Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 87-89).

Thesis (M.D.)--Boston University

Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes, including turnover of inositol phospholipids, activation of a number of kinases, and changes in intracellular pH (pHⁱⁿ) and calcium ([Ca²⁺]ⁱⁿ). Changes in both pHⁱⁿ and [Ca²⁺](in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e. secretion, neurotransmission, proliferation and differentiation. The intracellular pH changes that follow mitogenic activation are complex and may reflect several different H⁺ transporting mechanisms. There are at least three main systems involved in the regulation of pHⁱⁿ in eukaryotic cells: (a) the mitogen stimulated Na⁺/H⁺ exchange, which electroneutrally raises pHⁱⁿ and can be inhibited by amiloride and its derivatives; (b) a variety of HCO₃⁻-based mechanisms which can alkalinize or acidify the cytosol, and can be inhibited by stilbene disulfonate derivatives; (c) and a plasma membrane H⁺-ATPase, which represents the least understood mechanism of pHⁱⁿ regulation. Under non-pathological conditions, pHⁱⁿ regulation is generally achieved by Na⁺/H⁺ exchange and HCO₃⁻-based mechanisms. Missexpression of a H⁺-ATPase in the plasma membrane can lead to a chronically high pHⁱⁿ in some tumor cells and might contribute to carcinogenesis. Chapter I explains the dissertation format and the relationship of the manuscripts included in three appendices. This chapter also indicates my contribution to each of these manuscripts. Chapter II is a summary of the most important findings in these manuscripts. Appendix I deals with the role of Na⁺/H⁺ exchange and Cl⁻/HCO₃⁻ exchange in the regulation of pHⁱⁿ. Appendix II deals with the role of H⁺-ATPase in the maintenance of a chronically high pHⁱⁿ and its possible involvement in tumorigenesis. Appendix III describes a technique to simultaneously measure pHⁱⁿ and [Ca²⁺]ⁱⁿ by fIuorescence spectroscopy. This appendix also describes the application to study the role of pHⁱⁿ and Ca²⁺ in the regulation of cell growth and progesterone secretion.

Thesis (Ph.D.)--University of Texas at Dallas, 2008. / Includes vita. Includes bibliographical references (leaves 115-126)

Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references (leaves 166-205). Also available via World Wide Web.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 107-121). Also available in electronic version.

Due to the diverse functions of Fc eRII, such as its roles in cellular adhesion, growth and differentiation of B and T lymphocytes, rescue of B cells from apoptosis and release of cytotoxic mediators, it is clear why it is believed to be a central molecule in allergic response. Because of its important role in the regulation of IgE production, FceRII may be the primary cause of certain allergic conditions. This study attempted to express and purify a recombinant human soluble FceRII to test its effect on a B-lymphoblastoid (RPMI 8866) and a monocytic (U937) cell line. The protein was expressed in Escherichia coli inclusion bodies, before being refolded and purified in a single gel chromatography step. This pure protein was then tested for biological activity by testing its IgE binding func tion. Once proven functional, it was used to test its effect on the cell lines at three concentrations for its apoptotic rescue properties and its cytokine effects. The recombinant protein did not seem to have any significant effect on the apoptotic rescue of either cell line. While the recombinant sFceRII appeared to have a slight effect on the stimulation of IL-1ß and TNFa in the RPMI 8866 cells, there was no apparent effect on the production of NF?B. In U937 cells, the protein did not seem to have any effect on the stimulation of IL-1ß, TNFa or NF?B. However, the cytokine effects of the recombinant protein were tested on isolated PBMCs from a healthy individual and a hyper-IgE syndrome patient. The recombinant protein was able to stimulate the production of cytokines in both individuals’ PBMCs, proving that it has the same effect as the natural protein. The upregulation of these cytokines indicates that the recombinant protein is able to stimulate the immune system. Therefore, this recombinant soluble FceRII protein could possibly be used for immune therapy.

Note:

Cloudman S91 melanoma cells respond to a variety of endocrine factors, including melanotropins and steroid hormones. The murine S91 melanoma cell line, CCL 53.1, responded to α-melanocyte-stimulating hormone (α-MSH) in a dose- and a time-dependent manner, by increased tyrosinase activity. The minimal effective dose of α-MSH required to stimulate tyrosinase activity was 10⁻⁹M. By prolonging the exposure period of the cells to the hormone from 24 to 48, to 72 hours, the magnitude of tyrosinase stimulation was increased, but the minimal effective dose was not changed. α-MSH action involved elevation of intracellular cyclic AMP levels, and did not require the influx of extracellular calcium. Three melanotropin analogues, [Nle⁴, D-Phe⁷]-α-MSH, Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₁-NH₂, and Ac-[Nle⁴, D-Phe⁷]- α-MSH₄₋₁₀-NH₂, have been shown to be more active than (alpha)-MSH in dispersing melanosomes within amphibian and reptilian integumental melanophores. These analogues also demonstrate prolonged activities and resistance to enzymatic degradation. The unique properties of these melanotropin analogues led to investigate their effects on the phenotypic expression and on the proliferation of S91 melanoma cells. The relative potency and the possible prolonged actions of the three [Nle⁴, D-Phe⁷] -substituted analogues were investigated and compared to those of α-MSH. The melanotropin analogues proved to be 100-1000 fold more active than α-MSH in stimulating tyrosinase activity. These analogues elicited significant tyrosinase activation following brief contact times with the cells, and maintained their stimulatory effects for days after removal from the culture flasks, and after the α-MSH effect had totally dissipated. Contrary to previous reports that melanotropins inhibit the proliferation of melanoma cells, α-MSH and [Nle⁴, D-Phe⁷] -α-MSH stimulated, rather than inhibited, the proliferation of CCL cells under culture conditions that allowed optimal phenotypic expression. Also, the effects of the steroids, β-estradiol, progesterone, and dexamethasone, on CCL cells were studied. Dexamethasone had the most remarkable effects, which involved stimulation of tyrosinase activity and inhibition of proliferation in monolayer culture and in soft agar. Furthermore, this study defined some of the factors that influence the endocrine responsiveness of melanoma cells. The results of this study have important implications for the regulation of phenotypic expression and of proliferation in S91 melanoma cells, and for the properties of the melanoma melanotropin receptor.

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