B cell chronic lymphocytic leukemia (B-CLL) is a hematologic neoplasm characterised by the proliferation and accumulation of sIgM+/D+ B cells that fail to progress to the final stages of B cell development. The malignant cells in B-CLL also express the pan-T cell antigen CD5, suggesting that CLL is a malignancy of the CD5+ subset of B cells. Additional characteristics of the malignant clone include a low proliferative index, enhanced in vivo survival and constitutive expression of the anti-apoptosis oncoprotein bcl-2. The behaviour of leukemic CD5 B cells in vitro contrasts their arrested in vivo state. That is, despite the majority of cells being arrested in the G0 phase of the cell cycle, the leukemic B cells are not irreversibly frozen as they can be induced to differentiate to Ig-secreting cells under appropriate in vitro conditions. Furthermore, leukemic CD5 B cells rapidly undergo death by apoptosis following in vitro culture. This thesis describes the requirements for in vitro activation of leukemic CD5+ B cells, the characterisation of the events involved in apoptosis of these cells as well as the identification of various growth factors capable of modulating these events. Stimulation of unfractionated peripheral blood lymphocytes (PBLs) from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and 1gM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of 1gM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis were also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on leukemic CD5 B cells was independent of T cells. In contrast, activation and differentiation of the leukemic CD5 B cells into 1gM-secreting cells following culture with mitogens did not occur in the absence of T cells. Interestingly, co-stimulation of leukemic CD5+ B cells with PMA and anti-Ig induced cellular responses that exceeded those induced by either activator alone. Thus, leukemic CD5+ B cells from patients with B-CLL can be activated in vitro and differentiate in response to stimulation via both T cell-dependent and T cell-independent mechanisms. Apoptotic cell death was characterised in purified leukemic CD5 B cells obtained from six B-CLL patients. All leukemic CD5 B cell populations entered an apoptotic pathway in vitro as evidenced by a reduction in cell size, loss of cell viability and fragmentation of DNA into multimers of -180 base pairs. Following 24 hours of in vitro culture 24.0±16% of DNA was fragmented. After 8 days, the majority of DNA was fragmented, and fewer than 10% of cultured cells were viable. Examination of bcl-2 expression in the malignant B cells by flow cytometry revealed a unimodal pattern of expression in greater than 85% of cells from each B-CLL patient prior to culture. During in vitro culture, bcl-2 expression became bimodal such that the B cells displayed a bcl-2hjgh and bcl-2iow phenotype. The level of expression by the bCl2hjgh cells was similar to that observed prior to in vitro culture, indicating that bcl-2 is down-regulated in apoptosing cells. Interestingly, despite this downregulation, the overall number of cells positive for bcl-2 remained constant. This suggests that the enhanced survival of leukemic CD5+ B cells in vivo is mediated by the sustained expression of bcl-2 and that additional mechanisms exist capable of overriding the protective effect of bcl-2 when bcl-2 is present at reduced levels. Leukemic B cell apoptosis has previously been reported to be delayed or prevented by IL-4, IFN-y and IFN-a. These results were confirmed in this study where it was found that culture of leukemic CD5 B cells with IL-4 or IFN-y enhanced cell viability and delayed apoptosis in 6/6 and 5/6 populations of leukemic B cells, respectively. This function was also found to be shared by IL-2, IL-6, IL-13 and TNF-a as these cytokines enhanced cell viability and delayed apoptosis in some of the cell populations examined at a level similar to that observed for IL-4 and IFN-y. These cytokines may mediate their effect via the expression of bcl2 as culture in the presence of IL-2, IL-4, IL-6, IL-13, IFN-y or TNF-a resulted in a higher percentage of cells displaying the bcl-2high phenotype, compared to unstimulated cells. Taken together, these results suggest that autocrine and/or paracrine growth loops may play a role in the pathogenesis of B-CLL and that cytokines that prevent apoptosis in vitro may be targets for treatment of this B cell malignancy.

No description available.

Bibliography: leaves 154-185 / iv, 185 leaves, [15] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Science, 1987

During mammalian development a small number of pluripotent cells proliferate and differentiate to give rise to all the mature cell types of the organism. Among the earliest differentiation events is the process of gastrulation, in which pluripotent primitive ectoderm cells form the three germ lineages, mesoderm, ectoderm and endoderm under the control of complex signalling and environmental cues. This process can be modelled using embryonic stem cells, which have proven to respond to embryologically relevant signals during in vitro differentiation and promise to uncover additional insights into the process of germ lineage specification. This thesis describes the differentiation of mouse ES cells to committed cell types via a second intermediate population of pluripotent cells termed Early Primitive Ectoderm-Like (EPL) cells. The similarity of EPL cells to primitive ectoderm and the rapid acquisition of lineage specific markers and loss of pluripotent characteristics upon differentiation of EPL cells suggest they are an excellent model for the cells in the embryo that undergo germ lineage commitment. EPL cells can be differentiated as EPLEBs, which are highly enriched in mesodermal cell types and contain essentially no ectodermal derivatives and no visceral endoderm. Here it is shown that EPLEBs can be generated from EPL cells grown either adherently or in suspension culture provided the cells are reduced to a single cell suspension before reaggregation as EPLEBs. Since EPLEBs are a rich source of mesoderm and contain less non-mesodermal cell types than traditional ESEBs, they were assayed for definitive blood formation, however none was detected. Alternately, EPL cells can be differentiated in the presence of MEDII in aggregates termed EBMs, which are restricted to ectodermal cell fates. Here it is demonstrated that the switch from mesodermal to ectodermal differentiation observed in ELPEBs and EBMs relies on two variables; a mesoderm suppressing activity within MEDII and the pro-mesodermal activity of cell dissociation as undertaken during EPLEB formation. Evidence has been presented that interventions that modulate the epithelial identity of EPL cells are capable of influencing subsequent differentiation such that protection of the epithelial cell state favours ectoderm while disruption favours mesoderm. Staurosporine (SSP) is a kinase inhibitor that has been shown to induce an epithelial to mesenchymal transition in chick neural tube. Here it was added to EPL cells with the result that mesodermal differentiation was enhanced at the expense of ectoderm. DAPT is a potent inhibitor of ƴ-secretase, which cleaves a number of protein targets including the adherens junction component E-cadherin. Addition of DAPT to differentiating EPL cells has the opposite effect to SSP, with an increase in ectodermal differentiation at the expense of medoderm. It is proposed that DAPT is acting by preventing E-cadherin cleavage and thus stabilising the epithelial state. Modulation of epithelial contacts between pluripotent cells represents a novel way to control lineage induction and as such the incorporation of these findings into methodologies for directed differentiation in defined culture conditions is likely to provide improved outcomes in the production of desired cell types. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

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Graduation date: 1998

Graduation date: 1997

This thesis deals with different forms of strategic complementarities in industrial organization problems. Chapter 2 is an attempt to develop a unified approach to endogenous heterogeneity by constructing a general class of two-player symmetric games that possess only asymmetric pure-strategy Nash equilibria. These classes of games are characterized in some abstract sense by two general properties: payoff non-concavities and some form of strategic substitutability. While the second characteristic allows to show the existence of pure strategy Nash equilibria, the second precludes these equilibria to be symmetric. Other two classes of games that always possess asymmetric, but never symmetric, pure-strategy equilibria, although they are not of strategic substitutes are also studied. This chapter also generalizes a number of models dealing with two-stage games, with long term investment decisions in the first stage and product market competition in the second stage. Chapter 3 investigates the effects of forward looking behaviour in technology adoption. The setup is an overlapping generations model where agents choose between two alternative networks taking into consideration both the installed base and the expected base. The latter element is the distinctive feature of the approach. It is shown that a unique equilibrium exists, on which agents coordinate their expectations. While exhibiting hysteresis, the equilibrium adoption path does not comply with technologies locking in. Network choices are characterized both in terms of their long run properties and the expected time of adoption. Chapter 4 studies the problem of a monopolist who produces a good with network externalities and faces the possibility of selling a new higher quality. Within the vertical product differentiation it identifies the necessary and sufficient conditions for quality improvement to take place when a good, produced by a monopolist, exhibits positive network externalities. When network effects are not very strong, the monopolist produces both the high and the low quality and thus quality improvement takes place. In this case, he will use an introductory pricing strategy for the quality that benefits from network externalities, not maximizing however the network size. As the network effect becomes more important, the monopolist will have an incentive to practise introductory pricing and produce both qualities. Finally, if the network externality is higher than the intrinsic quality differential, quality improvement does not take place. Chapter 5 deals with the problem of an incumbent producing a low quality good with network externalities that faces the threat of entry by a higher quality good. In the framework of a vertical product differentiation model, it is identified a necessary and sufficient condition under which quality improvements are spontaneously adopted along, in spite of the existence of network effects. This condition says that the intensity of network effects on consumers' preferences should not exceed twice the differential of intrinsic qualities existing between the two variants. Finally, chapter 6 is concerned with the optimal path of prices of a monopolist who operates in a network industry for a finite horizon. Agents obtain intrinsic utility from the good and from the fact that in the past there have been other consumers using it. It is observed that the monopolist has an incentive to introduce the good at initially low prices and to increase the price as the time goes by. This chapter concludes with a necessary and sufficient condition under which the initial price, and only the initial one is zero. This condition is related both with the intensity of the preferences for the network and with the time horizon of the monopolist.

Human mesenchymal stem cells (MSCs) are being largely studied for their differentiation potential and immunological properties. In the present study, we evaluated the ability to reliably differentiate mesenchymal lineages into hepatocyte-like cells both in vitro and in vivo. For this purpose, we handled several tissue sources and compared typical MSCs from bone marrow (BM) or umbilical cord, to liver-derived mesenchymal-like cells and to fibroblasts. We observed that hepatocyte differentiation of BM-MSCs was incomplete and variable with elective expression of some specific markers. These mesenchymal-derived hepatocyte-like cells (MDHLCs) were also chimerical in their phenotype as they expressed mesenchymal markers while these were down-regulated. We therefore designed differentiation cocktails with an aim to improve MDHLC phenotype and some unexpected results were obtained with LIF cytokine whose action on stem cells for hepatocyte differentiation was not documented. Nevertheless, we observed a limitation in the acquisition yield of hepatic features. Furthermore, the hepatocytelike phenotype of MDHLCs completely disappeared when the cells were incubated into growth medium. However, we showed that hepatic functionality of these cells, as urea secretion and gluconeogenesis, could be increased under specific conditions, suggesting the potential to improve MDHLC phenotype. In vivo, MSCs were able to express hepatic markers into SCID-mice livers while their chimerical phenotype remained. In contrast, MDHLCs down-regulated their hybrid phenotype after transplantation suggesting a beneficial influence of in vitro differentiation step. MSCs were also able to engraft and even partially differentiate into wild-type mice which was a strong argument for their low immunogenicity. Surprisingly, fibroblasts showed highly similar potential than MSCs to differentiate into hepatocyte-like cells both in vitro an in vivo and these results underlined the difficulty to accurately distinguish between both cell types using current techniques. Umbilical cord-derived stem cells (UCMSCs) and adult-derived human liver stem cells (ADHLSCs) were different in nature and displayed a native hybrid phenotype while their differentiation allowed high levels of hepatocyte-like feature acquisition. Together all these data suggest the current possibility to engineer mesenchymal-derived hepatocyte-like cells owning specific features acquisition while remaining limited in their commitment. This highlights the need for further investigations to evidence the usefulness of these mesenchymal lineages for liver cell therapy.

Sim2s is a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor. In Drosophila, the Sim2 homolog, sim, is necessary for cell fate determination during central nervous system (CNS) development. In mammals, both Sim2 isoforms are involved in development of various tissues, including muscle, cartilage, and mammary gland. Loss-of-function studies revealed a role for Sim2s in specifying epithelial cell fate during mammary development and inhibiting growth and invasion of aggressive breast cancer cells. This study determined the role of Sim2s in mammary epithelial cell differentiation. Our hypothesis is that Sim2s is sufficient to promote lactogenic differentiation in vivo, characterized by expression of lactation-specific genes. Two models were used to test this hypothesis: (1) a transgenic mouse, expressing Sim2s under control of the MMTV-LTR, and (2) the mouse mammary epithelial cell line HC11. Together, these models allow analysis of the effect of Sim2s on global mammary gland differentiation and the mechanism through which it accomplishes this in a relatively homogenous population of cells. We determined that precocious expression of Sim2s in vivo is associated with upregulation of a subset of milk protein genes in nulliparous females. During early pregnancy, Sim2s regulation of lactogenic differentiation extended to a larger group of genes. Following pup removal, Sim2s appears to promote survival of alveolar epithelial cells. In vitro, Sim2s expression is necessary for maximal Csn2 expression, as determined by loss-of-function studies. Overexpression of Sim2s is sufficient to enhance prolactin-mediated Csn2 expression. Chromatin immunoprecipitation assays performed in HC11 cells revealed enhanced recruitment of Stat5a and RNA Polymerase II (RNAPII) to the regulatory region of Csn2 in the presence of Sim2s. In addition, Sim2s and RNAPII were found in a complex that was localized to both the promoter and coding region of the Csn2 gene. These studies support the idea that Sim2s is upregulated in a developmental stage-specific manner in the mouse mammary gland to promote the survival and differentiation of alveolar epithelial cells expressing high levels of milk protein genes. Further, Sim2s may regulate the function of a specific subset of alveolar cells by targeting the RNAPII holoenzyme complex to genes expressed during lactogenic differentiation.