A novel truncate of Influenza A M2 protein (residues 22-62), incorporated into a uniquely tailored proteoliposome proton uptake assay, demonstrated proton flux more characteristic of an ion transporter than a traditional ion "channel." The liposome paradigm was essential for testing the conductance activity of this M2 truncate at a range of extraphysiological pHs appropriate for channel vs. transport function determination. In addition to transporter-typical proton flux, M2(22-62) showed the key characteristics of functional integrity: selective proton uptake into liposomes and block of uptake by amantadine. Two sets of proteoliposome proton flux assays were carried out, Set 1 at pH values of 6.5, 6.0. 5.5, 5.0, and 4.5; Set 2 at pH values of 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, and 4.75. Observed flux rates followed a proton transport saturation curve similar to that observed in mouse erythroleukemia cells1. Proton transport was maximal at pH 5.5 in Set 1 (139 H+/second/tetramer) and at pH 5.75 in Set 2 (43 H+/second/tetramer). Amantadine block was strongest at pH 5.5 in Set 1 and 6.25 in Set 2, and apparent desensitization of the protein severely reduced proton flux and amantadine sensitivity below pH 5.5 in both sets of experiments. Decreased external pH increased proton uptake with an apparent pKa of 6 (Set 1) or 6.5 (Set 2). These data indicate acid activation of M2(22-62) between pH 5.5-6, optimal amantadine block between pH 5.5-6.25, and a loss of peptide functionality between pH 5.9-4.7.

This study investigated the effects of the pharmacological manipulation of noradrenergic activities on dopaminergic phenotypes in aged rats. Results showed that the administration of L-threo-3,4-dihydroxyphenylserine (L-DOPS) for 21 days significantly increased the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum and substantia nigra (SN) of 23-month-old rats. Furthermore, this treatment significantly increased norepinephrine/DA concentrations in the striatum and caused a deficit of sensorimotor gating as measured by prepulse inhibition (PPI). Next, old rats were injected with the α2-adrenoceptor antagonist 2-methoxy idazoxan or β2-adrenoceptor agonist salmeterol for 21 days. Both drugs produced similar changes of TH and DAT in the striatum and SN. Moreover, treatments with L-DOPS, 2-methoxy idazoxan, or salmeterol significantly increased the protein levels of phosphorylated Akt in rat striatum and SN. However, although a combination of 2-methoxy idazoxan and salmeterol resulted in a deficit of PPI in these rats, the administration of 2-methoxy idazoxan alone showed an opposite behavioral change. The in vitro experiments revealed that treatments with norepinephrine markedly increased mRNAs and proteins of ATF2 and CBP/p300 and reduced mRNA and proteins of HDAC2 and HDAC5 in MN9D cells. A ChIP assay showed that norepinephrine significantly increased CBP/p300 binding or reduced HDAC2 and HDAC5 binding on the TH promoter. The present results indicate that facilitating noradrenergic activity in the brain can improve the functions of dopaminergic neurons in aged animals. While this improvement may have biochemically therapeutic indication for the status involving the degeneration of dopaminergic neurons, it may not definitely include behavioral improvements, as indicated by using 2-methoxy idazoxan only.

Sphingosine 1-phosphate (S1P) is a lipid chemoattractant molecule. Once formed, S1P can be transported extracellularly by S1P transporters spinster homolog 2 (Spns2) or major facilitator domain containing 2B (mfsd2b). In the extracellular space, S1P can bind to S1P-specific G-protein coupled receptors (S1PR), which initiate many signaling pathways. A critical role of extracellular S1P is its ability to cause lymphocyte egress, which can have implications for inflammatory and autoimmune diseases. For this reason, there has been a growing interest in exploring potential spns2 inhibitors to further elucidate their therapeutic potential. Initial screenings confirmed that fused heterocycles, including phthalimide and benzoxazoles, demonstrated moderate inhibition of Spns2 using a HeLa cell assay. An extensive structure-activity relationship (SAR) study of these scaffolds was performed to analyze the impact of various amine head groups, regioisomers, and alkyl tails on performance. It was determined that 2-aminobenzoxazoles with secondary amines were potent inhibitors of the transporter. Additionally, the position of the lipophilic tail moiety played a large role in activity. From these modifications, SLB1122168 (2.44p) was found to be our lead compound. It was determined that (2.44p) had an IC50 of 94 ± 6 nM and was shown to be efficacious in decreasing lymphocyte count by 55% in a dose-dependent manner in both rat and mice models. The discovery of (2.44p) can serve as a novel chemical tool to investigate Spns2 biology and use it as a probe to determine the potential of Spns2 as a drug target. Organoboron compounds are useful synthetic intermediates in forming C-X, C-C, and C-H bonds. One way to synthesize these compounds is through copper catalysis. Copper is favorable to other transition metals because it is an Earth-abundant, low-cost metal that can be utilized in regio- and stereoselective reactions. Conjugated 1,3-enynes are important functional groups that iii are found in active natural products, organic synthetic intermediates, and materials. Previous methods used rare transition metals, designer ligands, or harsh acidic conditions to synthesize such compounds. In this dissertation, we developed a stereoselective one-pot copper-catalyzed semi-reduction of 1,3-diynes to produce (Z)-1,3-enynes. This method uses Cu(OAc)2, HBpin and Xantphos to successfully synthesize (Z)-1,3-enynes that were tolerated well over a broad substrate scope, including heterocyclic, alkyl, and aryl substituents. It was determined that this reaction went through a 2-boryl intermediate which was facilitated by a CuH species. / Doctor of Philosophy / Autoimmune diseases are caused by immune cells attacking healthy cells. The signaling lipid sphingosine-1-phosphate (S1P) plays a major role in trafficking immune cells, in which immune cells follow the S1P gradient from low concentrations (secondary lymphoid tissues) to high concentrations (lymph). In the case of multiple sclerosis, immune cells can attack healthy neurons that cause a myriad of symptoms. Currently, there are four drugs approved by the Food and Drug Administration (FDA) targeting the S1P pathway for multiple sclerosis. In all cases, these drugs act as S1P-receptor (S1PR) functional antagonists, which decreases the amount of extracellular S1P, which in turn decreases the immune cells in the lymph that can attack healthy cells. Unfortunately, all four drugs exhibit on-target cardiovascular side effects. To circumvent the on-target side effects seen in current FDA-approved drugs, other nodes of the S1P pathway have been assessed for multiple sclerosis. One node of interest is spinster homolog 2 (Spns2), a transporter of S1P, whose inhibition has also been shown to decrease extracellular S1P. In this dissertation, we will be assessing various inhibitors for their in vitro and in vivo properties. 1,3-Enynes are a functional group found in medicinally relevant compounds and can be used as intermediates to make more complex compounds. Current methods to make this functional group use expensive rare metals or harsh acidic conditions. We developed new methods that utilized copper, an abundant metal, and boron, an atom whose empty p orbital allows for unique reactivity. Utilizing a copper-hydride species allowed us to semi-reduce 1,3-diynes to (Z)-1,3-enynes, where water was used instead of acid to allow for the semi-reduction to occur. This reaction was shown to tolerate a wide range of substrates and gave good to excellent yield.

In addition to being involved in protein biosynthesis and metabolism, the amino acid glycine is the most important inhibitory neurotransmitter in caudal regions of the brain. These functions require a tight regulation of glycine concentration not only in the synaptic cleft, but also in various intracellular and extracellular compartments. This is achieved not only by confining the synthesis and degradation of glycine predominantly to the mitochondria, but also by the action of high-affinity large-capacity glycine transporters that mediate the transport of glycine across the membranes of presynaptic terminals or glial cells surrounding the synapses. Although most cells at glycine-dependent synapses express more than one transporter with high affinity for glycine, their synergistic functional interaction is only poorly understood. In this review, we summarize our current knowledge of the two high-affinity transporters for glycine, the sodium-dependent glycine transporters 1 (GlyT1; SLC6A9) and 2 (GlyT2; SLC6A5) and the alanine–serine–cysteine-1 transporter (Asc-1; SLC7A10).

A gene x environment interaction exists in the expression of anxiety for both human and nonhuman primates, such that individuals who are carriers of the (s) allele of the serotonin transporter genotype ( 5-HTT LPR) and exposed to early life stress are more at risk for exhibiting anxiety. The hypothalamic-pituitary-adrenal (HPA) axis has also been implicated in anxiety disorders but the relationship between early life/genotype, HPA activity, and anxiety is not well understood. Further, studies linking the HPA axis to anxiety have relied on "point" samples (blood and salivary cortisol) which reflect moments in time rather than long-term activity. The purpose of this dissertation was three fold: (1) to examine anxious behavior in monkeys with different 5-HTT LPR genotypes and rearing environments across the first two years of life, (2) to compare long-term HPA activity (as measured with hair cortisol) with acute HPA activity (as measured with salivary cortisol) in the same period, and (3) to determine which measure of HPA activity predicts anxiety and/or mediates the rearing/genotype influences on anxious behavior. Infant rhesus monkeys ( Macaca mulatta , N=61) were mother-peer-reared (MPR, n=21), peer-reared (PR, n=20), or surrogate-peer-reared (SPR, n=20) for 8 months, then all relocated into a large social housing situation for the next 18 months. Monkeys were genotyped for 5-HTT LPR and hair and saliva samples were collected for cortisol analysis at months 6, 12, 18, and 24. Behavior was recorded twice per week per subject from 2-24 months and analyzed for the duration of anxiety, social play, and grooming. Regression analysis established predictors of these behaviors. Rearing condition and sex were significant predictors of anxiety across the two years, and HPA activity added significant predictive power in the first six months only. Mediation of the rearing/anxiety relationship by the HPA axis was not evident. Interestingly, hair (but not salivary) cortisol early in life was positively correlated with later anxious behavior. These findings demonstrate the detrimental effects of adverse early life experience on behavioral development and shed light on the interplay between environment, adrenocortical activity, and anxiety. They further demonstrate the usefulness of a long-term measure of HPA activity in predicting later behavior.

Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.

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