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B lymphopoiesis is regulated by cytokines, chemokines and cell surface proteins that initiate signal transduction pathways necessary for maturation to proceed. Many of these factors are expressed by cells in the surrounding bone marrow (BM) microenvironment, which also form the niches that support development. Interleukin-7 (IL-7) is an essential cytokine for progenitor B cells and is important in providing survival, proliferation and maturation signals. By growing BM B cells for extended periods of time in vitro with IL-7 it is possible to select for cells that possess the ability to grow indefinitely, and these cultures can be used to generate cell lines. Data presented herein describe the generation and characterization of IL-7-dependent B cell lines as well as their utility in investigating aspects of B cell development. As B cells mature they lose responsiveness to IL-7, yet retain IL-7 receptor expression. I demonstrate that a B cell’s ability to respond to IL-7 is controlled by the expression of suppressor of cytokine signaling (SOCS) proteins, which are regulated by a variety of signaling pathways including those initiated by IL-7. Development of progenitor B cells to mature immunoglobulin secreting B cells is mediated in part by surface proteins present on stromal cells as well as on B cells themselves. Heparan sulfate and CD19 play important roles in regulating this transition and I provide data that demonstrates how their ability to regulate Erk activation downstream of the pre-B cell receptor (pre-BCR) alters the proliferation and maturation of developing B cells.

B lymphopoiesis is regulated by cytokines, chemokines and cell surface proteins that initiate signal transduction pathways necessary for maturation to proceed. Many of these factors are expressed by cells in the surrounding bone marrow (BM) microenvironment, which also form the niches that support development. Interleukin-7 (IL-7) is an essential cytokine for progenitor B cells and is important in providing survival, proliferation and maturation signals. By growing BM B cells for extended periods of time in vitro with IL-7 it is possible to select for cells that possess the ability to grow indefinitely, and these cultures can be used to generate cell lines. Data presented herein describe the generation and characterization of IL-7-dependent B cell lines as well as their utility in investigating aspects of B cell development. As B cells mature they lose responsiveness to IL-7, yet retain IL-7 receptor expression. I demonstrate that a B cell’s ability to respond to IL-7 is controlled by the expression of suppressor of cytokine signaling (SOCS) proteins, which are regulated by a variety of signaling pathways including those initiated by IL-7. Development of progenitor B cells to mature immunoglobulin secreting B cells is mediated in part by surface proteins present on stromal cells as well as on B cells themselves. Heparan sulfate and CD19 play important roles in regulating this transition and I provide data that demonstrates how their ability to regulate Erk activation downstream of the pre-B cell receptor (pre-BCR) alters the proliferation and maturation of developing B cells.

B lymphocytes develop from pluripotent stem cells, and differentiate to plasma cells (PCs) in reaction to signals from the supportive microenvironment. Different sets of signals, which are derived from multiple sources such as soluble cytokines and cell-cell contacts, are required at different stages of development. For instance, murine B cell progenitors require the action of interleukin-7 (IL-7) in the early phase of their development in the bone marrow (BM). The necessity for IL-7 decreases as the cell matures, and this event is correlated with the appearance of CD22. The first two chapters of this thesis focus on the early stages of B cell development that take place in the BM. In chapter 1, I examine the IL-7 response and, although I do not show a specific role for CD22 in the loss of sensitivity to IL-7, my data suggest that cis interactions involving sialic acids might modulate the IL-7 response. This section is followed by the analysis of the effect of IL-21 on B cell progenitors in the BM. IL-21 is known to regulate the terminal stages of B cell differentiation. In collaboration with Dr. Danijela Konforte, I present evidence that B cell progenitors in the BM also express a functional IL-21 receptor and that stimulation of this receptor with IL-21 accelerates the maturation pace of B cells. I further demonstrate that proB cells stimulated with IL-21 and anti-CD40 can differentiate into immunoglobulin (Ig)-secreting cells, and discuss the possibility that IL-21 plays a role during inflammation for the development of B cell progenitors in peripheral lymphatic organs. Finally, in the last chapter, in collaboration with the laboratory of Dr. Gommerman, I investigate how the microenvironment can shape the development of B cells. It has been demonstrated by my collaborators that IgA+ PCs present in the gut produce iNOS and display traits commonly associated to the myeloid lineage, and in this chapter, I describe a co-culture system with BM and gut stroma to study the conditions that sustain the generation of IgA+iNOS+ cells. In particular, I show that the presence of microbial products is one of the key factors required for their development.

B lymphocytes develop from pluripotent stem cells, and differentiate to plasma cells (PCs) in reaction to signals from the supportive microenvironment. Different sets of signals, which are derived from multiple sources such as soluble cytokines and cell-cell contacts, are required at different stages of development. For instance, murine B cell progenitors require the action of interleukin-7 (IL-7) in the early phase of their development in the bone marrow (BM). The necessity for IL-7 decreases as the cell matures, and this event is correlated with the appearance of CD22. The first two chapters of this thesis focus on the early stages of B cell development that take place in the BM. In chapter 1, I examine the IL-7 response and, although I do not show a specific role for CD22 in the loss of sensitivity to IL-7, my data suggest that cis interactions involving sialic acids might modulate the IL-7 response. This section is followed by the analysis of the effect of IL-21 on B cell progenitors in the BM. IL-21 is known to regulate the terminal stages of B cell differentiation. In collaboration with Dr. Danijela Konforte, I present evidence that B cell progenitors in the BM also express a functional IL-21 receptor and that stimulation of this receptor with IL-21 accelerates the maturation pace of B cells. I further demonstrate that proB cells stimulated with IL-21 and anti-CD40 can differentiate into immunoglobulin (Ig)-secreting cells, and discuss the possibility that IL-21 plays a role during inflammation for the development of B cell progenitors in peripheral lymphatic organs. Finally, in the last chapter, in collaboration with the laboratory of Dr. Gommerman, I investigate how the microenvironment can shape the development of B cells. It has been demonstrated by my collaborators that IgA+ PCs present in the gut produce iNOS and display traits commonly associated to the myeloid lineage, and in this chapter, I describe a co-culture system with BM and gut stroma to study the conditions that sustain the generation of IgA+iNOS+ cells. In particular, I show that the presence of microbial products is one of the key factors required for their development.

BLNK is an adaptor protein that functions in B-cell receptor signalling, and is vitally necessary at signalling checkpoints of mammalian B-cell development. However, its importance to avian B-cell development remains unclear. To explore the function of BLNK in chickens, shRNA-mediated RNA interference was delivered to a chicken B-cell line in vitro by replication-competent avian retrovirus (RCAS), and effective shRNA were determined. To observe an shRNA phenotype on chicken B-cell development, we simultaneously explored whether RCAS penetrance was correlated between red blood cells (RBC) and bursal B-cells by infecting chicken embryos with RCAS expressing a fluorescent tag. We found that RCAS penetrance was correlated between RBC and B-cells, which provides a system to observe any in vivo effects of BLNK shRNA on B-cell development. Furthermore, this system for observing BLNK function may be complemented by genetically-modified BLNK, particularly variants resistant to RNA interference.

BLNK is an adaptor protein that functions in B-cell receptor signalling, and is vitally necessary at signalling checkpoints of mammalian B-cell development. However, its importance to avian B-cell development remains unclear. To explore the function of BLNK in chickens, shRNA-mediated RNA interference was delivered to a chicken B-cell line in vitro by replication-competent avian retrovirus (RCAS), and effective shRNA were determined. To observe an shRNA phenotype on chicken B-cell development, we simultaneously explored whether RCAS penetrance was correlated between red blood cells (RBC) and bursal B-cells by infecting chicken embryos with RCAS expressing a fluorescent tag. We found that RCAS penetrance was correlated between RBC and B-cells, which provides a system to observe any in vivo effects of BLNK shRNA on B-cell development. Furthermore, this system for observing BLNK function may be complemented by genetically-modified BLNK, particularly variants resistant to RNA interference.

The work presented in this thesis is concerned with photovoltaic cells formed by plating CdS single crystals and thin films, and Cd(_y) Zn(1 _ y)S single crystals, with copper sulphide. An electroplating technique has been used to control the phase of copper sulphide by changing the electric field during its formation. Different phases of Cu(_x)S have been identified directly using Reflection High Energy Diffraction (RHEED), and indirectly from spectral response measurements. A dramatic change in the spectral response accompanying the reduction in the covellite response associated with an increase in that from chalcocite following argon heat treatment has been achieved. The change from the djurleite phase to that of chalcocite has also been obtained by using argon heat treatment for 5 minutes at 200 C. This effect was found to be reversible in that layers of chalcocite were converted to djurleite when air was used as the ambient for the heat treatment. C-V measurements have demonstrated that with increasing plating bias the donor concentration decreases at first before it assumes a constant value. This led to the effect of decreasing the junction capacitance as the width of the depletion region changed. The problem of the stability of the CdS-Cu(_2)S photovoltaic devices formed by wet plating" is addressed by studying the combined effects of the substrate onto which the CdS is deposited and the ambient used during annealing. Thin film cells have been prepared on both Ag/Cr and SnO substrates, and the device characteristics for each have been investigated as a function of annealing ambient. The results have shown that devices formed on Ag/Cr substrates were more stable following annealing in air than in argon, while the converse was true for cells fabricated on SnO(_x) substrates. The degradation effects of CdS-Cu(_2) S photovoltaic cells have been investigated. While devices stored in the dark showed little or no degradation, those maintained under illumination exhibited a significant deterioration in all operational parameters over a four week period. As far as the combined effect of temperature and ambient on the stability of cells are concerned, it was found that the ageing of devices in argon at room temperature in the dark was negligible, and moreover the fill factor was observed to improve marginally. When the devices were stored in the same ambient conditions at 50 C, they showed a significant improvement in the fill factor, but simultaneously exhibited a considerable reduction in the short circuit current. This process was reversible, since the sensitivity of degraded devices could be restored by annealing them in a hydrogen/nitrogen mixture. By comparing Electron Spectroscopy for Chemical Analysis (ESCA) studies with solar cell device characteristics, it has been shown that the formation of copper oxide on the Cu(_2)S surface plays a significant role in the degradation of CdS-Cu(_2) S devices. The extent of the cross-over between the dark and light J-V characteristics is a function of the period of etching used prior to junction formation. The variation of current and diode factor has been established as a function of the bias value. The dependence of forward current on the temperature at fixed forward voltage has also been investigated. Finally this work has shown that an increase in V(_oc) can be achieved when Cd(_0◦8)Zn(_0◦2)S is used as a base material for solar cells instead of CdS. Different traps were identified through a photocapacitance investigation. An important trap was found at 0.78eV below the conduction band. It has been demonstrated that the effect of this level was found to be diminished much more slowly when the annealing was carried out in argon rather than in air. This level may play an important role in the Cd(0◦8) Zn(0◦2)S-Cu(_2)S solar cell properties.

Induction of the cytochrome P-450 mixed-function oxidase and specifically the cytochrome P-450 IVA1 isoenzyme by seven phenoxy acid herbicides in rat liver and kidney, have been studied. results using liver microsomes demonstrated that the 12-hydroxylation of lauric acid was significantly induced by all compounds (3-8-fold), 4-chlorophenoxyacetic acid (CPA) (300 mg/kg) being the weakest and 2,4,5-trichlorophenoxypropionic acid (2,4,5-TP) (200 mg/kg) the most potent inducers respectively. This increase in lauric acid 12-hydroxylase-activity was accompanied by an increase in the hepatic content of cytochrome P-450 IVA1 as assessed by both a qualitative Western blot procedure and a quantitative ELISA method. Furthermore, there was a parallel increase in cytochrome P-450 IVA1 mRNA and a similar increase in peroxisomal B-oxidation subsequent to exposure to these compounds. In addition, benzphetamine-N-demethylase, a marker of cytochrome P-450 IIBl and IIB2 activities, was not affected by any of the herbicides, whereas cytochrome P-450 IA1 and IA2, as assayed by ethoxyresorufin-O-deethylase activity, was significantly increased (up to 2.2-fold) by some of the compounds. Kidney microsomal parameters were not affected by any of these compounds. My in vivo studies using antipyrine, pentobarbital and zoxazolamine indicated that the metabolism of these substrates was marginally affected by only some of the compounds. In order to highlight the possible involvement of a metabolite of the chlorophenoxy acids in the induction of cytochrome P-450, I investigated four related chlorophenols. There was no significant change in cytochrome P-450 isoenzyme levels in rat liver and kidney microsomes nor was there any increase in peroxisomal beta-oxidation. Taken collectively, the results presented in this thesis indicate that the chlorinated phenoxy acid herbicides studied preferentially induce the cytochrome P-450 IVA1 isoenzyme and peroxisomal beta-oxidation in a pattern similar to the typical inducers of this isoenzyme such as clofibrate. A scheme is presented whereby induction of catalytically competent cytochrome P-450 IVA1 is required for the phenomenon of peroxisome proliferation by these chlorophenoxy acid derivatives.

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