Introdução: A Leucemia Linfóide Aguda (LLA) e o Linfoma não-Hodgkin (LNH) são os tipos de câncer mais incidentes em crianças e a ingestão alimentar pode ser diminuída pela quimioterapia. A sensação do gosto é resultante da detecção e resposta ao estímulo doce, salgado, azedo, amargo e umami. Esse último, identificado pelo glutamato monossódico (MSG), é relacionado ao aumento da palatabilidade, o que pode colaborar para a melhora da aceitação alimentar em crianças com câncer. Objetivo: Identificar os limiares de detecção do gosto umami e a qualidade da alimentação em crianças portadoras de LLA e LNH. Metodologia: Foi aplicado teste de sensibilidade de Threshold para determinar o limiar do gosto umami, com 6 concentrações crescentes de água deionizada e MSG. Aplicou-se recordatório 24 horas e questionário de frequência alimentar para avaliar o consumo alimentar. O peso e altura foram mensurados e IMC utilizados para classificação do estado nutricional, segundo o National Center for Health Statistics (2000). Caracterizou-se a amostra através da distribuição de frequência das variáveis, com auxílio do pacote estatístico Epinfo Versão 6.0. As análises estatísticas e gráficas foram feitas no software R, versão 2.6.2. Foi realizado teste de Cluster para caracterizar a amostra. Resultados: Dos 102 pacientes, 94 eram sensíveis ao umami. 54,3 por cento do sexo masculino e 45,7 por cento do feminino. 78,4 por cento portadores de LLA e 21,6 por cento de LNH. 91,0 por cento em fase de manutenção. Quanto à idade, 38,3 por cento entre 6 e 7 anos; 20,6 por cento entre 8 e 9; 15,7 por cento entre 10 e 11; 15,7 por cento entre 12 e 13 e 9,8 por cento 14 anos. 8,5 por cento apresentaram baixo peso, 66,0 por cento eutrofia e 25,5 por cento sobrepeso ou obesidade. O produto rico em glutamato monossódico mais consumido foi macarrão instantâneo. O molho inglês e de soja foram os menos consumidos. Os alimentos preferidos foram salgadinhos e macarrão instantâneo, e o que menos gostavam era a mostarda. Não houve diferença estatisticamente significante entre os limiares de sensibilidade ao umami e as variáveis em estudo. O agrupamento da amostra caracterizou 4 grupos: Grupo 1, composto por crianças mais sensíveis ao umami, mais jovens, maioria eutrófica e do sexo masculino; Grupo 2, maioria eutrófica, do sexo feminino, com menor consumo de carboidrato e maior de proteína e lipídeo; Grupo 3, composto por crianças mais velhas, maioria eutrófica e do sexo masculino, com maior consumo calórico e de carboidrato; Grupo 4, com crianças menos sensíveis ao umami, eutróficas e com sobrepeso, maioria do sexo masculino e com ingestão calórica mais baixa. Conclusão: As crianças são sensíveis ao gosto umami e essa característica independe do sexo, idade, estado nutricional, fase de tratamento, ingestão calórica e de macronutrientes. A qualidade da alimentação e a idade foram variáveis determinantes das similiradades entre os grupos. O teste de sensibilidade para detecção do gosto umami é de grande interesse para conhecer o comportamento alimentar e auxiliar na melhora da aceitação dos alimentos / Introduction: The Acute Lymphoblastic Leukemia (ALL) and non-Hodgkin Lymphoma (NHL) are the most frequent cancers in children and food intake can be reduced by chemotherapy. The sense of taste is a result of the detection and response to the sweet, salty, sour, bitter and umami stimulus. The latter is identified by monosodium glutamate (MSG) and is related to the increase of palatability, which may contribute to improve food acceptance in children with cancer. Objective: identification of the thresholds of detection of umami taste and food quality in children with LLA and LNH. Methodology: the threshold sensitivity test was applied in order to determine the threshold of the umami taste using six increasing concentrations of deionized water and MSG. A 24-hour recall and food frequency questionnaire were applied to check food intake. Weight and height were measured and the BMI was used to determine the nutritional status, according to the National Center of Health Statistics (2000). The sample was characterized by the distribution of the frequency of the variables, with the help of the Epinfo Version 6.0 statistical package. The statistical and graphical analyses were done using the R statistical software, version 2.6.2. The Cluster test was applied to characterize the sample. Results: from the 102 patients, 94 were sensitive to umami taste. 54,3 per cent were male and 45,7 per cent were female. 78,4 per cent had ALL and 21,6 per cent had NHL. 91 per cent were in the maintenance stage. Regarding age, 38,3 per cent were between 6 and 7 years old; 20,6 per cent were between 8 and 9 years old; 15,7 per cent were between 10 and 11 years old; 15,7 per cent were between 12 and 13 years old and 9,8 per cent were 14 years old. 8,5 per cent were underweight, 66 per cent were eutrophic and 25,5 per cent were overweight or had obesity. The most consumed product was instant noodles, rich in monosodium glutamate. The tabasco and soy sauces were the least consumed. The favorite food was snacks and instant noodles and mustard was the food they liked the least. There was no statistically significant difference between the thresholds of sensitivity to umami and the variables in study. The gathering of the sample characterized four groups: Group 1 formed by younger children, most male and eutrophic, more sensitive to the umami taste; Group 2 formed by most eutrophic and female children, showing lower intake of carbohydrates and higher intake of proteins and lipids; Group 3 formed by older children, most eutrophic and male, showing higher caloric and carbohydrate intake; Group 4 formed by eutrophic and overweight children, most male and with lower caloric intake, less sensitive to the umami taste. Conclusion: children are sensitive to the umami taste and this characteristic does not depend on the sex, age, nutritional status, treatment stage, caloric and macronutrients intake. The food quality and age were determinant variables of the similarities among the groups. The test of sensitivity for the detection of the umami taste is of great interest in the knowledge of food intake behavior and in the increase of food acceptance

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg·HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg·Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg·Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg·Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg·Glu on cell viability was compared against NaCl, Arg·HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg·Glu in the cell culture models studied, therefore Arg·Glu has the potential as an excipient as it reduces aggregation and is nontoxic. Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.

Psychotic symptoms are prevalent in a wide variety of psychiatric and neurological disorders. Yet, despite decades of research, the neurobiological mechanisms via which these symptoms come to manifest themselves remain to be elucidated. I argue in this thesis that using a mechanistic approach towards understanding psychosis that borrows heavily from the predictive coding framework, can help us understand the relationship between neurobiology and symptomology. In the first results chapter I present new data on a biomarker that has often been cited in relation to psychotic disorders, which is glutamate levels in the anterior cingulate cortex (ACC), as measured with magnetic resonance spectroscopy. In this chapter I aimed to replicate previous results that show differences in glutamate levels in psychosis and health. However, no statistically significant group differences and correlations with symptomology were found. In order to elucidate the potential mechanism underlying glutamate changes in the anterior cingulate cortex in psychosis, I tested whether a pharmacological challenge of Bromocriptine or Sulpiride altered glutamate levels in the anterior cingulate cortex. However, no significant group differences were found, between medication groups. In the second results chapter I aimed to address a long-standing question in the field of computational psychiatry, which is whether prior expectations have a stronger or weaker influence on inference in psychosis. I go on to show that this depends on the origin of the prior expectation and disease stage. That is, cognitive priors are stronger in first episode psychosis but not in people at risk for psychosis, whereas perceptual priors seem to be weakened in individuals at risk for psychosis compared to healthy individuals and individuals with first episode psychosis. Furthermore, there is some evidence that these alterations are correlated with glutamate levels. In the third results chapter I aimed to elucidate the nature of reward prediction error aberrancies in chronic schizophrenia. There has been some evidence suggesting that schizophrenia is associated with aberrant coding of reward prediction errors during reinforcement learning. However it is unclear whether these aberrancies are related to disease years and medication use. Here I provide evidence for a small but significant alteration in the coding of reward prediction errors that is correlated with medication use. In the fourth results chapter I aimed to study the influence of uncertainty on the coding of unsigned prediction errors during learning. It has been hypothesized by predictive coding theorists that dopamine plays a role in the precision-weighting of unsigned prediction error. This theory is of particular relevance to psychosis research, as this might provide a mechanism via which dopamine aberrancies, might lead to psychotic symptoms. I found that blocking dopamine using Sulpiride abolishes precision-weighting of unsigned prediction error, providing evidence for a dopamine mediated precision-weighting mechanism. In the fifth results chapter I aimed to extend this research into early psychosis, to elucidate whether psychosis is indeed associated with a failure to precision-weight prediction error. I found that first episode psychosis is indeed associated with a failure to precision-weight prediction errors, an effect that is explained by the experience of positive symptoms. In the sixth results chapter I explore whether the degree of precision-weighting of unsigned prediction errors is correlated with glutamate levels in the anterior cingulate cortex. Such a correlation might be plausible given that psychosis has been associated with both. However, I did not find such a relationship, even in a sample of 137 individuals. Thus I concluded that anterior cingulate glutamate levels might be more related to non-positive symptoms associated with psychotic disorders. In summary, a mechanistic approach towards understanding psychosis can give us valuable insights into the disease mechanisms at play. I have shown here that the influence of expectations on perception is different across disease stage in psychosis. Furthermore, aberrancies in prediction error mechanisms might explain positive symptoms in psychosis, a process likely mediated by dopaminergic mechanisms, whereas evidence for glutamatergic mediation remains absent.

Hyper-activation of N-methyl-D-aspartate receptors (NRs) is associated with excitotoxic cell death during secondary injury following traumatic brain injury (TBI). The efficiency of the NR is dependent on the location of receptors in membrane raft microdomains that provide a platform for coupling of NRs and effector proteins. In many neurodegenerative diseases, activation of the autophagy pathway has been suggested to contribute to glutamate excitotoxicity, but whether increased autophagy signaling contributes to pathology after TBI has not been defined. In these studies, I investigate whether membrane rafts mediate NR signaling and autophagy in cortices of adult male rats subjected to moderate TBI and in sham-operated controls. These studies demonstrate that membrane rafts of the normal rat cortex contain a novel multi-protein signaling complex that links the NR2B glutamate receptor and the autophagic protein Beclin 1. TBI caused a rapid disruption of this complex in which NR2B and pCaMKII were recruited to membrane microdomains. Alteration in NR2B-Beclin 1 association in membrane rafts resulted in activation of autophagy as demonstrated by increased expression of key autophagic proteins Beclin 1, ATG 5 and ATG 7, and significant increases in autophagic vacuoles in neurons of traumatized brains. Administration of the NR2B antagonist RO 25-6981 significantly blocked TBI-induced redistribution of NR2B signaling intermediates and Beclin 1 and delayed the increase in autophagy protein expression in traumatized cortices. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin 1 in membrane rafts after TBI.

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that orchestrates key events of development, disease and repair in the central nervous system (CNS). To investigate the effects of chronically producing TGF-β1 on synaptic structure and synaptic transmission, I performed immunohistochemistry and immunoblot of brain tissues from transgenic mice (TGF-β1 mice) that over-express active form of TGF-β1 from astrocytes in the CNS. Immunohistochemical assays showed that synaptophysin increased in the CA3 subfield whereas calbindin-D28K decreased in the mossy fibres. Immunoblot analysis revealed that several α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunit proteins were up-regulated in the hippocampus of TGF-β1 mice. To examine the direct effect of TGF-β1 alone on glutamatergic synaptic activity, cultured hippocampal neurons were treated with or without TGF-β1. Electrophysiological recordings displayed that TGF-β1 significantly increased the amplitude of glutamate-evoked current (p<0.05). Taken together, these data suggest that TGF-β1 modulates hippocampal glutamatergic synaptic protein expression and regulates synaptic transmission.

Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that orchestrates key events of development, disease and repair in the central nervous system (CNS). To investigate the effects of chronically producing TGF-β1 on synaptic structure and synaptic transmission, I performed immunohistochemistry and immunoblot of brain tissues from transgenic mice (TGF-β1 mice) that over-express active form of TGF-β1 from astrocytes in the CNS. Immunohistochemical assays showed that synaptophysin increased in the CA3 subfield whereas calbindin-D28K decreased in the mossy fibres. Immunoblot analysis revealed that several α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunit proteins were up-regulated in the hippocampus of TGF-β1 mice. To examine the direct effect of TGF-β1 alone on glutamatergic synaptic activity, cultured hippocampal neurons were treated with or without TGF-β1. Electrophysiological recordings displayed that TGF-β1 significantly increased the amplitude of glutamate-evoked current (p<0.05). Taken together, these data suggest that TGF-β1 modulates hippocampal glutamatergic synaptic protein expression and regulates synaptic transmission.

The synthesis of arborescent poly(gamma-benzyl L-glutamate) (PBG) molecules was achieved through successive grafting reactions of linear PBG chains. These linear PBG building blocks were obtained by the ring-opening polymerization of gamma-benzyl L-glutamic acid N-carboxyanhydride initiated with n-hexylamine. Cleavage of a fraction of the benzyl ester groups on a linear PBG substrate, followed by coupling with linear PBG side chains via standard peptide coupling techniques, yielded a comb-branched or generation zero (G0) arborescent PBG. Further cycles of partial deprotection and grafting reactions led to arborescent PBG molecules of the subsequent generations (G1-G3). Molecular weights reaching over 106 were obtained for G3 arborescent PBG, while maintaining narrow molecular weight distributions (Mw/Mn ≤ 1.06) for each generation. The arborescent PBG molecules displayed α-helix to randomly coiled chain conformation changes from N,N-dimethylformamide to dimethylsulfoxide. Amphiphilic copolymers were obtained by grafting the arborescent PBG substrates randomly with side chains of either poly(glycidol acetal), poly(ethylene oxide), or poly(γ-tert-butyl L-glutamate) via the same peptide coupling techniques used to generate arborescent PBG. Copolymers were also synthesized by a chain end grafting method, whereby the linear chain segments were coupled exclusively with the chain termini of the arborescent PBG substrates. Water-soluble species were obtained by removal of the acetal and tert-butyl protecting groups from the poly(glycidol acetal) and poly(γ-tert-butyl L-glutamate) side chains, respectively, while the copolymers with poly(ethylene oxide) side chains did not require further modifications. Dynamic light scattering (DLS) measurements on the arborescent copolymers in aqueous solutions revealed that unimolecular micelles were the dominant species for the chain end grafted arborescent copolymers, whereas the randomly grafted arborescent copolymers were either insoluble or displayed significant aggregation. The synthesis of arborescent copolymers with PBG cores was also achieved through “click” chemistry, using the copper-catalyzed azide-alkyne Huisgen cycloaddition (CuAAC) reaction. To that end, polyglycidol, poly(ethylene oxide), and poly(2-trimethylsilylethyl acrylate) chains terminally functionalized with azide groups were grafted onto either randomly or chain end alkyne-functionalized arborescent PBG substrates. DLS analysis revealed solubility trends similar to the arborescent copolymers obtained by the peptide coupling method. The CuAAC reaction enables the incorporation of a broader range of polymers into arborescent copolymer structures derived from PBG substrates.

Zebrafish, Danio rerio, have become an important model for developmental studies and have several advantages over other model systems. These advantages include (1) the easy accessibility of zebrafish embryos for direct observation of their development and (2) their suitability for systematic mutagenesis studies for the identification of genes regulating the development of various tissues and organs, including the skeletal system. Recently, it has been reported that glutamate receptors are expressed in many types of bone cells and regulate bone physiological functions. In the present study, we have examined the effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist¡Xdextromethorphan¡Xon the development of the axial skeleton in zebrafish embryos by using calcein stain. Our results revealed that dextromethorphan significantly attenuates the formation of the axial skeleton and that it is inhibited on pretreatment with glutamate. Moreover, immunohistochemical analysis revealed protein level expression of the NMDA subunit NR1 in the axial region of zebrafish. Our results also indicate that attenuation of NMDA receptor activity-induced change in the axial skeleton may be related to heat-shock protein and extracellular signal-regulated kinase (ERK) signalings. In conclusion, we suggest that the NMDA receptor plays an important role in the development of the axial skeleton. However, further studies are required on the cellular mechanisms of glutamate regulated bone formation.

We have two topics, In the first study, we synthesized three low-molecular-weight poly(glutamate)s¡Xpoly( £^-methyl l-glutamate) (PMLG), poly( £^-ethyl l-glutamate) (PELG), and poly( £^-benzyl l-glutamate) (PBLG)¡Xthrough living ring-opening polymerization of their £\-amino acid-N-carboxyanhydride derivatives and then blended them with phenolic resin to control the secondary structures of these polypeptides. Each of the three binary blends exhibited a single glass transition temperature (differential scanning calorimetry) and solid state nuclear magnetic resonance (NMR) spectroscopy], characteristic of a miscible system. The strength of the inter-associative interactions depended on the nature of the hydrogen bond acceptor groups, increasing in the order phenolic/PELG > phenolic/PMLG > phenolic/PBLG, as evidenced through analyses using Fourier transform infrared (FTIR) spectroscopy and the Painter¡VColeman association model. The fractions of £\-helical conformations (measured using FTIR and solid state NMR spectroscopy) of PMLG and PELG decreased initially upon increasing the phenolic content, but increased thereafter; in contrast, the fraction of £\-helical conformations of PBLG increased continuously upon increasing the phenolic contents. Using variable-temperature infrared spectroscopy to investigate the changes in the conformations of the secondary structures of the peptide segments in these three binary blends, we found that the £\-helical conformation in these three blend systems correlated strongly with the rigidity of side chain groups, the strength of the intermolecular hydrogen bonding with the phenolic resin, the compositions of phenolic resin, and the temperature. More interestingly, the content of £\-helical conformations of the polypeptides in these phenolic/PBLG blends increased upon increasing the temperature. The second topic is synthesized low-molecular-weight poly( £^-benzyl l-glutamate) (PBLG) through living ring-opening polymerization of their £\-amino acid-N-carboxyanhydride derivatives and blended them with poly( styrene¡^(PS), poly (acetoxystyrene) (PAS) and poly(vinyl phenol) (PVPh) to control the secondary structures of these polypeptides. DSC have been used to investigate the miscibility of. FTIR spectroscopies and wide-angle X-ray diffraction (WXRD) spectroscopic analyses provided evidence for the change and specific interactions between (PS, PAS and PVPh) and PBLG. That the secondary structures of polypeptides can be altered through blending with other different Specific Interactions, mediated by hydrogen bonding, dipole¡Vdipole, and £k¡X£k Interaction, we investigate strong Specific interactions was found between the side-chain esters of PAS, PVPh, but not found between PBLG and PS, because more weakly with the aromatic rings of PS through intermolecular £k¡X£k interactions, so that this latter system is phase separated.