Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-98). Also available for download via the World Wide Web; free to University of Oregon users.

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

　　Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5’-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells. 　　One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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Η geminin δρα ως ρυθμιστής του κυτταρικού πολλαπλασιασμού και διαφοροποίησης συμμετέχοντας στην οργάνωση της χρωματίνης και των μεταγραφικών προγραμμάτων των πρόγονων κυττάρων μέσω της αλληλεπίδρασης της με διάφορους παράγοντες. Ο αυστηρός έλεγχος του κυτταρικού πολλαπλασιασμού και της διαφοροποίησης είναι βασικός για την παραγωγή του κατάλληλου αριθμού και τύπου κυττάρων που συμμετέχουν στην οργανογένεση. Επιπλέον η ικανότητα αυτό-ανανέωσης των πρόδρομων κυττάρων βασίζεται στον πολλαπλασιασμό τους, χωρίς διαφοροποίηση και είναι απαραίτητη για την ανανέωση των ιστών. Ωστόσο η απορρύθμιση των μηχανισμών που ελέγχουν τον κυτταρικό πολλαπλασιασμό είναι επιζήμια για τον οργανισμό αφού είναι το πρώτο βήμα κατά την καρκινογένεση. Η κυτταρική γήρανση έχει προταθεί ότι είναι ένας ογκοκατασταλτικός μηχανισμός. Στην παρούσα μελέτη διερευνήσαμε το ρόλο της geminin στην ρύθμιση των φυσιολογικών κυτταρικών διαδικασιών της διαφοροποίησης και της γήρανσης. Για αυτό το σκοπό, χρησιμοποιήσαμε ποντικούς στους οποίους η geminin απενεργοποιείται ειδικά στην λεμφοειδή κυτταρική σειρά. Δείξαμε ότι η απουσία της geminin οδηγεί σε μείωση του συνολικού αριθμού κυττάρων του θύμου και του σπλήνα. Επιπρόσθετα οι CD4 και CD8 Τ κυτταρικοί πληθυσμοί επηρεάστηκαν από την απενεργοποίηση της geminin. Για την εύρεση ενός πιθανού ρυθμιστικού ρόλου της geminin στην επαγωγή κυτταρικής γήρανσης χρησιμοποιήσαμε ινοβλάστες ποντικού που εκφράζουν μειωμένα επίπεδα της πρωτεΐνης. Δείξαμε ότι οι ινοβλάστες με μειωμένα επίπεδα της geminin παρουσιάζουν φαινότυπο γήρανσης νωρίτερα από τους ινοβλάστες αγρίου τύπου όπως καθορίστηκε από τη χρώση για σχετιζόμενη με γήρανση β-γαλακτοσιδάση. / Geminin acts as a coordinator of proliferation and differentiation by regulating the chromatin organization and transcription programs of progenitor cells through its interaction with several partners. The tight control of proliferation and differentiation, is essential in the generation of mature cells of the proper number and type necessary for organ formation. Moreover the self-renewing capacity of progenitor cells depends on proliferation without differentiation of the cells and is indispensable in tissue regeneration. However the deregulation of the mechanisms that control the proliferation capacity of a cell has deleterious effects for the organisms since it is the first step of carcinogenesis. Cellular senescence was proposed to be a tumour-suppressive mechanism. In the present study we addressed the role of geminin in the physiological processes of cellular differentiation and senescence. We have used a mouse line in which geminin is specifically inactivated in the lymphoid lineage. We showed that geminin’s absence leads to reductions in the thymic and splenic cellularities. In addition to that, CD4 and CD8 T cell populations of the thymus and the spleen of conditional KO animals, were affected by geminin’s ablation. To study a potential regulatory role of geminin in the induction of cellular senescence we used mouse fibroblasts that express reduced levels of the protein. We found that the fibroblasts with reduced levels of geminin’s expression present senescent phenotype earlier than the WT fibroblasts as determined by SA-β-gal staining. 9

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Abies concolor (white fir) is one of the many conifers often planted as an ornamental tree. Like so many other conifers, Abies is difficult to vegetatively propagate. Recent successes in using the techniques of tissue culture as a means of vegetatively propagating plants prompted the present study of applying these techniques to Abies.A review of the literature indicated that no conifer had ever been grown into plantlets by using the techniques of tissue culture. Only a very few reports exist for cellular differentiation although nearly thirty species of conifers have been grown in-vitro as callus.The review of literature also indicated that a variety of growth regulators, supplements and conditions of growth were utilized in promoting differentiation and the development of plants from callus. Since no consistent patterns emerge as guidelines for achieving differentiation, a proposal was made to test the effects of several growth regulators on callus tissue of Abies in hopes of causing differentiation and organogenesis.Terminal stem sections from the lower branches of a mature tree of Abies concolor were removed, sterilized and and placed on experimental solid media containing mineral salts, growth regulators and organic supplements. Growth of a callus occurred as a cream-colored friable mass on several of the media. When examined, however, the growth was found to consist of only parenchymatous cells. When transferred to a medium (G7) containing coconut milk, casein hydrolysate and 2 mg/l kinetin, compact, hemispherical nodules developed over a period of six weeks.A number of these nodules were fixed, embedded,'sectioned and stained with safranin and fast green. These were examined with a microscope and comparative photographs between this type of growth and the friable callus were made.Sections of the nodules showed a more compact arrangement of cells and an incomplete ring of growth consisting of cells arranged in radial files as if produced by a cambium. The center of the incomplete ring contained parenchymatous cells primarily, but scattered tracheid-like cells with bordered pits were also noted. Photographs of the well organized incomplete ring of growth and the tracheid-like cells were made.It can be concluded that differentiation of Abies concolor callus did occur on the G7 medium and that this represents a positive step toward organogenesis using the techniques of tissue culture.

Macrophages are involved in many aspects of both the innate and acquired immunity, and participate in tissue homeostasis, bone remodelling, wound healing, and tissue repair. When their function becomes dysregulated, they contribute to the initiation and progression of inflammatory diseases. Genes expressed in activated macrophages are likely to play an important role in inflammation and/or immunity. One family of genes that is highly expressed in activated macrophages is the Schlafen (Slfn) gene family. Given that very little is known about the function of this family, particularly in macrophages, this study focused on the regulation and function of one family member, namely Slfn-4. The transcriptional regulation of Slfn-4 was characterised in murine macrophages. The expression of Slfn-4 was transiently down regulated during macrophage differentiation and dramatically up regulated in response to activation signals including lipopolysaccharide (LPS) and dsRNA Poly(I:C). A potential association with inflammation was further suggested by the enhanced expression of Slfn-4 in a mouse model of rheumatoid arthritis. Further investigations into transcriptional regulation of Slfn-4 revealed that it belongs to the subset of genes that are type I interferon (IFN)-inducible. This finding is consistent with the predicted transcription factor binding sites in the putative promoter of Slfn-4, and suggests a role for Slfn-4 in the antiviral response. To gain further insight into the function of Slfn-4 in macrophage biology, in vitro over-expression approaches were undertaken and its cellular localisation in macrophages was characterised. In resting and activated macrophages, Slfn-4 exhibited a cytoplasmic and strong perinuclear localisation. Additional studies were carried out to investigate the in vivo biology of Slfn-4 in macrophages. For this purpose, the Csf1r-GAL4VP16/UAS-ECFP (referred to as “MacBlue”) transgenic mouse line was first characterised. Enhanced cyan fluorescent (ECFP) reporter expression in the MacBlue transgenic mice was specifically detected in cells of the mononuclear phagocyte system during embryonic development and adulthood. The MacBlue transgenic mouse line was next used to drive expression of Slfn-4 in cells of the myeloid lineage in vivo, and to examine the phenotype of this line. Specific over-expression of Slfn-4 in cells of the myeloid lineage in vivo altered the percentage of peripheral blood monocytes and caused extramedullary hematopoiesis. In summary, this thesis demonstrated that Slfn-4 expression is dynamically regulated during macrophage differentiation and activation, and the enforced Slfn-4 over-expression in cells of the myeloid lineage perturbs normal monocyte/macrophage development.