A correta classificação do tipo de DM leva mais precocemente ao tratamento adequado e atualmente é dividida em 4 categorias: DM tipo 1, DM tipo 2, Outros tipos e Diabete Gestacional. O DM tipo 1 é geralmente auto-imune, surge em geral antes dos 20 anos de idade e é dependente de insulina para impedir a cetoacidose. O DM tipo 2 é responsável por mais de 90% dos casos, acontece em geral após os 45 anos, com história familiar e associado à síndrome metabólica. Na categoria “outros tipos”, o Maturity Onset Diabetes of the Young (MODY) é um subtipo que inicia abaixo dos 25 anos, não-dependente de insulina e apresenta herança dominante. No entanto, apesar da classificação definir essas categorias através de características peculiares, pode existir uma sobreposição de quadros, principalmente no DM que inicia no adulto jovem. Assim, novos sistemas de classificação têm sido propostos, empregando a presença da auto-imunidade (anticorpos) e índices de função de célula β (peptídeo-C) para definir a patogênese e nomenclatura mais específicas. O objetivo desta revisão é descrever e analisar o desempenho destas ferramentas diagnósticas na classificação do DM. Os anticorpos evidenciam a auto-imunidade do DM 1. O IAA (insulin auto-antibody) está presente quando o início do DM se dá, principalmente, antes dos 5 anos de idade e o anti- GAD (glutamic acid decarboxylase) tem seu melhor desempenho nos indivíduos com início da doença acima dos 20 anos, sendo o teste que permanece positivo por mais tempo. A medida do peptídeo-C avalia a reserva pancreática de insulina e deve ser realizada com glicemia entre 70-200 mg/dl. A medida após estímulo é a mais estudada e <1,5 ng/ml define o paciente como DM 1. O estímulo com refeição mista é o recomendado pela ADA, mas o teste com 1 mg de glucagon é mais simples e igualmente acurado. Dados em relação à utilização da classificação baseada na medida de diferentes anticorpos dirigidos ao pâncreas classificação A (anticorpos) e β (peptídeo-C) pode ser adotada como um método acurado, relativamente simples e preciso de classificação de DM. / The correct classification of DM type leads to earlier appropriate treatment and is currently divided into 4 categories: type 1 DM, type 2 DM, Other types and Gestational DM. Type 1 DM is generally autoimmune, it usually appears before the age of 20 years, and depends on insulin to prevent ketoacidosis. Type 2 DM accounts for over 90% of the cases, it usually occurs after the age of 45, with a family history and metabolic syndrome. In the category “other types”, Maturity Onset Diabetes of the Young (MODY) is a subtype that begins before the age of 25, is non-insulin dependent and presents a dominant heritage. However, although the classification defines these categories through peculiar characteristics, there may be superimposed pictures, especially in the case of DM which begins in the young adult. Thus, new classification systems have been proposed, using the presence of autoimmunity (antibodies) and β cell (C-peptide) indexes to define the pathogenesis and more specific nomenclatures. The purpose of this review is to describe and analyze the performance of these diagnostic tools in the classification of DM. The presence of antibodies show the autoimmunity of type 1 DM. IAA (insulin auto-antibody) is present mainly before the age of 5 years, and anti-GAD (glutamic acid decarboxylase) performs best in individuals who begin the disease above the age of 20, and its test remains positive longest. The C-peptide measure evaluates the pancreatic reserve of insulin and should be performed with a glycemia between 70-200 mg/dl. Post-stimulus measuring is more widely studied and <1.5 ng/ml defines the patient as DM1. Stimulation with a mixed meal is recommended by ADA, but the test with 1 mg of glucagon is simpler and just as effective. Data on classification A (antibodies), β (Cpeptide) suggest that it may be adopted as an effective, relatively simple and precise method for DM classification.

O ácido ƴ-poliglutâmico (ƴ-PGA) é uma homopoliamida aniônica, biodegradável, comestível e atóxica, sintetizada por bactérias do gênero Bacillus, podendo ser utilizado nas indústrias alimentícia e de cosméticos, na medicina e no tratamento de águas residuais. Este trabalho teve como objetivo caracterizar e identificar potenciais aplicações para o ƴ-PGA obtido através do cultivo submerso de Bacillus subtilis BL53, conduzido sob condições otimizadas em trabalhos anteriores. Além disso, foi avaliado o efeito de diferentes inóculos e da adição de precursores da rota metabólica na produção do biopolímero. A melhor condição obtida foi testada em biorreatores com adição de polidimetilsiloxano (PDMS) como carreador de oxigênio, com o objetivo de aumentar a produtividade do biopolímrero. A massa molar média (Mw), obtida através de espalhamento de luz estático, na ordem de 106 g mol-1 não apresentou diferenças significativas para o biopolímero obtido após 48 e 96 h de cultivo. As análises reológicas conduzidas em viscosímetro rotacional indicaram que os polímeros obtidos após 48 e 96 horas apresentaram comportamento Newtoniano, sendo que após 96 horas a viscosidade absoluta foi maior. As análises térmicas (calorimetria diferencial exploratória e análise termogravimétrica) indicaram a temperatura de fusão (Tm) de 134 ºC e 128 ºC e o intervalo de degradação (Td) entre 120 ºC - 190 ºC e 120 ºC - 215 ºC, para os biopolímeros obtidos após 48 e 96 horas de cultivo respectivamente. O caldo LB apresentou-se como o melhor inóculo para a produção de ƴ-PGA. A adição dos precursores L-glutamina e ácido -cetoglutárico aumentou em 20 % a produção do biopolímero. A adição de 10 % de PDMS nos cultivos em biorreatores aumentou o coeficiente volumétrico de transferência de massa (KLa) e a produção e produtividade do ƴ-PGA, sendo produzidos 23.5 g L-1 do biopolímero em 24 horas de cultivo, uma produtividade aproximadamente 40 % superior às obtidas por outros autores utilizando o mesmo microrganismo. / Poly-ƴ-glutamicacid (ƴ-PGA) is an anionic, biodegradable, non-toxic and edible homopolyamide, synthesized by bacteria of the genus Bacillus, being used in food, cosmetics, medicine and waste water treatment. The aim of this study is to characterize and indentify potencial applicatiions for the ƴ-PGA obtained by submerged cultivation of Bacillus subtilis BL53, conducted under optimized conditions in previous studies. We also evaluated the effect of different inoculants and addition of precursors in the metabolic pathway of production of the biopolymer. The best condition obtained yet been tested in bioreactors with addition of polydimethylsiloxane (PDMS) as a carrier of oxygen in order to further increase the productivity of biopolymer. The average molecular weight (Mw) obtained by static light scattering, on the order of 106 g mol-1, showed no significant differences for biopolymer obtained after 48 and 96 h of cultivation. Analyses conducted in rotational viscometer indicated that biopolymers after 48 and 96 h have a Newtonian behavior, and the 96 hours had higher absolute viscosity. The thermal analysis (differential scanning calorimetry and thermo gravimetric analysis) indicated the melting temperature (Tm) as 134 ºC and 128 ºC and degradation temperature range (Td) of 120 ºC - 190 ºC and 120 ºC - 215 ºC, after 48 and 96 hours respectively. It was found that the best inoculum medium for biopolymer production was the LB broth. The addition of the precursors L-glutamine and -ketoglutaric acid increased in 20% the ƴ-PGA production. The addition of 10% of PDMS in bioreactors cultures increased the mass transfer volumetric coefficient (KLa) and the production and productivity of ƴ-PGA, being produced 23.5 g l-1 of the biopolymer in 24 hours of cultivation, a productivity about 40 % higher than those obtained by other authors using the same microorganism.

INTRODUÇÃO: O transtorno obsessivo-compulsivo (TOC) apresenta prevalência ao longo da vida ao redor de 2%. O tratamento farmacológico de primeira linha para o TOC é feito com inibidores da recaptura de serotonina (IRS). Aproximadamente metade dos pacientes tratados adequadamente com um IRS não apresenta resposta satisfatória. Resultados de estudos de neuroimagem, genéticos e de análise do líquido cefalorraquidiano de portadores de TOC sugerem o envolvimento da disfunção da atividade glutamatérgica na fisiopatologia do TOC. Medicamentos com efeito modulador da atividade glutamatérgica vem sendo testados em pacientes com TOC e alguns deles mostraram-se eficazes. A N-Acetilcisteína (NAC) é um agente modulador da atividade glutamatérgica e antioxidante. Este estudo tem como objetivo principal testar a eficácia da potencialização de IRS com NAC, em comparação com placebo, em pacientes com TOC resistente ao tratamento. MÉTODOS: Estudo duplo cego, randomizado e controlado com placebo, com duração de 16 semanas, conduzido num ambulatório especializado de um hospital terciário (maio/2012 - outubro/2014). Critérios de inclusão: 18-65 anos; diagnóstico principal de TOC, de acordo com os critérios do DSM-IV; falha terapêutica a pelo menos um tratamento farmacológico adequado para o TOC; escore inicial da escala Yale-Brown de Sintomas Obsessivo-Compulsivos (Y-BOCS) >= 16 ou >= 10, se apenas compulsões; e gravidade inicial do TOC pelo menos moderada, de acordo com a escala de gravidade da Impressão Clínica Global. Os medicamentos em uso no momento da randomização foram mantidos nas mesmas doses. A intervenção consistiu na associação de NAC (até 3000 mg/dia) ou placebo. Avaliações cegas foram realizadas antes e ao final da intervenção. Utilizamos como desfecho primário os escores da Y-BOCS. Utilizamos análise de variância não-paramétrica para medidas repetidas. Como desfechos secundários, consideramos a redução dos escores iniciais dos Inventários de Depressão e Ansiedade de Beck, da Y-BOCS Dimensional e da Escala Brown de Avaliação de Crenças. Registro do estudo: clinicaltrials.gov identifier: NCT01555970. RESULTADOS: Foram realizadas 145 consultas de triagem, 129 indivíduos preencheram os critérios de inclusão, 40 foram randomizados (NAC até 3000 mg/dia, n= 18; placebo, n= 22), 39 iniciaram a intervenção e 35 terminaram o estudo. Não houve diferença significativa entre os grupos quanto à taxa de perda (NAC: 1 em 17 [5,9%]; placebo: 3 em 22 [13,6%]; ?2 = 0,63; P= 0,43). Todos os indivíduos melhoraram significativamente ao longo do tempo, de acordo com a redução do escore da Y-BOCS, mas não houve diferenças significativas entre os grupos (NAC: 25,6 [DP= 4,4] para 21,3 [DP= 8,1]; placebo: 24,8 [DP= 3,8] para 21,8 [DP= 6,0]; F= 0,33; P= 0,92). A associação com NAC foi superior ao placebo em relação à melhora da gravidade dos sintomas ansiosos, indicada pela redução do escore do Inventário de Ansiedade de Beck (média [DP]: NAC= 7,8 [11,7]; placebo: -0,55 [7,9]; U= 89; P= 0,02). Não houve diferenças significativas entre os grupos quanto à melhora dos sintomas depressivos, das diferentes dimensões de sintomas de TOC e do nível de insight. CONCLUSÕES: A potencialização de IRS com NAC foi superior ao placebo em relação à melhora da gravidade dos sintomas ansiosos. Entretanto, não houve diferença entre os grupos na melhora da gravidade dos sintomas do TOC / INTRODUCTION: Obsessive-compulsive disorder (OCD) is a debilitating psychiatric disorder with a lifetime prevalence of 2-3%. Treatment guidelines recommend serotonin reuptake inhibitors (SRIs) as the first-line pharmacological treatment for OCD. However, approximately half of patients treated with an adequate trial of SRIs fail to fully respond to treatment. OCD is associated with hyperactivity in cortical-striatum-thalamus-cortical (CSTC) circuits. Cortico-striatal and thalamo-striatal afferents use the excitatory neurotransmitter glutamate, and there is evidence suggesting abnormal glutamate levels and/or homeostasis in OCD patients. Researchers have been testing glutamate-modulating medications in OCD, with some evidence for efficacy. N-Acetylcysteine (NAC), a glutamate-modulating agent, is being considered as an add-on strategy for treatment-resistant OCD. The main objective of this study was to determine if NAC is effective in treatment-resistant OCD patients after 16 weeks of SRI augmentation. METHODS: We conducted a randomized, double blind, placebo-controlled, 16-week trial in an OCD-specialized outpatient clinic at a tertiary hospital (May 2012-October 2014). Inclusion criteria: age between 18-65 years; DSM-IV primary diagnosis of OCD; failure to respond to at least one previous adequate pharmacological treatment for OCD; baseline Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) global score >= 16 or >= 10 if only compulsions are present; OCD symptoms of at least moderate severity on the Clinical Global Impression-Severity subscale. Medications in use at randomization were maintained at the same dose. Assessments were conducted at baseline and end of the study. The primary outcome measure was the Y-BOCS scores. To evaluate the variables group, time, and interaction effects for Y-BOCS scores at all time points, we used nonparametric analysis of variance (ANOVA) with repeated measures. The secondary outcomes were the mean reduction of the baseline Beck Anxiety and Depression Inventories, Dimensional Yale-Brown Obsessive-Compulsive Scale and Brown Assessment of Beliefs Scale scores. Trial registration: clinicaltrials.gov identifier NCT01555970. RESULTS: We assessed 145 patients for eligibility, 129 were eligible, 40 were randomized (NAC up to 3000 mg/day, n= 18; placebo, n= 22), 39 initiated the intervention and 35 completed the trial. Dropout did not significantly differ by treatment group (NAC: 1 of 17 [5.9%]; placebo: 3 of 22 [13.6%]; ?2 = .63; P= .43). Both groups significantly improved over the 16 weeks, as indicated by the reduction of baseline Y-BOCS scores, but there were no significant differences between groups (NAC: 25.6 [SD= 4.4] to 21.3 [DP= 8.1]; placebo: 24.8 [SD= 3.8] to 21.8 [SD= 6.0]; F= .33; P = .92). Adding NAC to SRI was superior to placebo in improving anxiety symptoms, as measured by the reduction of baseline Beck Anxiety Inventory score (mean [SD]: NAC= 7.8 [11.7]; placebo: -.55 [7.9]; U= 89; P= .02). There were no significant differences between groups in regards to the improvement of depressive symptoms, different dimensions of OCD symptoms and insight level. CONCLUSIONS: NAC augmentation of SRI was more effective than placebo in reducing the severity of anxiety symptoms in this sample of treatment-resistant OCD individuals. However, it was not better than placebo in reducing OCD severity

This thesis discusses the structure-function studies on triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf), directed towards understanding the roles of highly conserved residues by site derected mutagenesis. Chapter 1 provides an introductory overview to the relevant literature on triosephosphate isomerase. In addition, this Chapter provides an analysis of conserved residues in TIM, and amino acid diversity at specific positions in the structure using a dataset of 503 TIM sequences. Chapter 2 reports the work on the completely conserved residue, C126 in TIM, which is proximal to the active site. Five mutants, C126S, C126A, C126V, C126M and C126T have been characterized. Crystal structures of 3-phosphoglycolate (PGA) bound C126S mutant and the unliganded forms of the C126S and C126A mutants have been determined at a resolution of 1.7 Å to 2.1 Å. Kinetic studies reveal a ~5 fold drop in kcat for the C126S and C126A mutants, while a ~ 10 fold drop is observed for the other three mutants. All the mutants show reduced stability at lower concentration and higher temperature. Chapter 3 presents the kinetic and structural characterization for the E97Q and E97D mutants of Pf TIM. A 4000 fold reduction in kcat is observed for E97Q, 100 fold reduction for the E97D mutant, while a ~ 9000 fold drop in activity for the control mutant, E165A. A large conformational change for the critical K12 side chain is observed in the crystal structure of the E97Q mutant, while it remains unchanged in the E97D structure. The results are interpreted to invoke a direct role for E97 in the catalytic proton transfer cycle, eliminating the need to invoke the formation of the energetically unfavorable imidazolate anion at H95. Chapter 4 reports investigations with position 96 by the biochemical and structural characterization of single mutants, F96Y, F96A and the double mutants, F96S/S73A and F96S/L167V. F96Y showed ~100 fold drop in activity, F96A revealed ~10 fold drop in activity, while F96S/S73A showed 100 fold lower activity than that of the wild type enzyme. Interestingly, the double mutant F96S/L167V proved to be a partial pseudorevertant, showing 10 fold higher activity than the single mutant, F96S. Chapter 5 describes the cloning, and preliminary kinetic and biophysical characterization of the enzyme, Dm TIM. A survey of disease causing mutations in TIM and the relationship of these sites of mutation to the active site and the dimer interface of TIM is presented in this Chapter.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The development of polymers with the ability to transport and release drugs and bioactives has been growing rapidly because of their unique properties in protecting and enhancing solubilization of drugs in physiological media. In this work, polyesters derived from β-cyclodextrin were developed in two different systems: The former consisted of cyclodextrin cross-linked with citric acid and subsequently functionalized with glutamic acid. The latter was obtained from β-cyclodextrin esterified with gluthamic acid. The polyesters were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Solid-state Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance (CP/MAS 13C–NMR) and Thermogravimetric Analysis (TGA) for the confirmation of the formation of polymers and the esterification of glutamic acid with β-cyclodextrin molecules. The formation of inclusion complex of the polymers in solution with orange methyl was evaluated to verify the activation of β-cyclodextrin cavities. The polymers were used as reducer and stabilizer in the synthesis of silver nanoparticles (AgNPs). The characterization of the AgNPs was performed by UV-Vis spectroscopy, and bands between 350-500 nm evidenced of the obtainment of silver nanoparticles. Analyses by transmission electron microscopy revealed AgNPs with spherical morphology and diameter around ~ 5 to ~60 nm, corroborating with the results of UV-Vis. / O desenvolvimento de polímero com a capacidade de transportar e liberar fármacos e compostos bioativos vem crescendo rapidamente, devido suas propriedades únicas em proteger e aumentar a solubilização em meios fisiológicos. Neste trabalho, foram desenvolvidos poliésteres derivado de β-ciclodextrina em dois sistemas distintos. O primeiro sistema foi obtido a partir de β-ciclodextrina reticulado com ácido cítrico e posteriormente funcionalizado com ácido glutâmico. O segundo foi a partir de β-ciclodextrina esterificada com ácido glutâmico. Os poliésteres foram caracterizados por espectroscopia na região do infravermelho com transformada de Fourier (FTIR), ressonância magnética nuclear no estado sólido sob polarização cruzada e rotação com ângulo mágico (RMN 13C CP/MAS) e análise termogravimétrica (TG), para a confirmação da formação dos polímeros e a esterificação do ácido glutâmico com as moléculas de β-ciclodextrina. A formação de complexo de inclusão dos polímeros com o alaranjado de metila foi avaliada em solução, comprovando-se a ativação das cavidades de β-ciclodextrina. Os polímeros foram testados quanto a habilidade de promover a formação/estabilização de nanopartículas de prata (AgNPs) a partir de nitrato de prata em solução. A caracterização das AgNPs foi realizada por espectroscopia de UV-Vis, e as bandas observadas entre 350-500 nm são evidências da presença de nanopartículas de prata. As imagens de microscopia eletrônica de transmissão revelaram AgNPs de morfologia esférica com diâmetro em torno de ~5 a ~60 nm, corroborando com os resultados de UV-Vis. / São Cristóvão, SE

The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute and chronic activation of both ionotropic and metabotropic glutamate (glut) receptors is implicated in many neurodegenerative disorders including AD, dementia, epilepsy, stroke and neurotrauma. TRH and glut receptors (ionotropic & metabotropic) receptors are differentially coexpressed in granule and pyramidal neurons of the hippocampus. The author shows TRH to be protective when added to cultured pituitary adenoma (GH-3) cells and neuron-like pheochromocytoma (PC12) cells either prior to, during, or after glut-induced toxicity (Endo. Soc. Abs. 01), and also shows that the possible neuroprotective mechanism may involve heterologous downregulation of the metabotropic glut receptors, using superfused hippocampal slices and noting a reduction of Gαq/11 (SFN Abs. 02). He has also demonstrated that TRH protected against glut toxicity in fetal cortical cultures (Endo. Soc. Abs. 04). To extend these studies he used 14-day cultured rat fetal hippocampal neurons (Day E17) to determine if TRH is protective against toxicity induced by specific ionotropic and metabotropic glut agonists. Neuronal viability and integrity were assessed by trypan blue exclusion and LDH release after 18 hrs following 30 min exposure to glut agonists. Ten µM dihydroxyphenylglycine (DHPG, a Group 1 receptor agonist) + 30 µM N-methyl-D-aspartate (NMDA)-induced toxicity (42% vs contr. P<0.05); whereas, concurrent and continued treatment with 10 uM but not 1uM 3Me-HTRH resulted in less neuronal death and damage (86% vs contr P<0.05; 53% vs contr. P>0.05) respectively. DHPG treatment alone (10 µM) for 30 min. was non-toxic by both criteria (90% vs contr. P<0.05). The data suggest that TRH may be a selective modulator of glut-induced toxicity.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.

Poly-gamma-glutamic acid (γPGA), which is a biodegradable, non-immunogenic and unusual anionic amino-acid polymer consist of D- and L-glutamic acid units, was exploited for a wide array of useful applications. Bacillus are well known cellular system important for fermentation to synthesize γPGA, which is used as thickener, drugs carrier, cryoprotectant, humectant, biological adhesive, flocculants, or heavy metal absorbent. This study focused on the isolation of Bacillus spp. that is possible to produce γ-PGA from different soil samples from different places in Vietnam. Study the effect of precursors, temperature, carbon sources, times and pH on γ-PGA production. From 31 soil samples and 4 straws samples, strain 20.2 which produced the highest γ-PGA yields (riches 15.2 mg/ml), was identified as Bacillus sp. 20.2 by molecular biology method. The suitable conditions for growing of Bacillus sp. 20.2 strain to produce γ-PGA are at 37°C, pH 7 after 72 hours. Citric acid instead of glucose in a GSP medium is better for producing γ-PGA by strain Bacillus sp. 20.2. / Poly-gamma-glutamic acid (γ-PGA) là một polymer amino-acid gồm D và L-glutamic acid, có khả năng phân hủy sinh học, không gây miễn dịch, đã được ứng dụng rộng rãi trong công nghiệp, y học. Bacillus subtilis được biết đến là hệ thống tế bào ý nghĩa quan trọng trong quá trình lên men để tổng hợp γ-PGA. γ-PGA hòa tan trong nước, phân hủy sinh học và không độc đối với con người và môi trường. γ-PGA ổn định với nhiều protease vì các protease thường không nhận acid γ- glutamic (Obst et al., 2004). γ-PGA có cấu trúc đồng phân đơn giản, không gây miễn dịch. Do đó, γ-PGA đã được quan tâm ứng dụng trong các lĩnh vực như y học, công nghiệp thực phẩm, mỹ phẩm và đặc biệt là xử lý nước nhiễm kim loại nặng. Trong nghiên cứu này chúng tôi tập trung phân lập, tuyển chọn các chủng Bacillus có khả năng sinh tổng hợp PGA cao. Sau đó định danh và đánh giá khả năng sinh tổng hợp PGA từ chủng đã phân lập được. Kết quả cho thấy từ 34 mẫu rơm và đất, chúng tôi đã phân lập được chủng với mã số 20.2 có khả năng sinh PGA cao nhất đạt 15.2 mg/ml. Chủng này đã được định danh bằng phân tích trình tự gene 16S rRNA và thuộc loài Bacillus sp. Môi trường thích hợp sinh tổng hợp PGA là GSP ở điều kiện 37oC pH7 sau 72 giờ nuôi cấy.

L'étude qu'on a entreprise a pour but d'analyser les facteurs de virulence des espèces "Classiques" et "nouvelles" de Brucella. Dans cette perspective, on a analysé les génomes des espèces récemment découvertes : Brucella inopinata BO1 et Brucella inopinata-like BO2, isolés pour la première fois de patients humains sans réservoir animal connu. On a découvert que ces deux espèces possèdent des profils de restriction uniques. De plus, BO2 possède deux chromosomes de taille identique, un profil jamais décrit pour une autre espèce de Brucella. L'analyse de la réplication intracellulaire de ces deux espèces révèle que BO2 ne se réplique pas dans les macrophages humains et murins alors que BO1 se réplique d'une façon similaire à Brucella suis 1330, ce qui confirme la potentielle implication de BO1 dans la pathogenèse chez l'homme. Sur un autre niveau d'analyse, on a été à la recherche de facteurs de virulence potentiels dans d'autres espèces de Brucella notamment Brucella microti et Brucella suis sur les niveaux génomique et post-transcriptionnel. Sur le niveau génomique, on a découvert que le système GAD (glutamate decarboxylase) confère une résistance à l'acidité à Brucella microti lors de son passage dans l'estomac. Sur le niveau post-transcriptionnel, on a isolé, séquencé et identifié les petits ARNs noncodant associés à la protéine chaperone Hfq, qui joue un rôle important dans la virulence de Brucella. / We have undertaken in this study a multidimensional analysis of the virulence factors of "Classical" and new "Brucella species". In this objective, we have analysed the genomes of newly described species Brucella inopinata BO1 and Brucella inopinata-like BO2 isolated for the first time from human patients with no known animal reservoir. We found that these two species have unique restriction profiles. In addition, BO2 has a unique chromosomal distribution with two chromosomes of the same size, never seen before in Brucella. Analysis of the intracellular replication of these strains reveals that BO2 is unable to replicate in neither human nor mouse macrophages while BO1 successfully entered and replicated as efficiently as Brucella suis 1330 confirming the potential virulence of this species for humans. On an other level of analysis, we looked for potential virulence factors in other Brucella species including Brucella microti and Brucella suis at the genomic and post-transcriptional level. At the genomic level we discovered that the glutamate decarboxylase system confers resistance to acidity to Brucella miroti during its transit in the stomach. On the post-transcriptional level, we isolated, sequenced and identified small noncoding RNAs associated to the chaperone protein Hfq, known to play a role in the virulence of Brucella.