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nÃo hà / O trabalho realizado nesta tese visa a cristalizaÃÃo do Ãcido glutÃmico, favorecendo a obtenÃÃo da fase α (metaestÃvel) em relaÃÃo ao polimorfo β. ApÃs a obtenÃÃo dos cristais realizaram-se as medidas experimentais de fluorescÃncia, com vistas a comparaÃÃo com os dados teÃricos obtidos, considerando as limitaÃÃes do mÃtodo de simulaÃÃo ab-initio utilizado. Os cÃlculos de primeiros princÃpios foram realizados para estudar as propriedades do cristal do Ãcido glutÃmico nas modificaÃÃes α e β no que se refere ao aspecto estrutural, eletrÃnico (estrutura de bandas) e Ãptico (funÃÃo do complexo dielÃtrico), tudo isto em relaÃÃo ao polimorfismo conformacional. A teoria do funcional da densidade (TFD) à considerada usando uma base de ondas planas, pseudopotenciais ultramacios, e o potencial de correlaÃÃo-troca dentro do Generalized-Gradient Approximation (GGA). Os parÃmetros de rede estÃo de acordo com os resultados experimentais, embora na forma α à previsto ter um gap indireto entre os pontos de alta simetria  e X, igual a 4.69 eV, para a forma β nÃo pode-se tirar uma conclusÃo sobre o gap direto (-), devido a imprecisÃo do cÃlculo. A funÃÃo dielÃtrica de ambos os polimorfos sÃo muito semelhantes no caso de uma amostra policristalina, mas difere fortemente no caso da polarizaÃÃo da luz das faces do cristal 100 e 010. / Done the work accomplish in this thesis it seeks the crystallization of the glutamic acid, favoring the obtaining of the phase α (metastable) in relation to the polymorph β. After the obtaining of the crystals they did take place the experimental measures of fluorescence, with views the comparison with the obtained theoretical data, considering the limitations of the method of simulation used ab-initio. Were the calculations of first principles accomplished to study the properties of the crystal of the glutamic acid in the modifications α and β in what she refers to the aspect structural, electronic (structure of bands) and optical (function of the dieletric complex), everything this in relation to the polymorphism conformational. The theory of the functional of the density (TFD) is it considered using a base of plane waves, pseudo potential ultrasoft, and the correlation-change potential inside of the Generalized-Gradient Approximation (GGA). Are the net parameters in agreement with the experimental results, although in the form α is it foreseen to have an indirect gap between the points of high symmetry and X, equal to 4.69 eV, for the form β a conclusion cannot be reached on the direct gap (-), due to imprecision of the calculation. The function dieletric of both polymorph are very similar in the case of a sample polycrystal, but it differs strongly in the case of the polarization of the light of the faces of the crystal 100 and 010

Orientador: Ranulfo Monte Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-25T07:40:59Z (GMT). No. of bitstreams: 1 Brito_PriscilaNunes_M.pdf: 1128086 bytes, checksum: 7b2118f1fa20cddb36c2bd8dd3949e0f (MD5) Previous issue date: 2014 / Resumo: A utilização industrial de matérias-primas obtidas por fontes renováveis é essencial para o desenvolvimento sustentável da sociedade moderna. A necessidade de se utilizar de processos catalíticos "verdes", ecologicamente corretos para converter fontes renováveis em commodities químicos, dentre eles os biopolímeros está mobilizando pesquisadores do mundo inteiro. Dentre os diversos polímeros biossintetizados, encontra-se o gama-PGA, que é solúvel em água, aniônico, biodegradável e biocompatível, com amplas aplicações nas áreas química, médica, ambiental e de alimentos. Este trabalho buscou a otimização da obtenção do ácido gama-poliglutâmico (gama-PGA) por fermentação em estado sólido utilizando resíduo fibroso de soja como fonte de carbono. A espécie selecionada para este estudo foi Bacillus velezensis e Bacillus subtillis onde três linhagens foram testadas, selecionando-se a que apresentou melhor crescimento e produção de gama-PGA. Além disso, foram mensurados os fatores como: aeração e umidade do meio de fermentação. O estudo de otimização foi realizado utilizando a estratégia de planejamentos experimentais. O meio sólido que maximizou a produção foi fixado em 9:1 de sub-produto fibroso de soja e casca de arroz, possibilitando a máxima produção de 46,19 g de gama-PGA/ kg de meio sólido (b.s.), sem adição do precursor ácido glutâmico, sendo assim um meio viável para produção de gama-PGA com menor custo de processo / Abstract: The industrial use of raw materials obtained from renewable sources is essential to the sustainable development of modern society. The need to use catalytic processes "green" ecologically correct to convert renewable commodity chemicals, including biopolymers is mobilizing researchers worldwide. Among the various polymers biosynthesized, is the gama-PGA, which is water-soluble, anionic, biodegradable and biocompatible, with wide applications in the chemical, medical, environmental, and food. This study aimed to optimize the obtaining the ?-polyglutamic acid (gama-PGA) by solid state fermentation using soy fibrous residue as a carbon source. The species selected for this study was Bacillus subtilis and Bacillus velezensis where three strains were tested, selecting the one that showed better growth and production of gama-PGA. In addition, we measured factors such as aeration and moisture from the fermentation medium. The optimization study was performed using the strategy of experimental design. The solid medium which maximized the yield was fixed at 9:1 fibrous byproduct of soybean and rice hulls, allowing for maximum yield of 46.19 g of gama-PGA / kg of solid medim (bs) without addition precursor glutamic acid, and thus a viable means for producing gama-PGA with lower process cost / Mestrado / Engenharia de Alimentos / Mestra em Engenharia de Alimentos

Orientador: Alan Stewart Hazell / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T22:16:15Z (GMT). No. of bitstreams: 1 FaimFerreira_Samantha_M.pdf: 1383020 bytes, checksum: 557a29a1393de153dfad4f2c4815defa (MD5) Previous issue date: 2014 / Resumo: A neurotoxicidade por manganês (Manganismo) leva a uma disfunção neurológica caracterizada pelo desenvolvimento de ataxia, hipocinesia, rigidez e tremores. Evidências sugerem que os astrócitos desempenham um papel importante na disfunção cerebral nesta desordem, pois acumulam manganês e sequestram o metal na mitocôndria, o que inibe a oxidação fosforilativa. A exposição aguda ao manganês leva ao acúmulo focal do metal e perda neuronal no cerebelo. No entanto, a relação entre esta deposição localizada de manganês e as manifestações neurológicas não são claras. Neste estudo, foram caracterizados os efeitos do manganês na marcha e no equilíbrio após um tratamento subagudo em ratos adultos da linhagem Sprague-Dawley (CEMIB ¿ UNICAMP). Os animais foram divididos em três grupos: Controle; tratado com Mn e tratado com Mn + NAC (N - acetilcisteína), um anti-oxidante. Os ratos do grupo Mn receberam cloreto de manganês (II) (50 mg/kg de peso corporal, i.p.) diariamente durante 4 dias, enquanto os ratos do grupo Mn + NAC foram co-tratados diariamente com o cloreto de manganês (II) e NAC (163 mg/kg, i.p.). Na análise da marcha o grupo tratado com Mn demonstrou alteração na marcha, vizualizados pela diminuição da área da impressão plantar (comprimento x largura), do comprimento da passada e da base de suporte. Apesar das alterações observadas nesses parâmetros, os animais não apresentaram mudanças na pressão exercida pela pata durante a marcha. O grupo co-tratado com NAC não demonstrou essas alterações, apresentando-se semelhante ao controle. Nos estudos de imonohistoquímica, imunofluorescência, histoquímica e Western blotting o grupo tratado com Mn apresentou morte neuronal, aumento da reatividade astrocítica na camada granular, desarranjo na camada das células de Purkinje e aumento na expressão do transportador de glutamato GLT-1a. Estes resultados corroboram com as importantes alterações na função motora de animais tratados com Mn. O co-tratamento com o antioxidante NAC foi capaz de impedir parcialmente esses danos, exercendo uma ação protetora na área do cerebelo e na função motora. Em conclusão, demonstramos que a intoxicação por manganês gera alterações morfo-funcionais no cerebelo, as quais podem ser principalmente revertidos pelo uso do antioxidante NAC / Abstract: Manganese (Mn) neurotoxicity (Manganism) leads to a neurological disorder characterized by the development of ataxia, hypokinesia, rigidity and tremors. Evidence suggests that astrocytes play an important role in brain dysfunction in this disorder because accumulate manganese and sequester metal in the mitochondria, where it inhibits oxidative phosphorylation. Acute exposure to manganese leads to focal accumulation of the metal and neuronal loss in the cerebellum. However, the relationship between this localized deposition of manganese and neurological manifestations are unclear. In this study, we characterized the effects of manganese in gait and balance after a subacute treatment in Sprague - Dawley rats (CEMIB - UNICAMP). The animals were divided into three groups: control, treated with Mn and Mn + treated with NAC (N - acetylcysteine). The rats from manganese group were administered with manganese (II) chloride (50 mg / kg body weight , ip) daily for 4 days, whereas rats of the group Mn + NAC daily were co- treated with manganese chloride (II) and NAC (163 mg / kg , ip). In Catwalk test group treated with manganese showed changes in gait and balance, leading to a reduction of the area of the paw (length and width), the stride length and the base of support. Despite the changes observed in these parameters, the animals showed no changes in pressure exerted by the leg during gait. The group co-treated with NAC showed no such changes, keeping similar to the control group. In studies of immunohistochemistry, immunofluorescence, western blotting and histochemistry group treated with showed neuronal death, an increase in signal astrocytic reactivity in the granular layer, a derangement in the Purkinje cell layer and an increase in the expression of glutamate transporter GLT - 1a. These results suggest significant changes in motor function in animals treated with Mn. The co - treatment with the antioxidant NAC was able to partially prevent this damage, exerting a protective action in the area of the cerebellum and motor function. In conclusion, we demonstrated that manganese poisoning produces morphological and functional changes in the cerebellum, which can principally be reversed by the use of the antioxidant NAC / Mestrado / Fisiopatologia Médica / Mestra em Ciências

Orientador: Ranulfo Monte Alegre / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T06:43:41Z (GMT). No. of bitstreams: 1 Moraes_LuanaPereirade_D.pdf: 2084726 bytes, checksum: 11e04e791877c99a08c60405caa33111 (MD5) Previous issue date: 2014 / Resumo: O ácido y-poliglutâmico, também conhecido como y-PGA, é um polímero largamente estudado com capacidade de produzir polímeros e monômeros de ácido glutâmico quando hidrolisado. Por ser solúvel em água, biodegradável e atóxico, o y-PGA é aplicado em indústrias alimentícias, cosméticas e medicina. Com o objetivo de estudar a produção de y-PGA e sua hidrólise, foram avaliadas diferentes cepas de Bacillus spp e condições experimentais, além disso, meios de cultivos sem a presença do ácido glutâmico a partir de subprodutos industriais, como glicerol e melaço. O estudo iniciou com a escolha do meio de cultivo e aplicação de substratos alternativos e baixo custo. Foram determinadas as possíveis espécies do Bacillus produtoras de y-PGA, analisou-se os componentes do meio fermentativo e as condições de cultivo que interferem na produção de y-PGA através do planejamento experimental e aplicou-se um delineamento composto central rotacional (DCCR) para otimização das condições de cultivo. Por fim, avaliou-se o consumo de y-PGA e a hidrólise de y-PGA pela enzima presente no meio de cultivo. O micro-organismo Bacillus velezensis NRRL B-23189 foi o selecionado para a produção de y-PGA. A produção de y-PGA em meio de cultivo sem ácido glutâmico contendo glicerol (100 g/L) foi otimizado, obtendo-se o ponto ótimo em meio cuja composição foi de sulfato de amônio, 8 g/L, ácido cítrico, 20 g/L, temperatura de 27 °C, agitação de 200 rpm, pHinicial 6,5 em cultivo submerso conduzido por agitador orbital. Nas condições otimizadas a produção de y-PGA sem utilização do ácido glutâmico foi de 4,17 g/L. Ao substituir o glicerol por melaço, obteve-se uma produção de 4,82 g/L do y-PGA em meio contendo melaço, 200 g/L, sulfato de amônio, 8 g/L, ácido cítrico, 12,5 g/L , temperatura de 27 °C, agitação de 200 rpm, pHinicial 6,5 em cultivo submerso conduzido por agitador orbital. A avaliação das condições de cultivo em biorreator com capacidade de 5 L, agitação de 500 rpm, taxa de aeração de 2 VVM, 27 °C e as mesmas condições otimizadas, mostrou um aumento na produção do y-PGA quando o subproduto de glicerol foi utilizado, a produção de y-PGA foi de 28 g/L. Um aumento na produção de ?-PGA para 6,69 g/L foi determinado quando utilizou melaço. O Bacillus velezensis NRRL B-23189 utiliza o y-PGA como fonte de carbono quando outras fontes não estão no meio fermentativo. Para isto o microorganismo libera uma enzima no meio de cultura responsável pela hidrólise do y-PGA / Abstract: The poly(y-glutamic acid), also known as y-PGA, is a polymer that much research has been done on this metabolite and it produces glutamic acid polymers and monomers when hydrolyzed. y-PGA is a water-soluble, biodegradable and non toxic biopolymer with applications in medicine, food, cosmetic industries. This work aims to study the process of y-PGA production and hydrolysis. Bacillus strains and experimental conditions had been determined, as well as different culture conditions without glutamic acid as alternative industrial substrates by-products (glycerol and molasses). Firstly, culture media and alternative and cheap substrates were selected and Bacillus strains was evaluated. A fractional central composite design was utilized to evaluate the effects of fermentative compounds and culture conditions on y-PGA production. The optimization of culture conditions was determined using central composite design (CCD). Finally, consumption and enzymatic hydrolysis of y-PGA in culture media was studied. The Bacillus velezensis NRRL B-23189 strain was selected. The culture media without glutamic acid with 100 g/L of glycerol was optimized. The CCD showed the best y-PGA production in culture media with ammonium sulphate, 8 g/L, citric acid, 20 g/L, the temperature of 27 °C, the agitation of 200 rpm, the initial pH 6,5 in shaker. Under optimized conditions, the production of y-PGA was 4,17 g/L. The production of y-PGA was 4,82 g/L when the molasses, 200 g/L, was supplemented with ammonium sulphate, 8 g/L, citric acid, 12,5 g/L, temperature of 27 °C, agitation of 200 rpm, initial pH 6,5 in shaker. Experimental conditions of 5 L in stirred bioreactor with stirring rates of 500 rpm, aeration rate of 2 VVM, the temperature of 27 °C and same optimal conditions, showed higher y-PGA production than when used glycerol in culture media, 28 g/L. When it was used molasses the y-PGA production was 6,69 g/L. The Bacillus velezensis NRRL B-23189 uses y-PGA as only carbon source or when the carbon at the medium was not sufficient. Thus, the microrganism produces the enzyme that hydrolyses y-PGA / Doutorado / Engenharia de Alimentos / Doutora em Engenharia de Alimentos

Several biological abnormalities exist between the ruminant and nonruminant small intestine and influences carbohydrate assimilation. Two experiments were conducted to identify potential mechanisms to improve carbohydrate utilization in cattle. Experiment 1 evaluated the effects of duodenal starch infusions with casein or glutamic acid on post-ruminal carbohydrase activities. Experiment 2 evaluated the effects of dietary fructose on visceral organ development and expression of nutrient transporters and digestive enzymes involved in carbohydrate assimilation. In experiment 1, the results suggest that small intestinal starch digestion may be improved in cattle with increased small intestinal flow of casein through increases in post-ruminal carbohydrase activities. In experiment 2, dietary fructose supply influenced nutrient utilization, visceral organ growth, and digestive enzyme mRNA expression and activity in neonatal calves.

Glutamate is essential for proper brain function as it is our nervous systems principal excitatory neurotransmitter, a signal that stimulates nerve cells to send messages to other cells. Glutamate activates ionotropic glutamate receptors (iGluRs), which are linked to several neurological diseases in cases when they are improperly regulated. iGluRs are transmembrane channels that allow calcium, as well as other cations, into the post synaptic neuron upon binding of glutamate or other agonists.Interestingly, iGluR homologs in plants also mediate calcium signaling upon glutamate activation and were accordingly named glutamate receptor-like channels (GLRs). Cell signaling is critical for plant survival to mediate rapid response to growth, defense, and other environmental cues. GLRs are found in all plants and vital for their health, hardiness, and adaptation for growth and survival in unfavorable conditions, such as drought, nutrient poor soil, temperature extremes, pathogens, and predators. Plant research is important with vast applications. Firstly, crops are our primary source of nutrition. In addition, plants are used as sources of drugs that we employ for treating diseases. Some examples of plant-derived neuroactive compounds include caffeine in coffee beans, nicotine in tobacco, and opium from poppy plants. In short, optimizing plant growth is beneficial to maintaining our own survival and potentially achievable by understanding GLRs role in plant health and hardiness. Despite their importance for cell signaling and implication in plant defense and regeneration, the structural basis underlying the function of these channels remains ambiguous, representing a critical barrier to our understanding of GLR function. To address this problem, I dedicated my thesis work to study the structure of GLRs and gain insight into their function. There are 20 GLRs in the model plant organism, Arabidopsis thaliana, classified into 3 different clades (AtGLR1-3). To narrow down which AtGLRs to focus our structural studies on, we investigated clade 3 representatives, as many of these GLR3s have been extensively studied in different plant species, especially crops. For example, studying AtGLR3.4 could provide useful information to how the homolog in rice, OsGLR3.4, contributes to growth and production in rice. Studying AtGLR3.4’s structure may elucidate how agonistic or antagonistic targets bind and gate the channel, potentially revealing “druggable” targets to alter plant response for defense and regeneration. Without any structural information available for GLRs, I started my studies by first focusing on their mammalian homologs, iGluRs. I first designed multiple constructs for heterologous expression and purification from cell culture (for example HEK293S GnTI- cells). Then, I optimized protein extraction and purification to obtain pure protein samples. Purified proteins were then subjected to cryo-electron microscopy (cryo-EM) which eventually allowed us to solve the structure of AtGLR3.4, the first full-length GLR structure. AtGLR3.4’s structure revealed similarities to structures of its mammalian homologs, iGluRs. In comparison to iGluRs, our GLR structure also showed tetrameric subunit assembly, with a three-layer architecture that includes the ligand binding domain (LBD) in the middle, sandwiched between the extracellular amino terminal domain (ATD) at the top and the transmembrane domain (TMD) at the bottom. In contrast to the majority of iGluR structures, however, AtGLR3.4 displayed unique symmetry and domain arrangement with the non-swapped extracellular ATD and LBD domains. We also provided further evidence supporting ligand binding promiscuity that was previously revealed in isolated LBD crystal structures from other AtGLR3s. Surprisingly, we found endogenous glutathione bound to the ATDs and demonstrated its contribution to channel activity. It is important to fill the gaps in knowledge about GLR structure to understand how these channels are activated and gated. In doing so, we will learn more about iGluRs as well as better understand plant defense and growth, which has the potential to enhance crop production for food security and our overall survival.

No description available.

Myriad small molecule compounds targeting metabotropic glutamate receptors (mGluRs) have been investigated for the treatment of various neuropsychiatric diseases and displayed promise in preclinical studies. At the clinical level, many of these compounds have been well tolerated by human subjects but have eluded success as promising therapeutics. There are eight subtypes of mGluRs, which express as constitutive dimers. This dimerization can occur between identical (homodimerization) or different (heterodimerization) mGluR protomer subtypes, which are subject to pairing-specific signaling mechanisms. Subtype expression of mGluRs is heterogenous between brain regions and cell types, yielding probable cell-specific homo- and heterodimer combinations that respond differently to certain drugs. While G protein recruitment to active mGluR dimers has been studied extensively, little is known about arrestin recruitment to these receptors. I used bioluminescence resonance energy transfer (BRET) assays, which provide a quantitative measure of protein-protein proximity, to observe and quantify arrestin recruitment to specific mGluR subtype pairings upon ligand administration in heterologous cells. I studied how select allosteric ligands affect communication between protomers to enhance arrestin recruitment to dimers. My findings indicate that arrestin recruitment occurs only at select mGluR homodimers upon orthosteric stimulation but is frequently stimulated or enhanced by administration of activating allosteric ligands. Additionally, I found that trans-protomer communication is highly specific to mGluR protomer subtype pairings, the ligand administered,a nd inter-protomer signal direction. Lastly, my findings reveal a cooperative effect of mGluR2 and 3 heterodimerization on arrestin recruitment that is dependent on the functional ability of each protomer to bind orthosteric agonist and responds distinctively from homodimers to stimulation by certain allosteric ligands. Taken together, this work shows that mGluR signaling can be tuned using strategic pharmacology and energizes hope for future clinical success of mGluR-targeting ligands.

For glutamate and the developing optic pathway, glutamate and its ionotropic receptor subunits are expressed widely in retina and ventral diencephalon, and in cells that are related to the chiasm formation. These studies indicate that glutamate may act as a communicator or attractor to coordinate with other factors to affect the retinal axon pathfinding in the prenatal optic pathway. / Furthermore, for the function of glutamate, AMPARs and KARs in the optic chiasm formation, we did retinal explant culture experiment at E14 in vitro, with application of different concentration of L-glutamate (500muM -1mM), AMPAR antagonists: CP465022 hydrochloride (2-20muM) and GYK15466 dihydrochloride (25-150muM), and KAR antagonists: CNQX (50-500muM) and UBP301 (5-25muM). The results show that L-glutamate promotes retinal axon outgrowth; AMPA receptor antagonists inhibit that; and KAR antagonists have no effect on that. In the presence of different combinations of ionotropic receptor antagonists (including NMDAR antagonist), they suggest that the blockage of glutamate iontroptic receptors displays an obvious effect of inhibiting neurite outgrowth in E14 retinal explants. However, inhibiting kainate receptors show little effect on retinal neurite outgrowth which is different from that of blocking AMPARs. We also did E13 and E15 brain slice culture experiments, and found that blocking of glutamate ionotropic receptors affects crossed axon projection in the midline at early stage, but has no effect to the uncrossed one. / Glutamate is the dominant amino acid neurotransmitter in the central nervous system naturally occurring in the L-form. Glutamate ionotropic receptors can be further a-amino-3-hydroxy-5-methy1-4-isoxazole-propionate divided into three types by their ligand (AMPA, specificities: GluR1-4), N-methyl-D-aspartate (NMDA, NR1-3) and kainate (KA, GluR5-7 and KA1-2) receptors, which function as ligand-gated ion channels. In this study, we focus on the AMPARs and KARs which are expressed in the developing brain. / Here, we used semi-quantitative RT-PCR to analyze mRNA expression levels of AMPAR and KAR subunits in the mouse retina and ventral diencephalons at different developmental stages, and in adult retina. The results show that both AMPAR and KAR subunits can be detected in retina and ventral diencephalon at as early as E13. We also used specific antibodies to investigate glutamate, AMPAR and KAR subunit expression in the mouse retinofugal pathway. We found that: 1) Glutamate is expressed at as early as E13. In retina, it tends to localize in retinal ganglion cells (RGCs) and their axons; in ventral diencephalon, it is most intense in optic stalk, optic chiasm and optic tract. It is also localized with chiasmatic neurons, which are related to the formation of optic chiasm. 2) For the individual AMPAR and KAR subunits, all of them are expressed at as early as E13. The immunoreactive GluRl and GluR5/6/7 are distributed preferentially in the RGCs and their axons; the staining of GluR2/3 and GluR4 are largely found in RGCs and the supporting cells around the pathway, but for GluR4, its staining is weakly detected in optic fibers and strongly in the midline of chiasm. Although the staining patterns of these specific subunits are different, they are all localized in chiasmatic neurons in diencephalon. / Cheng, Xiaojing. / "November 2010." / Adviser: Sun On Chan. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.