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Access to abstract permanently restricted to Ball State community only. / Access to thesis permanently restricted to Ball State community only.

A double immunolabelling technique was developed to study microscopically the interactions between CD4, CD45 and the T cell receptor on the surface of rat T cells induced by the phenomenon of co-capping. It was found that both CD4 and CD45 passively co-cap with the actively capped T cell receptor, that the T cell receptor and CD45 passively co-cap with CD4, but that neither CD4 nor the T cell receptor co-cap with CD45. Co-crosslinking and active capping of CD45 with either the T cell receptor or CD4 prevented CD4 or the T cell receptor respectively, from passively co-capping. These experiments were extended to study the effects of particular antibody crosslinking conditions on T cell proliferation and tyrosine phosphorylation. A correlation was found to exist between receptor distribution and the effects of particular antibody combinations on proliferation and tyrosine phosphorylation. The significance of this with respect to T cell activation is discussed. Finally, an observation is reported concerning the failure of some cell lines to cap antibody-crosslinked surface molecules. Preliminary investgations into the nature and extent of the phenomenon are described.

The IL-6 family of cytokines includes IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1, and IL-11. Functioning in a pleiotropic and redundant manner, these cytokines play an important role in the regulation of complex cellular processes such as gene activation, proliferation and differentiation, by signalling through homo- or heterodimers of gp130. This thesis describes the characterization of the interactions between the cytokine oncostatin M (OSM) and the cytokine-binding homology region (CHR) of its receptor gp130. Three forms of OSM were expressed, the native form and two truncated forms. Both mutations were obtained by C-terminal truncation. The first, OSM185, has an 11 amino residue deletion and the second, OSM187, has a 9-residue deletion. A variety of biophysical techniques were applied to investigate the complex. Analytical ultra-centrifugation (AUC), surface Plasma Resonance (SPR) and isothermal titration calorimetry (ITC) studies indicated that the purified proteins were stable in monomeric form and can form a 1:1 complex with affinity in the 0.1 μM range. One of the C-terminal truncated forms, the 187 residues version, showed higher stability than the native OSM (196 residues), but still demonstrated similar binding properties to the gp130-CHR. A ¹⁵N and ¹³C double-labelled OSM187 sample was produced for NMR studies. Due to the size of these two proteins, OSM187 (21.5 kDa) and gp130-CHR (25.2 kDa), the NMR studies of the complex are challenging. Applying the TROSY technique, data were obtained from the labelled OSM187 when it is in complex with gp130-CHR. The data could be compared with the free form OSM187 and several shifted peaks were detected. The binding site mapping work has just begun. The characterized binding properties and methods established for sample preparation provide a solid starting point for later studies. The thesis also contains an exploratory study of interactions between interleukin-2 (IL-2) and the IL-2 receptor β chain.

No description available.

The normal function of the thyroid hormone (TH) system is essential for growth, development and metabolism in humans as well as in other species. The action of TH is dependent on its binding to thyroid hormone receptors (THR) found in the cell nucleus. In some situations, chemicals with structural similarities to TH can bind to these receptors and disrupt their normal function. It has been previously demonstrated that environmental contaminants including, carbamazapine, nonlyphenol (NP), bisphenol A (BPA), and several others are able to bind to the THR as either agonists or antagonists and modulate downstream biochemical responses. Municipal wastewater effluent (MWWE) is a major source of these contaminants entering aquatic environments. Recently extracts of MWWE have been shown to contain chemicals that are capable of binding to THRs. However, MWWE is a complex mixture of chemicals and the specific chemicals have not been identified. In this thesis, a proof of concept was developed for using an Effects Directed Assessment (EDA) approach to isolate thyroid receptor active compounds in MWWE. An EDA is a technique created to extract and identify chemicals from complex mixtures, using various fractionation methods. Once these chemicals have been identified, they are further reviewed for biological relevance. A competitive binding assay for THR was developed and applied to determine the relative binding affinity of known environmental contaminants to THR. Nuclear thyroid hormone receptors were isolated from rainbow trout liver by differential centrifugation. This method involved liver tissue homogenization and subsequent centrifugations to separate the nuclear fraction containing the receptors. The binding characteristics of the isolated THR were evaluated using the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in a competitive binding assay. Minimal binding affinity was present in this assay and future studies should validate the assay further and assure that it is comparable to literature values. Environmental contaminants, including BPA, NP were also tested to determine their relative binding affinity to the THRs compared to the endogenous hormones. High concentrations of both BPA and NP bound to the thyroid hormone receptor, displacing radiolabeled T3 from its binding site. The rainbow trout competitive binding assay was also used to test the binding affinities of extracts from two municipal wastewater effluents collected in the Grand River watershed in southern Ontario. Effluents were extracted using a solid phase adsorbent (HLB Oasis cartridge), eluted with methanol, taken to dryness then reconstituted in ethanol for use in the assay. Both effluent extracts displaced the binding of radiolabeled T3 to the thyroid receptors. The studies demonstrate that a competitive THR assay can be used to detect chemicals in complex mixtures with the potential to interact with THRs. The next step should be to apply the assay using an EDA approach to isolate and identify specific chemicals in effluents that are not yet known to bind to the THR. Interference with the normal function of the TH system has the potential to disrupt normal growth, development and metabolism in aquatic organisms in the receiving environments.

Thesis (MAppSc (Physiotherapy)) -- University of South Australia, 1994

Includes bibliographical references (17 leaves) / vii, 113, [89] leaves, [32] 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. / Generates and exploits activating point mutations in the common ?gb?s subunit (h?gb?sc) of the human GM-CSF, IL-3 and IL-5 receptors. Elucidates the mechanisms by which the isolated mutations confer constitutive activity on h?gb?sc. Identifies the putative all-specific signalling molecules and provides a map of the locations of activating point mutations in h?gb?sc, / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1998