Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 7 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (p. [167]-180).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 126-149).

5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) was normally recognised as a Cl- channel inhibitor, but its specificity is in question, since an inhibitory effect against K⁺ channels has been reported. To identify the significance of the molecules structural components, NPPB and related compounds, such as 2-(3-phenylpropylamino) benzoic acid (PPAB), 5- nitro-2-heptylamino benzoic acid (HANB) and 2-nitro-5-heptylamino benzoic acid (HANB-2) were synthesised by reductive amination using various aldehydes and amines. Using internodal cells of the giant green Characean algae, Nitella hookeri, the effects of NPPB and related compounds on cytoplasmic streaming and turgor regulation were determined. Previous experiments stated that cytoplasmic streaming was sensitive to NPPB, PPAB and HANB with IC₅₀ values of 24µmol/L, 455µmol/L, and 6.4mmol/L, respectively. In this report, the IC₅₀ values of purchased NPPB and niflumic acid were found to be 88.65µmol/L and 121.82µmol/L, respectively. Although the IC₅₀ value of purchased NPPB showed a slight difference from that of synthesised NPPB, the results of the cytoplasmic streaming experiment indicated the possibility of this analysis to be a simple assay system for analysing the effects of structural modification to ion channel inhibitors on their biological activity. Moreover, NPPB and PPAB seem to stimulate regulation of turgor pressure under hyperosmotic shock, which can be explained by a blockage of K⁺ efflux during osmotic stress leading to faster recovery of turgor regulation. Additionally, the results of cytosolic free Ca²⁺ analysis using aequorin technology also suggested that the possibility of this analysis to be used as a more direct measure of the inhibitory effect, while the cytoplasmic streaming analysis is a more indirect method. The preliminary results from this research suggest the significance of the simple assay systems for analysing the effects of structural modification ion channel inhibitors, which can be used for future study regarding ion channel structures.

Introduction Human CD36 is a class B scavenger receptor expressed in a variety of cell types including macrophages and endothelial cells. This heavily glycosylated membrane protein has a large ectodomain (ED) responsible for binding a variety of ligands. These include oxidised LDL and long chain fatty acids, which link CD36 to the development of atherosclerosis and insulin resistance, respectively. CD36 has also been implicated in the phagocyte-uptake of apoptotic cells, anti-angiogenic effects in endothelial cells and development of a variety of fibrotic diseases, through interaction with thrombospondin-1. The main objective of this study was to express, purify and characterise the ligand binding ectodomain of CD36 with a view to better understanding how CD36 binds multiple ligands. Methods A baculovirus expression system was used to express and secrete CD36 ED with a 12-His tag from insect cells using an N-terminal secretion sequence. Subsequent purification was performed using nickel affinity chromatography with functionality of the ED assessed through the ability to bind modified LDL in a solid phase binding assay. The N-glycosylation status of the purified ED was explored through use of the N-glycosidase PNGase F and mass spectrometric analysis. Initial studies to measure binding kinetics involved use of surface plasmon resonance (SPR) and binding of commercially available antibodies, mAb1955, mAb1258 and mAbFA6-152, to purified CD36 ED immobilised on an NTA SPR sensor chip. Results The N-glycosylation status of CD36 ED produced in Sf21 and Hi5 insect cells was different with heterogeneous N-glycosylation being identified at individual glycosylation sites. Solid-phase ligand binding revealed that both glycosylated and deglycosylated ED retain affinity for modified LDL. The purified CD36 ED was found to homo-oligomerise particularly at higher concentrations. MAb1955 and mAb1258 were found to bind to the nickel on the sensor chip precluding microkinetic analysis of binding to CD36. However, mAbFA6-152 was found to bind to the immobilised CD36 ED with high avidity and two-step binding kinetics. Conclusions and future direction This study shows for the first time that N-glycosylation is not important for the binding of modified LDL to CD36. Furthermore it was demonstrated that native CD36 ED produced in Sf21 insect cells can be deglycosylated without denaturation, which may be critical for the success of future crystallisation trials. The characterisation of mAbFA6-152 binding by SPR is proof-of-principle that CD36 ED can be studied using this technique paving the way for future biophysical analysis of ligand binding.

Thyroid hormones (TH) exert their multitude of effects on body development, growth and metabolism largely via transcriptional regulatory pathways. TH-induced transcription is controlled by receptors present in the cell nucleus, therefore extracellular TH must first cross the plasma membrane to gain entry into the cell. The exact mechanisms of TH transport across the plasma membrane are only beginning to be clarified, but it is likely that transport may be an important control step for the effects of TH on transcription. Members of the recently cloned organic anion transporting polypeptide (OATP) family have been shown to transport TH. Inhibitor studies indicate that both the aromatic amino acid System T-type transporter, and the broad scope neutral amino acid transporter System L are mediators of TH uptake into various cell types. However cloned amino acid transporters have not been studied to demonstrate directly whether they can accept TH as substrates.

The causes of autism spectrum disorder (ASD) are not all known, but it is suspected that the serotonin transporter (SERT) plays an important role for some subjects with ASD. Mutations in the SLC6A4 gene, that encodes SERT, including the Ala56 mutation (Gly56Ala), have been found in some autism patients. This mutation makes the transporter more active and reduces the probability of serotonergic neurotransmission in the brain, which is linked to behavioral changes that are associated with core domain deficits of ASD 1. Depression also has been linked to decreases in the availability of serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system (CNS), and is associated with reduced hippocampal neurogenesis. Selective serotonin reuptake inhibitors (SSRIs), drugs used to block SERTs, are used to treat depression and/or anxiety by inhibiting SERT to increase synaptic 5-HT levels. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection