<p> Inappropriate expression of nicotinic acetylcholine receptors in the central nervous system is associated with nicotine addiction, Alzheimer&rsquo;s disease, Parkinson&rsquo;s disease and other disorders. Modulators (drugs) have the potential to restore circuit properties that arise from inappropriate expression of nicotinic receptor&rsquo;s. Compounds that interact with allosteric sites have a distinct advantage over agonists and partial agonists, in that, they retain normal activation patterns by allowing binding of the endogenous ligand. Positive allosteric modulators boost the receptors ability to respond to agonist. Studies of these modulators constitute a first step toward the identification and development of better compounds that minimize signaling errors at cholinergic synapses. We have used single molecule methods to investigate the action of a novel positive allosteric modulator, desformylflustrabromine (dFBr), on nicotinic receptors. Our studies were focused on the &alpha;4&beta;2 subtype of nicotinic receptors in the brain. These receptors exist in two forms with low sensitivity (&alpha;4₂&beta;2₃) or, alternatively, high sensitivity (&alpha;4₂&beta;2₃) to agonist. Our experiments allowed us to develop detailed gating models for high and low sensitivity receptors, as well as gain new insights regarding the mechanisms that underlie potentiation by allosteric modulators. We found that dFBr potentiates low sensitivity receptors by destabilizing desensitized states of the receptor. In contrast, potentiation of high sensitivity receptors arises from a synchronization of openings following an application of agonist due to an increase in the opening rate. Based on our results we now have a better understanding of the advantages of dFBr on high and low sensitivity receptors.</p>

<p> Phenylketonuria (PKU) is an inborn error of metabolism characterized by a loss of phenylalanine hydroxylase activity; an enzyme that metabolizes phenylalanine to tyrosine. Phenylalanine ammonia lyase (PAL) is currently being evaluated as a possible therapy for the management of PKU. PAL catalyzes the conversion of phenylalanine to transcinnamic acid (TCA). Our proposed therapy involves encapsulation of PAL enzyme in ethyl cellulose microcapsules using the spray drying method. PAL activity is markedly reduced due to product inhibition. We hypothesized that the addition of albumin to the PAL reaction mixture would bind the TCA and prevent it from inhibiting PAL. In the first phase of our research, we developed an HPLC assay to quantitate phenylalanine and TCA in the presence of albumin. In the second phase, we determined that albumin completely alleviated product inhibition and enhanced PAL activity. Subsequent ultrafiltration studies showed that albumin acted by extensively binding to and sequestering TCA. PAL microcapsules will be taken orally. In the final phase, we studied the activity of encapsulated PAL in simulated GIT conditions to evaluate the ability of microcapsules to protect PAL enzyme against pH and protease mediated degradation. The activity of encapsulated PAL was lower than an equivalent amount of free PAL possibly due to diffusional limitations to the entry of phenylalanine into the microcapsules. Encapsulation of PAL in ethyl cellulose microcapsules did not protect against acidic pH mediated reduction of PAL activity or pepsin mediated proteolytic degradation. </p><p>

<p> Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (Z_HER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of Z_HER2:342 has limited its pharmaceutical application. Human serum albumin (HSA), as the main protein in plasma, has been commonly used to extend the small peptides serum half-life. Its high solubility, stability and excellent ability to carry multiple ligands in blood stream make it a good candidate for drug delivery.</p><p> Two HSA and Z_HER2:342 fusion proteins, one with a single Z_HER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and the other with two tandem copies of Z_HER2:342 (rHSA-(ZHER2)₂), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)₂, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)₂ treatment has been observed. However, the inhibition effect on HER2-overexpressing cells is cell linedependent. Fusion protein rHSA-(ZHER2)₂ showed preferred accumulation in tumor tissues in xenograft model.</p><p> HSA fusion proteins are easy and economical to express, purify, and formulate. Two formulation strategies have been explored, one is to complex the fusion protein with FA modified chemo drugs, and the other is to make them into nanoparticles. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified fluorescein isothiocyanate (FITC) could be efficiently taken up by cells. FA-Taxol/albumin formulation showed its advantages over Taxol/albumin treatments on <i>in vitro</i> cell growth inhibition. Nanoparticles containing rHSA-ZHER2 produced by desolvation method displayed optimal size distribution, satisfactory stability and preferred binding/uptake on HER2-overexpressing cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.</p><p>

<p> The aim of this thesis is to evaluate the impact of somatostatin receptor subtype-4 (SSTR4) actions on microglia cell viability, towards the understanding and advance of pharmacological treatments for Alzheimer&rsquo;s disease (AD). As of March 2016, there were 5 million people living in the United States with AD. Current drugs for AD have highly variable effects from patient to patient and are palliative at best. This thesis project focuses on the study of the BV2 cell line and the compound NNC 26-9100 (NNC). BV2 cells are immortalized microglial cells that maintain most of their morphology. The data collected suggests that BV2 cells can phagocytize amyloid-? peptides (A?), respond to lipopolysaccharide (LPS), and have the somatostatin receptor subtype-4 (SSTR4). NNC is a selective agonist of the SSTR4 and we have found that it causes BV2 cells to increase in number. The effects of NNC were able to be reduced with a somatostatin receptor pan-antagonist, cyclosomatostatin, and the adenyl cyclase activator forskolin. NNC can alter BV2 numbers by binding to the SSTR4, creating an intracellular cascade that results in the inhibition of adenyl cyclase and an increase in cell count. Collectively, a potential therapeutic mechanism for AD is increasing the number of microglial cells to increase both amyloid beta (A?) phagocytosis and degradation of A? by neprilysin.</p>