction and stem cell science; b) the increasing phenomenon of stem cell tourism in the country, requiring the adoption of ethical and legal measures to avoid potential physical and financial harm to desperate patients who seek stem cell treatments.In conclusion, I argue that it is plausible to advance a permissive model of governance for the area of stem cell science. This thesis is supported by the evidence gathered from stakeholders’ opinions, added to the data emanating from the analysis of the country case study. As a result, it is possible to propose as an initial strategy the adoption of significant regulatory features of the paradigmatic system of governance which applies in the United Kingdom. The law is up to date as of 19 June 2012.

No description available.

Pluripotency is defined as the ability of a cell to give rise to all the cell types of the adult organism. In vivo this property is possessed transiently by the cells of the epiblast in the developing embryo but it can be maintained indefinitely by deriving embryonic stem (ES) cells. How the pluripotent state is established in the cells of the early embryo and how it is ‘captured’ and maintained in the form of ES cells is a fascinating question for biology with practical implications. It is hoped that ES cells will be of use in biomedical research and cell replacement therapy. Our understanding of their biology and our ability to manipulate the cells in vitro will be of great importance if these hopes are to be realised. The starting point for the work presented in this thesis was the development of a novel culture condition for the derivation and maintenance of mouse ES cells (Q-L. Ying and J. Nichols). The media is formed by the addition of three small molecule inhibitors to a previously described serum-free media, N2B27, and is termed 3i (three inhibitors).The inhibitors are SU5402, PD184352 and CHIRON99021, and they inhibit the FGF receptor, mitogen activated protein/extracellular signalregulated kinase (ERK) kinase (MEK), and glycogen synthase kinase 3 (GSK3) respectively. I attempt to further our understanding of pluripotency and self-renewal in ES cells by genetic and biochemical examination of ES cells cultured in 3i. Analysis of intracellular signalling pathways together with descriptions of genetic mutants for the targets of the inhibitors validates the mode of action and the specificity of the three inhibitors. Self-renewal of mouse ES cells is considered dependent on activation of STAT3 through provision of the cytokine leukaemia inhibitory factor (LIF). I demonstrate unequivocally that this pathway is not required for self-renewal in 3i by characterising Stat3-null ES cells. Further experiments reveal that preventing activation of ERK downstream of the growth factor FGF4, produced by the ES cells themselves, is key to preventing differentiation. Pleiotropic effects of GSK3 inhibition are observed and candidate GSK3 targets with known or predicted effects on self-renewal are investigated as potential downstream effectors. I propose that activation of canonical Wnt signalling, together with a global derepression of biosynthetic capacity, mediate the pro-self-renewal effects of GSK3 inhibition. The description of culture conditions that function independently of signalling pathways previously thought essential for self-renewal provides fresh insight into the nature of ES cell self-renewal and the relationship of ES cells to the pluripotent cells of the developing embryo. There are practical implications for ES cell biology as there is reason to hope that the new conditions will translate more readily to other mammalian species to facilitate the derivation of ES cells and will provide an optimal platform for differentiation of ES cells into somatic cell types of interest.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder associated with premature aging in various tissues and organs of the afflicted individuals, including accelerated skeletal muscle atrophy. Classical HGPS manifests due to single-base substitution in the LAMNA gene which encodes Lamin A/C proteins. As a result of the mutation, a truncated form of Lamin (known as Progerin) is produced which undergoes persistent farnesylation during post-translational modification. Accumulation of Progerin in the nucleus has been linked to various cellular abnormalities including abnormal nuclear morphologies and altered chromatin organization, among others. However, the exact molecular mechanisms leading to skeletal muscle atrophy have not yet been elucidated. In this study, the iPSC approach was implemented in order to study the skeletal muscle phenotype of HGPS by generating and characterizing a population of Pax7 positive skeletal muscle precursor cells (SMPs). During the course of this project, we have demonstrated the need for excessive optimization of the previously developed directed differentiation protocol for successful application on induced Pluripotent Stem Cells. Furthermore, we have successfully modified the protocol to allow for a more rapid expansion of the SMPs through regular passaging of the myogenic cells starting on day 20 of differentiation. Additionally, this new method produced more uniform distribution of the myogenic cells and allowed for successful freezing/thawing of the myogenic cells. When compared to the controls, the HGPS-derived SMPs did not appear to be defective in formation, proliferation or differentiation. Abnormal nuclear morphology and DNA damage, documented in HGPS fibroblasts and vascular smooth muscle cells, were not detected the in myogenic cells. Furthermore, we were not able to detect Progerin protein accumulation in the generated myogenic cultures, offering an explanation for the absence of these phenotypes in the skeletal muscle system.

Tsang Kam Sze Kent. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 277-339). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Haematopoietic stem cells (HSCs) are capable of differentiation into all mature haematopoietic lineages, as well as long-term self-renewal and are consequently able to sustain the adult haematopoietic system throughout life. Currently, in the mouse, HSCs are understood to first appear in the aorta-gonad-mesonephros (AGM) region at embryonic day 11 via a process of maturation from precursors (pre-HSCs). This maturation within the AGM region involves the complex interplay of signalling between cells of the niche and maturing precursor cell populations, but is relatively little understood at a molecular level. Recently our understanding of the AGM region has been refined, identifying the progression from E9.5 to E10.5 and the polarity along the dorso-ventral axis as clear demarcations of the supportive environment for HSC maturation. In this thesis, I investigated the molecular characteristics of these spatio-temporal transitions in the AGM region through the application of RNA-sequencing. This enabled the identification of molecular signatures which may underlie the supportive functionality of the niche. I further compared these expression signatures to the transcriptional profile of an independent cell type, also capable of supporting HSC maturation, the OP9 stromal cell line. By combining this transcriptional information with an ex vivo culture system, I screened a number of molecules for their ability to support HSC maturation from early precursors, leading to the discovery of a novel regulator of HSC maturation: BMPER. Further characterisation of this molecule enabled the identification of its specific cellular source and the proposal that through its action as an inhibitor of BMP signalling it facilitates the maturation of precursors into HSCs. These results lend further detail and support to the role of BMP signalling in the regulation of HSC maturation as well as demonstrating the potential of these transcriptional profiles to yield novel mechanistic insight.

In recent years, research in the area of cancer stem cells has spiked tremendously. Numerous investigators have found that several types of cancers contain a subpopulation of tumor cells that display many defining characteristics of normal tissue stem cells, including multipotent differentiation potential, long-term self-renewal capacity, and expression of molecular markers of stemness. Most importantly, these cancer stem cells (CSCs) have very high tumor initiating potential, a finding that has led to the development of the cancer stem cell model for tumor progression. This model suggests that tumors are organized in a developmental hierarchy (similar to healthy tissue), with long-term tumor progression being driven by self-renewing CSCs at the top of the hierarchy. The CSC model represents a significant shift in our understanding of tumor progression, and as such, it may be possible to expand our knowledge of other aspects of tumor biology by re-examining them in the context of the CSC model. My work focuses on investigating interactions between CSCs and the tumor vasculature. Previous work has demonstrated heterogeneity in the proangiogenic potential of cells in a tumor. Considering the possibility that angiogenesis may be driven by specific subsets of tumor cells, I investigated the contribution of the CSC fraction to tumor angiogenesis. Comparing tumors with low or high CSC fractions, I have found that CSCs contribute to tumor vascular development through promotion of endothelial cell activity and recruitment of bone marrow-derived proangiogenic cells, mediated in part by vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF1). Since some tissue stem cells are known to reside in a vascular niche, I investigated the possibility that CSCs may also be supported by blood vessels in the tumor microenvironment, and that consequently CSCs may be targeted by disruption of tumor vasculature with antiangiogenic therapy. By testing multiple antiangiogenic therapeutic strategies, I have found that antiangiogenic therapy sensitizes CSCs to the effects of cytotoxic chemotherapy. Taken together, my work demonstrates a bi-directional relationship in which CSCs promote tumor vascular development, and tumor vasculature supports and protects CSCs. This work has implications for our understanding of CSC biology, tumor angiogenesis and antiangiogenic therapy, and provides insight into strategies for targeting the critical CSC population.

In recent years, research in the area of cancer stem cells has spiked tremendously. Numerous investigators have found that several types of cancers contain a subpopulation of tumor cells that display many defining characteristics of normal tissue stem cells, including multipotent differentiation potential, long-term self-renewal capacity, and expression of molecular markers of stemness. Most importantly, these cancer stem cells (CSCs) have very high tumor initiating potential, a finding that has led to the development of the cancer stem cell model for tumor progression. This model suggests that tumors are organized in a developmental hierarchy (similar to healthy tissue), with long-term tumor progression being driven by self-renewing CSCs at the top of the hierarchy. The CSC model represents a significant shift in our understanding of tumor progression, and as such, it may be possible to expand our knowledge of other aspects of tumor biology by re-examining them in the context of the CSC model. My work focuses on investigating interactions between CSCs and the tumor vasculature. Previous work has demonstrated heterogeneity in the proangiogenic potential of cells in a tumor. Considering the possibility that angiogenesis may be driven by specific subsets of tumor cells, I investigated the contribution of the CSC fraction to tumor angiogenesis. Comparing tumors with low or high CSC fractions, I have found that CSCs contribute to tumor vascular development through promotion of endothelial cell activity and recruitment of bone marrow-derived proangiogenic cells, mediated in part by vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF1). Since some tissue stem cells are known to reside in a vascular niche, I investigated the possibility that CSCs may also be supported by blood vessels in the tumor microenvironment, and that consequently CSCs may be targeted by disruption of tumor vasculature with antiangiogenic therapy. By testing multiple antiangiogenic therapeutic strategies, I have found that antiangiogenic therapy sensitizes CSCs to the effects of cytotoxic chemotherapy. Taken together, my work demonstrates a bi-directional relationship in which CSCs promote tumor vascular development, and tumor vasculature supports and protects CSCs. This work has implications for our understanding of CSC biology, tumor angiogenesis and antiangiogenic therapy, and provides insight into strategies for targeting the critical CSC population.