Reduced activity of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine site on the NR1 subunit that may be a promising therapeutic target for psychiatric illness. Recently, D-serine has been discovered to be a high-affinity endogenous activator of the NMDAR glycine site. Levels of D-serine in the brain are controlled by its synthesis enzyme serine racemase (Srr) and its catabolic enzyme D-amino acid oxidase (DAO). This work investigates the NMDAR glycine site, D-serine, and D-serine-regulatory enzymes Srr and DAO in the pathophysiology and treatment of symptomatology relevant to schizophrenia and other psychiatric disorders. Pharmacological and genetic mouse models were used to alter glycine site function and D-serine availability. Behavioral responses in these models were assessed. Administration of exogenous D-serine and the glycine transporter 1 (GlyT-1) inhibitor ALX-5407 improved performance of C57BL/6J mice in behavioral tests examining prepulse inhibition (PPI) or latent inhibition (LI). These compounds also reversed impairments induced by the NMDAR antagonist MK-801, and produced similar beneficial effects to the classical atypical antipsychotic clozapine. Mice carrying a point mutation that leads to diminished NMDAR glycine site function demonstrated abnormally persistent LI and deficits in social approach and spatial recognition that were reversible by D-serine or clozapine administration. Similarly, mutant mice that lacked Srr function and had a severe reduction in D-serine displayed impairments in sociability, PPI, spatial recognition and memory. Behavioral deficits in mice without Srr were exacerbated by MK-801 and rescued by treatment with D-serine or clozapine. A genetically-induced loss of DAO function in mice resulted in the elevation of brain D-serine levels, and produced improvements in spatial reversal memory and extinction of a learned response in the Morris water maze, consistent with the effects of exogenous D-serine application in wild-type mice. Thus, deficiencies in NMDAR glycine site function and D-serine availability produce behavioral disturbances that are relevant to the negative and cognitive symptoms of schizophrenia. Activation of the NMDAR glycine site by D-serine, GlyT-1 inhibition, or diminished DAO activity may be beneficial for the treatment of schizophrenia and other psychopathologies involving cognitive dysfunction and persistent repetitive behaviors.

In this study, firstly, bioprocess characteristics for Serine Alkaline Protease (SAP) production, using recombinant Bacillus subtilis carrying pHV1431::subC, were examined. The cell concentration, substrate concentration, SAP activity and SAP synthesis rate profiles demonstrated that the system reaches to a steady state in terms of cell growth and SAP synthesis between t=15-25 h, therefore, this time interval is appropriate to employ both metabolic flux analysis and metabolic control analysis, which apply strictly to steady state systems. After that, three separate perturbations were introduced by addition of aspartate to the production medium at a certain time of the bioprocess. The response of the cells were observed and / by comparing the changes in intracellular reactions of aspartate pathway, Asn, Thr and Ile productions were determined to be the bottlenecks in aspartate pathway and the branchpoints splitting from Asp and AspSa were identified to be weakly rigid branchpoints. Lastly, metabolic control analysis principles were applied to determine the elasticity and flux control coefficients of the simplified aspartate pathway. Aspartate formation reaction and Lys, Thr, Ile, Met producing group share the control of asparagine synthesis. The results revealed that lysine producing branch flux dominates the other branch fluxes, therefore to eliminate bottlenecks and increase SAP production, the activity of the branches leading to the formation of Asn, Thr and Ile should be increased while decreasing the activity of lysine synthesizing branch. This could be achieved either by genetic manipulation or by addition of specific inhibitors or activators to the system.

Serine alkaline protease (SAP, EC 3.4.21.62) produced by Bacillus species, that are the microbioreactors within the bioreactors, is one of the major industrial enzymes. In this study, after production by Recombinant Bacillus subtilis (BGSC-1A751), carrying pHV1431::subc gene in the complex media and separation of solids, SAP was dried by using a spray drier. Experiments were performed to investigate the stabilization of SAP during spray drying and subsequent storage. Initially, the effect of air inlet temperature of the spray drier on SAP activity was evaluated. For this purpose, SAP solutions were spray dried in the absence of any protective agents at five different air inlet temperature (700C, 900C, 1100C, 1200C, 1300C). As a result, increasing air inlet temperature lead to an increase in activity loss of SAP during drying. Thereafter, the effect of protective additives, glucose and maltodextrin (0.5%, 1%, 2% w/v), on SAP activity was investigated during spray drying. The activity loss of SAP was completely inhibited in the presence of glucose at 70, 90 and 1100C. However, the addition of maltodextrin was better than glucose for activity preservation of SAP at 1300C. Among the obtained results, 1% glucose addition was the best to preserve activity of SAP during spray drying. Then, structural change of SAP during drying was investigated. FTIR-ATR spectrum was used to evaluate the change in physical structure of the dried SAP powders in the presence of 1% glucose. From infrared images, at 900C more native-like structure for dried SAP powders was observed. In the last stage of this study storage stability of obtained SAP powders at 40C for a long period (6 months) was investigated. When considering all conditions, 0.5% maltodextrin addition was the best for stabilizing SAP powders along storage time.

Sudden changes in nutrient resources are common in the natural environment. Cells are able to adapt and propagate under changing environmental conditions by making adjustments in their cellular processes. These cellular adaptations involve genome-wide transcriptional reprogramming that results in the induction or repression of metabolic pathways. Specific enzymes are then synthesised and activated to maximise the use of the newly available nutrient sources. L-serine is one of the twenty proteinogenic amino acids, and can be synthesised in yeast by the glycolytic and gluconeogenic pathways when growing on fermentable or non-fermentable carbon sources or taken up from the environment when available. L-serine is metabolically linked to glycine and is a predominant donor of one-carbon units in one-carbon metabolism. L-serine is also a source of pyruvate and ammonia and contributes to other cellular processes including the biosynthesis of cysteine and phospholipids. Previous work has shown that yeast cells exhibit transcriptional induction of the one-carbon pathway and the genes involved in the synthesis of purine and methionine after the addition of 10 mM glycine. Here it is shown that addition of 10 mM L-serine did not, however, elicit the same transcriptional response. This is primarily due to differences in the uptake of glycine and L-serine in yeast. High concentrations of extracellular L-serine were required for yeast to show an increase in intracellular L-serine concentration of the magnitude required to trigger a noticeable cellular response. Despite L-serine and glycine being interconvertable via the SHMT isozymes and being a one-carbon donor, the genome-wide transcriptional response exhibited by cells in response to L-serine addition was markedly different to that seen for glycine. The predominant response to an increase in intracellular L-serine was the induction of the general amino acid control system and the CHA1 gene encoding the serine (threonine) dehydratase. Unlike glycine, addition of L-serine triggered only minor induction of the one-carbon pathway. A large portion of intracellular L-serine was converted to pyruvate and ammonia in the mitochondrion as the result of induction of CHA1. The high intracellular concentration of L-serine stimulated the cell to increase the production of oxaloacetate and to increase the biosynthesis of L-aspartate. Transient increases in the intracellular L-glutamate and L-glutamine were also observed after the addition of L-serine. The work presented in this study shows that large increase in the intracellular concentration of amino acid is required to trigger a significant transcriptional response. Yeast cells exhibit different transcriptional and metabolic responses to the addition of L-serine and glycine even though these two amino acids are closely metabolically linked. Addition of L-serine provokes the GAAC response, expression of the CHA1 gene and stimulates the biosynthesis of L-aspartate in yeast whereas addition of glycine induces the one-carbon pathway which leads to the biosynthesis of the purine nucleotides.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 134-154).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves 113-139).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 192-203). Also available in print.