Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 92-105).

Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Description based on contents viewed Jan. 29, 2007; title from title screen. Includes bibliographical references (p. 51-54).

Thesis (Ph. D.)--University of Hong Kong, 2006. / Also available online.

No description available.

Cardiovascular disease is a leading cause of death worldwide and becomes increasingly prevalent in the elderly population. Independent of etiopathogenesis, heart failure (HF) is the final common stage of numerous heart diseases. Cardiac stem cell (CSC) therapy has emerged as a promising cell-based strategy for treatment of HF. However, cell replacement is not able to fully restore a structurally damaged myocardium in advanced and end-stage HF. The objective of this project was to test the following hypotheses: that a bioengineered heart extracellular matrix (ECM) with preserved intact geometric structure could be generated using decellularization by coronary perfusion; and that autologous CSCs, to repopulate this ECM, could be isolated and expanded from the adult heart, with the caveat that autologous CSCs are depleted and impaired by both aging and chronic dilated cardiomyopathy. This will help to develop a possible therapeutic approach for advanced HF, using a combination of CSCs and engineering technique. Resident CSCs were isolated from explant-derived cells (EDCs) and expanded into cardiosphere-derived cells (CDCs) via cardiosphere formation. The CDCs expressed CSC markers (c-kit and Sca-1), pluripotent markers (Oct3/4 and Sox2), and the cardiac lineage-committed marker (Nkx2.5), and showed clonal expansion, self-renewal, and cardiomyogenic potential in vitro. In tissue engineering experiments, CDCs survived and proliferated within biomaterial alginate scaffolds for up to 7 weeks. An engineered bioartificial ECM scaffold was successfully produced from a whole rat heart using retrograde coronary perfusion and possessed an intact 3D architecture with functionally perfusable vascular network. Compared with ventricles, cultures derived from atria produced significantly higher number of c-kit+ and Sca-1+ CSCs (c-kit: 13% vs. 3.4%; Sca-1: 82% vs. 53%, respectively) and exhibited greater clonogenic and proliferative capacity. CDCs could be grown from young and aged mice, but the yield of CSCs significantly declined with age, as did cell migration and differentiation potential. In comparison to wild-type mice, atrial-CDCs from dystrophic mice showed no significant differences in CSC subpopulations and characteristics, despite confirmation of cardiac dysfunction using MRI. In conclusion, CDCs could be considered to be a viable cell candidate for cardiac therapy and may be used to treat HF at various stages, in combination with myocardial tissue engineering.

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.

The hemopoietic system Is organized as a hierarchy of hemopoietic cell populations distinguished by differences in their proliferation and differentiation potential. Studies using short-term in vitro and in vivo assays based on colony formation in semi-solid medium, or in the spleens of lethally irradiated mice, respectively, have shown that these procedures detect primarily lineage-restricted progenitor types and have provided much information about the characteristics and regulation of such cells. Assessment of lymphoid and myeloid tissue reconstitution after more prolonged periods following transplantation has established the existence of a more primitive stem cell type; however, the retrospective nature of these complex analyses has Impeded characterization and purification of these cells. My first objective was to develop a procedure for the selective isolation of stem cells with short-term in vitro and in vivo multilineage differentiation potential. For this I devised a single-step, four-parameter fluorescence activated cell sorting procedure In which cells were selected according to their forward and orthogonal light-scattering properties, and their surface expression of the Thy-1 and H-2K antigens. Application of this procedure to marrow cells from mice treated 4 days previously with 150 mg/kg of 5-fluorouracil showed that it could be used to sort a subpopulation that was enriched 100-fold in CFU-GEMM and in which 1 in 4 cells was a day 12 CFU-S. To determine the extent to which stem cells with long-term lympho-myeloid repopulating potential had been copurified, I undertook to develop a quantitative procedure that might allow this primitive cell population to be measured and hence characterized on a routine basis. This required an assay that would detect donor-derived hemopoiesis exclusively, and that was sensitive enough for the detection of limiting numbers of cells with long-term lympho-myeloid repopulating potential. This was shown to be possible using a competitive repopulation assay in which lethally irradiated female recipients were transplanted with male "test" cells together with a second suspension of female cells with adequate short-term repopulating activity but greatly diminished long-term repopulating potential. These sex differences were then used to specifically identify the 5 week progeny of stem cells in the test suspension. Assessment of the sorted day 4 5-FU marrow population revealed that it was capable of repopulating all hemopoietic organs after transplantation and that an enrichment of 30-fold over unseparated, 5-FU-treated marrow had been achieved. My second objective was to determine whether the competitive long-term lymphoid and myeloid repopulation obtained with these sorted cells was due to the activity of Individual stem cells with a dual potential for lymphopoiesis and myelopoiesis. For this I used retroviral-infection to uniquely mark sorted cells In vitro, and then transplanted them in sufficiently low numbers to allow individual regenerated clones to be detected and analyzed. In some mice, distribution of cells with the same unique integration marker in different lymphoid and myeloid cell populations established the presence of lympho-myeloid stem cells in the original sorted population. In addition, clones with restricted tissue distributions were also documented. My final objective was to investigate whether the competitive repopulation assay was in fact able to serve as a procedure for the exclusive quantitation of long-term lympho-myeloid repopulating stem cells. A limiting dilution approach was used to compare the frequency of hemopoietic stem cells (competitive repopulating units, CRU) in marrow obtained from a variety of sources, using >20% repopulation by male cells at 5 or 10 weeks post-transplantation as the end point. The results obtained were largely independent of the time of analysis, and whether repopulation of recipient marrow or thymus was evaluated, suggesting that either can be used in this assay to quantitate a hemopoietic stem cell with the potential to regenerate both lymphoid and myeloid systems. These studies have provided procedures for the detection, quantitation and selective enrichment of the most primitive stem cells in the murine hemopoietic system which have competitive long-term lympho-myeloid repopulating ability. The availability of these procedures should facilitate the development of additional purification steps leading to the isolation of these cells as homogeneous suspensions, and their further use as targets for retrovirus-medilated gene transfer to determine the genetic basis of their activation, determination and neoplastic transformation. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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.

The overall goal of the work, described in this thesis was to investigate the molecular mechanisms that regulate normal pre-B cell proliferation and how these may be altered in transformed pre-B cells. Monoclonal antibodies and molecular biological techniques have allowed a number of stages of pre-B cell differentiation to be defined but little is known about mechanisms controlling their proliferation. Studies of pre-B cell production in animal models and in long-term cultures that support pre-B cell proliferation have suggested that stromal cells play a key role in this regard. As a first step to investigate the mechanisms involved, a number of pre-B cell supportive murine stromal cell lines were isolated and characterized. A number of pre-B cell lines were also isolated, cloned and characterized. From these, spontaneous and Abelson murine leukemia virus transformants were derived. These cell lines were then used in co-culture experiments to demonstrate that stromal cells constitutively secrete a pre-B stimulating factor. Characterization of the pre-B cell stimulating activity produced by one stromal cell line (M2-10B4) showed it to be a 10 Kd molecule sensitive to freezing and different from any cloned hemopoietic growth factor described to date. The possibility that extracellular matrix components might be involved in stromal cell-mediated control of pre-B cell growth was also investigated. It was found that pre-B cells attach specifically to fibronectin and that although fibronectin by itself cannot support pre-B cell proliferation, it contributes to stromal cell stimulation of pre-B cell growth. Both of these mechanisms were found to be affected in malignantly transformed pre-B cell populations irrespective of the mode of transformation. Transformed pre-B cells were found to have acquired the ability to secrete a novel 3 Kd autocrine factor that is also capable of stimulating normal pre-B cells. In addition transformed pre-B cells showed a greatly decreased ability to adhere to fibronectin and had become insensitive to the synergistic stimulating effect of fibronectin. It will be of interest to determine in the future whether these findings have a counterpart in human malignant pre-B cell populations. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Thesis advisor: Michael Russell / This paper describes the construct of fascination in science, a non-cognitive trait combining interest, curiosity, and mastery skills, and the particular relevance of fascination in science for students during middle school. Grounded in the theory of Science Learning Activation and employing data from the longitudinal Activated Learning Enables Success study of 2014 (ALES:14), cohorts of sixth and eighth graders were measured on fascination five times over two school years, allowing for an investigation of change over time. Multilevel models were constructed for each grade-level cohort in an effort to determine patterns of change, while also testing for relationships with several student-level characteristics and class-level instructional variables. Results suggest discontinuous patterns of change in fascination, with declining fascination scores in grade 6 boosted over the summer break and declining fascination scores in grade 8 rising the following school year. While the impact of instructional variables was negligible, relationships with several individual covariates were observed, primarily indicating the importance of family support for science. Future research should focus on context-specific elements of in-school activities, along with additional out-of-school factors that may influence fascination. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Lynch School of Education. / Discipline: Educational Research, Measurement and Evaluation.