DISC1, a strong genetic candidate for psychiatric illness, is a molecular scaffold residing in multiple subcellular compartments, where it regulates the function of interacting proteins with key roles in neurodevelopment and plasticity. Both common and rare DISC1 missense variants are associated with risk of mental illness and/or brain abnormalities in healthy carriers, but the underlying mechanisms are unclear. In this thesis, I initially examine the effect of a panel of common and rare single amino acid substitutions on DISC1 subcellular targeting, establishing that the rare mutation R37W and the common variant L607F disrupt DISC1 nuclear targeting in a dominant-negative fashion. This finding predicts that DISC1 nuclear expression is severely impaired in 37W and 607F carriers. In addition, I show that the L607F substitution results in aberrant cytoplasmic and cytoskeletal distribution of DISC1. In the nucleus, DISC1 interacts with the transcription factor ATF4, which is involved in the regulation of cellular stress responses and memory consolidation. Here I show that at basal cAMP levels, wild-type DISC1 strongly inhibits the transcriptional activity of ATF4, and this effect is ablated by 37W and 607F, most likely as a consequence of their defective nuclear targeting. 607F additionally reduces DISC1/ATF4 interaction, which likely contributes to its weakened inhibitory effect. I also demonstrate that DISC1 modulates transcriptional responses to endoplasmic reticulum stress, and that this modulatory effect is also ablated by 37W and 607F. By providing evidence that single amino acid substitutions of DISC1 associated with psychiatric illness impair its regulatory function on ATF4-dependent transcription, I highlight a potential mechanism by which these protein variants may impact on molecular pathways underlying cognition and stress responses, two processes of direct relevance to psychiatric disease.

The Solute Carrier (SLC) superfamily is the largest group of membrane-bound transporters, currently with 456 transporters in 52 families. Much remains unknown about the tissue distribution and function of many of these transporters. The aim of this thesis was to characterize select SLCs with emphasis on tissue distribution, cellular localization, and function.       In paper I, we studied the leucine transporter B0AT2 (Slc6a15). Localization of B0AT2 and Slc6a15 in mouse brain was determined using in situ hybridization (ISH) and immunohistochemistry (IHC), localizing it to neurons, epithelial cells, and astrocytes. Furthermore, we observed a lower reduction of food intake in Slc6a15 knockout mice (KO) upon intraperitoneal injections with leucine, suggesting B0AT2 is involved in mediating the anorexigenic effects of leucine.     In paper II, we studied the postnatal, forebrain-specific deletion of Slcz1, belonging to the SLC18 family, in conditional KO mice (cKO). We observed a decreased response to diazepam and a higher neuronal activity in cortex and hippocampus of cKO mice, as well as an impairment in short-term recognition memory. Intracellular expression was found in neurons but not astrocytes with IHC, indicating SLCZ1 is implicated in neuronal regulation of locomotion and memory.    In paper III, we performed the first detailed histological analysis of PAT4, a transporter belonging to the SLC36 family, involved in the activation of mTOR complex 1 on lysosomes. We found abundant Slc36a4 mRNA and PAT4 expression in mouse brain, using ISH and IHC. We used IHC to localize PAT4 to both inhibitory and excitatory neurons and epithelial cells. We also found both intracellular- and plasmalemmal expression and partial colocalization of PAT4 with lysosomal markers.    Lastly, in paper IV, we provided the first tissue mapping of orphan transporter MCT14 (SLC16A14). Using qPCR, we detected moderate to high Slc16a14 mRNA in the central nervous system and kidney. We found widespread Slc16a14 and MCT14 in mouse brain using ISH and IHC. We also found MCT14 to have intracellular and plasmalemmal expression in mainly excitatory but also inhibitory neurons, as well as epithelial cells. We found MCT14 to be most closely related to MCT8, MCT2 and MCT9, suggesting a similar role for this transporter.

The amino acid requirements of growing horses are currently unknown, and studies suggest that threonine is a limiting amino acid in common horse diets. Thus, the objective of this study was to determine the threonine requirement of growing horses fed two different forage to concentrate ratios using the indicator amino acid oxidation method. The study consisted of a high concentrate phase (HC; 60% concentrate and 40% forage) and a high forage phase (HF; 25% concentrate and 75% forage). Within each phase, 6 female yearling Thoroughbred horses were randomly assigned each of 6 dietary treatments in a 6 x 6 Latin square design. All 6 treatments were identical, apart from varying equimolar ratios of threonine to glutamate. After 6 days of adaptation, blood samples were collected before and after the morning meal for plasma urea nitrogen (PUN) and amino acid analysis. On day 7, horses underwent the IAAO protocol, during which regular breath and blood samples were collected. Phenylalanine flux, oxidation, non-oxidative disposal, and release from body protein, as well as total carbon dioxide production were calculated using plateau enrichment of samples. There was a significant linear effect of threonine intake on plasma threonine concentrations, and PUN had a significant linear response during the HC phase. There was no significant effect of treatment on phenylalanine oxidation during either phase (P ≥ 0.05). It is unlikely that threonine was limiting in the experimental diets.

Microvitellogenin is a female specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly (A)⁺ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by screening the cDNA library with antiserum against microvitellogenin. The results of Northern blot analysis and hybrid selection indicated that the cDNA clone was specific for microvitellogenin. The complete nucleotide sequence of the 834 base pair cDNA insert has been determined by the dideoxy chain termination method. The deduced amino acid sequence was compared with the N-terminal sequence determined by Edman degradation, an amino terminal extension of 17 amino acids appeared to be a signal peptide. The cDNA sequence predicts that the mature microvitellogenin is a protein of 232 amino acids with a calculated molecular weight of 26,201. A comparison of the translated amino acid sequence with the sequences in National Biomedical Research Foundation protein library did not establish any sequence similarity with known proteins. The microvitellogenin gene begins to be expressed in the fat body on the first day of the wandering (prepupal) females as determined by using the cDNA insert as a probe to hybridize with the mRNA for microvitellogenin. The cDNA probe was also used to screen a genomic library of M. sexta, yielding three genomic clones for microvitellogenin. One of them was characterized and it contained the complete microvitellogenin gene. The gene sequence was determined. Comparison to the cDNA sequence showed that the microvitellogenin gene contains an intron near the 5'-end of the non-coding region. The 5'-flanking sequence of the gene has been compared to the same regions of yp genes of Drosophila and vitellogenin genes of locust, some similar sequences have been observed and discussed.

No description available.

The host-guest chemistry between cucurbit[7]uril (CB[7]) and cationic organic guests of medicinal and biological interest are described in this thesis. In the first part, three cationic steroidal neuromuscular blockers (SNMBs) were studied, along with guests that model their monocationic N-alkyl-N-methylheterocyclic (morpholinium, pyrrolidinium and piperidinium) terminal groups of the SNMBs, and dicationic guests in which the two N-methylheterocyclic rings are linked by a decamethylene chain, modelling a variety of NMBs. Other cationic drugs related to acetylcholine processes in neuromuscular blockage were also studied. In the second part, the amino acids lysine, and its mono-, di- and trimethylated and acetylated Nε derivatives, and arginine, and mono- and (symmetric and asymmetric) dimethylarginine, were investigated as guests, along with analogs of arginine. The nature and strength of the complexation between CB[7] and these guests in aqueous solution were determined by 1H NMR spectroscopy and ESI mass spectrometry. The CB[7] showed high binding affinity (KCB[7] = 106-109 M-1) towards the N-alkyl-N-methylheterocyclic cations with a trend of piperidinium > pyrrolidinium > morpholinium, which reflects the relative hydrophobicities of the guests. The CB[7] forms 1:1 and 2:1 host-guest complexes with dicationic model guests, with the CB[7] initially encapsulating the decamethylene chain. The second CB[7] binds to a terminal site, resulting in electrostatic repulsions with the first CB[7], which are resolved by the translocation of the first CB[7] to the opposite terminal site. This 2:1 binding mode is also observed with CB[7] and the SNMBs, and the trend in KCB[7] with these SNMB terminal sites is comparable to that observed for the monocationic model guests. The other cationic drugs also form stable host-guest complexes with CB[7], and the binding constants displayed dependences on the size, charge, and hydrophobicity of the guests. The CB[7] exhibits significant selectivity towards different lysine and arginine derivatives, which can be related to the relative hydrophobicity afforded by the methyl substituents and the positioning of the guest within the CB[7] cavity. The 3500-fold selectivity for Nε,Nε,Nε-trimethyllysine over lysine by CB[7] is the highest observed for a synthetic macrocyclic receptor, while a modest selectivity of symmetrical over asymmetrical dimethylarginine by CB[7] is observed. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-09-26 14:33:40.063

Low molecular weight gelators (LMWGs) are interesting materials whose applications are as diverse and wide ranging as their molecular structures. These materials self-assemble through the formation of non-covelent intermolecular forces and interactions to form supramolecular assemblies that trap solvent within their matrices. Because of the non-covalent nature of the forces of self-assembly, the gelation process is typically thermally reversible. In addition, low molecular weight gelators can also be modified to respond to various stimuli, such as change in pH, presence of enzymes or metal cations, or exposure to light. The design of low molecular weight gelators is often difficult, and most new classes of low molecular weight gelators are discovered by serendipity. As such, it is often useful to use structural templates in the design of LMWGs. Biomolecules, such as steroids, amino acids and peptides, and carbohydrates make excellent templates due to their inherent propensity to self assemble. A review of the current literature regarding the use of biomolecules as templates for the design and synthesis of LMWGs will be presented in chapter 1. Our research group has been active in the research of carbohydrate-based LMWGs for several years, and these results are also briefly reviewed in the related chapters. The synthesis and characterization of ester derivatives of D-galactose, D-glucose, and amide derivatives of D-glucosamine will be discussed in chapters 2-4, along with their evaluation for gelation in aqueous and organic solvents, such as hexane, ethanol, water, and aqueous DMSO or ethanol mixtures.

Thesis advisor: Evan R. Kantrowitz / The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of cellular regulation can be related in some manner to protein conformational changes such as the quaternary conformational changes of allosteric enzymes that alter enzyme activity to regulate metabolism. The control of metabolic pathways by allosteric enzymes is analogous to a molecular valve with "on" and "off" positions. In the "off" position, flow through the pathway is severely hindered, while in the "on" position the flow is normal. For a comprehensive understanding of allosteric regulation we must elucidate in molecular detail how the allosteric signal is transmitted to the active site to alter enzyme activity. In this work we use unnatural amino acid mutagenesis to introduce a fluorescent amino acid into the allosteric binding site of aspartate transcarbamoylase (ATCase), the enzyme responsible for regulation of pyrimidine nucleotide biosynthesis. The fluorescence from the amino acid is exquisitely sensitive to the binding of the allosteric effectors ATP, CTP, UTP, and GTP. In particular we show how the asymmetric nature of the allosteric sites of the enzyme are used to achieve regulatory sensitivity over a broad range of mixed heterotropic effector concentrations as is observed in the cell. Furthermore, employing the method of random sampling - high dimensional model representation (RS-HDMR) we derived a model for how ATCase is regulated when all four nucleotides are present at fluctuating concentrations, consistent with physiological conditions. We've discovered the fundamental requirements to induce the allosteric transition to the R state by showing that although ATCase can accept L-asparagine as an unnatural substrate, the transition to the R allosteric state requires the correct positioning of the alpha-carboxylate of its natural substrate L-aspartate. However, linking the functionalities of L-asparagine and carbamoyl phosphate into a single molecule is sufficient to correctly position the bi-substrate analog in the active site to induce the allosteric transition to the R-state. The cooperative nature of ATCase was further investigated through the isolation of a unique quaternary structure of ATCase consisting of two catalytic trimers linked covalently by disulfide bonds. By relieving the quaternary constraints imposed by the bridging regulatory subunits of the native holoenzyme, the flexibility of the c6 subunit significantly enhanced enzyme activity over the native holoenzyme. Unlike the native c3 catalytic subunit, the c6 species displays homotropic cooperativity for L-aspartate demonstrating that, when two catalytic trimers are linked, a binding event at one or more active sites can be transmitted through the molecule to the other active sites in the absence of regulatory subunits. The catalytic reaction of ATCase follows an ordered sequential mechanism that is complicated by the transition from the T state to the R state upon the binding of the second substrate L-aspartate. Acquiring X-ray crystal structures at each step along the pathway has advanced our understanding of the catalytic mechanism, yet R-state structures are difficult to obtain. Using a mutant version of ATCase locked in the R-allosteric state by disulfide bonds we captured crystallographic images of ATCase in the R state bound to the true substrates (CP and Asp), products (CA and Pi), and in the process of releasing the final product (Pi) prior to reversion of the molecule to the T state. These structures depict the steps in the catalytic cycle immediately before the catalytic reaction occurs, immediately after the reaction, and after the first product has been released from the active site. This work also focuses on developing allosteric inhibitors of the enzyme fructose-1,6-bisphosphatase (FBPase), one of the enzymes responsible for regulation of the gluconeogenesis pathway. Inhibitors of FBPase could serve as potential therapeutic agents against type-2 diabetes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

With the increasing demand for assisted reproduction, more knowledge and understanding towards health requirements of oocytes and their inner workings are required. With current IVF success rates of approximately 40%, oocyte and embryo culture conditions in vitro can be improved by first understanding the finer details of oocyte function. As such, there is a need to better understand the mechanisms through which oocytes can acquire certain nutrients. This thesis focuses on the amino acid serine, which has been shown to improve outcome in developing embryos and also plays a variety of roles in the body that may carry over to oocyte health as well. Using radiolabeled [3H] serine, we measured uptake of serine as a function of time throughout growth and meiotic maturation in mouse oocytes. Serine transport appeared in oocytes during growth and became absent in mature eggs. With a competition assay using substrates diagnostic for several different amino acid transporter systems and culture with and without sodium in the external medium, I identified Na+-dependent SNAT7 of the System A/N (SLC38) family to be the most likely transporter in oocytes. Quantitative RT-PCR was consistent with this result. Transporter activity is also not activated by progression of meiotic maturation, as indicated by unperturbed transport when dbcAMP was provided to maintain meiotic arrest. However, a biological regulator of arrest, NPPC, resulted in enhanced transport activity in vitro. This may be due to signalling mechanisms of the NPPC pathway affecting regulation of serine uptake, which presents a direction for future research.

The linear peptide bisebromoamide was isolated by the Suenaga group in 2009 from the marine cyanobacterium Lyngbya sp. It exhibits antiproliferative activity at nanomolar levels against a wide range of cell lines. Current SAR data indicates that there is some flexibility in the structure with respect to stereochemistry, but the range of modifications that have been biologically tested is limited, as reviewed in Chapter 1. Bisebromoamide contains a number of non-commercial amino acids and an oxopropyl pyrrolidine moiety which had not been found in a natural product previously. Several new synthetic routes towards the non-commercial amino acid fragments have been developed, as described in Chapter 2, including two ring-closure-based approaches to the substituted proline derivative 4-methyl proline (4-MePro). While the presence of six amide bonds makes solid phase peptide synthesis (SPPS) an appealing approach to the synthesis of bisebromoamide, the 4-MePro moiety is attached to a thiazoline and it is well documented that the α-position of an amino acid will racemise, under both acidic and basic conditions, when attached to a thiazoline or oxazoline. Previous reports indicated that the methyl group of the thiazoline was not essential for biological activity and so to increase stability it was replaced with a thiazole. The total synthesis of a series of novel bisebromoamide analogues, via an SPPS approach which enables facile modification of the final structure, is described in Chapter 3. The simple and adaptable SPPS route developed lends itself to SAR studies and allows modifications such as an alanine scan, truncations and incorporation of modified proline derivatives to be achieved rapidly. The promising anticancer activity of bisebromoamide makes the biological activity of these analogues of particular interest and the results of current biological testing are reported in Chapter 4.