Proteins have been known for years as the most important constituent of living matter. Their great abundance in all forms of life have made them the subject of chemical and alchemical investigations since the dawn of science. It was found during the nineteenth century, however, that these relatively complex protein polymers could be decomposed by various means to form their monomers- the amino acids. This process, in fact, takes place during the digestion and ingestion of proteins as food matter. Today, the most important use of the amino acids is as a food. With the scarcity of food so prevalent throughout the world today, it would seem desirable to devise a means of synthesizing a foodstuff from ordinarily non-edible material. Glutamic acid, one of the more common amino acids, occurs to a large extent in any agricultural products, principally core, soy-beans and beet-sugar waste. In addition to its use as a food, the sodium salt of this compound-monosodium glutamate- is very widely used as a flavoring. Moreover, the pure acid has been found recently to have brain stimulating properties and to increase the intelligence of persons taking it medicinally. There are many methods known for the synthesis of this compound. But their disadvantage is that the starting materials are themselves often uncommon and costly items. It was thought from a study of the structure of the acid, that the glutamic acid molecule could be built up from relatively simple materials, since it is only a combination of carbon, water and ammonia. One of the most practical methods devised was first, the production of glutaric acid from propane, via chlorination, followed by the amination of glutaric acid to form the amino acid - glutamic acid. The purpose of this thesis, therefore, was to develop a practical method for the synthesis of glutamic acid, starting with propane, and through the processes of chlorination, nitrilization, hydrolysis and amination, producing this acid. It was further proposed to design a pilot plant for the further carrying out of this work. / Master of Science

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica / d by in vivo micro dialysis. Based on these observations, we conclude that calpain cleavage of fGAD65 occurs under pathological conditions. / by Chandana Buddhala. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Fast excitatory neurotransmission is critical for learning and memory, and its dysregulation is linked to numerous neurological diseases. These include developmental diseases such as fragile X syndrome, psychiatric disorders like schizophrenia, and chronic neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Throughout the central nervous system, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors (AMPARs) mediate the fastest excitatory neurotransmission. In response to the neurotransmitter glutamate, AMPARs open their ion channels and allow cation flux through the post-synaptic membrane. This initiates rapid depolarization and signaling in the post-synaptic neuron. Nearly all AMPARs exist as complexes with auxiliary subunits, which are regulatory proteins that modulate receptor assembly, trafficking, pharmacology and function. These auxiliary subunits determine brain region-specific AMPAR signaling, and aberrancies in complex formation or function lead to neuropathologies. Despite their importance for CNS signaling and implication in neurologic disorders, the structural bases underlying the function of AMPARs and AMPAR complexes remain ambiguous, representing a critical barrier to our understanding of excitatory neurotransmission. As a consequence, structure-based design of neuro-therapeutics is largely undeveloped: there is only a single FDA-approved drug targeting AMPARs. To address these problems, I wanted to dedicate my thesis work to study AMPAR synaptic complexes across an array of functional states and provide a new foundation for our structural understanding of AMPAR signaling. First, I designed a covalent-fusion construct approach to guarantee assembly and expression of AMPAR synaptic complexes in heterologous cells (HEK293). Then, I developed purification protocols allowing me to obtain chemically homogenous and pure complex protein. Since synaptic signaling is highly dynamic, complexes of AMPARs with auxiliary subunits are conformationally heterogeneous and are not amenable to X-ray crystallography. Cryo-electron microscopy (cryo-EM) enabled me to approach these complexes structurally, where I could collect data and parse out heterogeneity through image classification. With cryo-EM, I solved the structure of an AMPAR bound to the auxiliary subunit stargazin, which promotes AMPAR activation. This work provided the first structural information on how AMPARs form complexes with regulatory subunits. In a following study, I solved the structure of an AMPAR in complex with a functionally distinct auxiliary subunit, GSG1L. In contrast to stargazin, GSG1L promotes inactivation and desensitization of AMPARs, thus having a neuroprotective effect. To further characterize the function of these auxiliary subunits, I designed chimeras between stargazin and GSG1L and examined their function electrophysiologically. This experiment revealed that AMPAR auxiliary subunits have a modular design, where variable extracellular domain regions, supported by a conserved transmembrane α-helical bundle, distinctly regulate function of the core AMPAR. This study provided the first evidence of how brain region-specific expression patterns of similarly-structured auxiliary subunits may contribute to unique AMPAR functions. More recently, I’ve taken advantage of the modulatory effects of stargazin on AMPARs and I applied cryo-EM to an AMPAR-stargazin complex. This study determined how AMPARs are activated by the neurotransmitter glutamate, and revealed a novel mechanism by which glutamate binding induces opening of AMPAR ion channels. Our data show that two-fold symmetric kinking of ion channel helices allows cation flux into neurons, which triggers neurotransmission. Importantly, this study also provides insights into how mRNA editing and patient-derived disease mutations in the transmembrane (i.e., resulting in aberrantly firing of receptors during epilepsy) reshape AMPAR function and excitatory neurotransmission. Collectively, the findings from my thesis work provide a new paradigm for the molecular-level understanding of glutamatergic neurotransmission throughout the CNS. These studies lay the groundwork for new directions in precision-medicine design of therapeutics targeting brain region-specific AMPAR synaptic complexes in neurological diseases.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-21T18:03:11Z No. of bitstreams: 1 FranciscoMarceloVictor.pdf: 2391704 bytes, checksum: 4edeacd54e236a943cfebb1187a37c40 (MD5) / Made available in DSpace on 2017-09-21T18:03:11Z (GMT). No. of bitstreams: 1 FranciscoMarceloVictor.pdf: 2391704 bytes, checksum: 4edeacd54e236a943cfebb1187a37c40 (MD5) Previous issue date: 2017-06-30 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / In this work, DL-glutamic acid monohydrate crystals (DLAGM) were grown by the technique of slow evaporation of the solvent. Initially the crystal was characterized by Xray diffraction using the powder method and its structural parameters adjusted by the Rietveld method, and thus, its orthorhombic crystal structure of space group Pbca (D2h), with eight molecules per unit cell. Raman scattering measurements were performed and the vibrational normal modes of DLAGM crystal were tentatively assigned using, mainly, glutamic acid polymorph crystals. The DLAGM crystal was studied by Raman spectroscopy under conditions of high hydrostatic pressures up to 6.9 GPa, as well as by X-ray diffraction in the range of 3.0-15.0 GPa. Changes in the lattice modes show that the crystal undergoes a phase transition in the pressure range of 5.1-5.7 GPa characterized by the disappearance and splitting of bands as well as the discontinuity in the slope dω/dP of these vibrational modes, modifications in vibrational internal modes (NH3 +, CO2-, H2O) and appearance of new bands. In addition, decompression at ambient pressure shows that the phase transition is reversible. For pressure XRD measurements, a phase transition was observed in the same pressure range as in the Raman experiment (5.0-5.8 GPa), characterized mainly by the appearance of new peaks at 5.8 GPa. Comparing such behaviora with those of L-glutamic hydrochloric, α-glutamic and β-glutamic acids, a great stability is observed for DL-glutamic monohydrate. / Neste trabalho, cristais de DL-ácido glutâmico monohidratados (DLAGM) foram crescidos pela técnica de evaporação lenta do solvente. Inicialmente o cristal foi caracterizado por meio de difração de raios X usando o método do pó e seus parâmetros estruturais ajustados pelo método de Rietveld, e assim, constatando sua estrutura cristalina ortorrômbica de grupo espacial Pbca (D2h), com oito moléculas por célula unitária. Medidas de espalhamento Raman foram realizadas e os modos normais de vibração do cristal foram atribuídos tentativamente usando-se, principalmente, cristais de polimorfos do ácido glutâmico. O cristal de DLAGM foi estudado por espectroscopia Raman em condições de altas pressões hidrostáticas em até 6,9 GPa, bem como por difração de Raios X no intervalo de 3,0-15,0 GPa. Mudanças nos modos de rede mostram que o cristal sofre uma transição de fase no intervalo de pressão de 5,1-5,7 GPa, caracterizada pelo desaparecimento e splitting de bandas, bem como a descontinuidade na inclinação dω/dP destes modos vibracionais, modificações nos modos internos (NH3 + , CO2 - , H2O) e aparecimento de novas bandas. Além disso, a descompressão à pressão ambiente mostra que a transição de fase é reversível. Para as medidas de DRX com pressão, foi verificado uma transição de fase na mesma faixa de pressão daquela observada no experimento Raman (5,0-5,8 GPa), caracterizada principalmente pelo aparecimento de novos picos em 5,8 GPa. Comparando tais comportamentos com os do ácidos L-glutâmico hidrocloridrico, α-glutâmico e β-glutâmico percebe-se uma grande estabilidade para DL-glutâmico monohidratado.

Thesis (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Elizabeth Tietz. Includes abstract. Title from title page of PDF document. Bibliography: pages 88-94, 130-136, 178-189, 218-266.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 117-119). Also issued on the Internet.

published_or_final_version / Physiology / Master / Master of Medical Sciences

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by eight bacteria (B. subtilis 23856, B. subtilis 23857, B. subtilis 23858 B. subtilis 23859, B. subtilis natto, B. licheniformis 1525, B. licheniformis 6816 and B. licheniformis 9945a) in GS and E media. B. subtilis natto and B. licheniformis 9945a have been investigated extensively for γ-PGA production, however, the remaining six have not previously been used. Using the eight bacteria, yields of up to 22.3 g/l were achieved in shake flasks. On characterization, it was observed that γ-PGA with different properties (crystallinity, acid/salt form and molecular weights ranging from 3,000 Da to 871,000 Da) was produced. Production of γ-PGA by B. subtilis natto in GS medium was scaled up using a fermenter and was tested for novel probiotic applications. The survival of probiotics during freeze drying, storage and ingestion was improved by combining them with a γ-PGA matrix. For L. paracasei, 10% γ-PGA protected the cells significantly better (P < 0.05) than 10% sucrose during freeze drying, whereas for B. longum and B. breve, it showed comparable cryoprotectant activity (P > 0.05) to 10% sucrose. This study also demonstrated the potential use of a non-dairy foodstuff (orange juice) for delivery of probiotics. Two Bifidobacteria strains protected with γ-PGA survived significantly better (P < 0.05) in orange juice for 39 days, with a log reduction in viability of less than 2.99 CFU/ml, when compared to unprotected cells, which showed complete loss in viability by day 20. In addition, γ-PGA protection improved survival of Bifidobacteria in a solution mimicking the environment of the stomach. γ-PGA-protected Bifidobacteria showed little (< 0.47 log CFU/ml) or no loss in viability when stored in simulated gastric juice (pH 2.0) for four hours, whereas unprotected cells died within two hours.