Résumé : Grâce à des caractéristiques physiques particulières, le [indice supérieur 64]Cu (T[indice inférieur 1/2]= 12.7 h; β[indice supérieur+], 0.65 MeV [17.8 %]; β[indice supérieur −], 0.58 MeV [38.4 %]) est un candidat idéal pour l’imagerie TEP et la radiothérapie ciblée du cancer. Son utilisation est actuellement limitée par la disponibilité de chélateurs bi-fonctionnels (CBFs) offrant une résistance élevée aux réactions de transmétallation in vivo. Récemment nous avons développés deux nouveaux CBFs cycliques, DOTHA[indice inférieur 2] et NOTHA[indice inférieur 2], portant des ligands hydroxamates pour la complexation au [indice supérieur 64]Cu. Ces CBFs possèdent une cinétique de marquage rapide dans des conditions très douces, une stabilité élevée in vivo et un profil de biodistribution favorable avec une clairance rapide. Nous proposons maintenant d’étendre notre approche à la préparation de CBFs acycliques plus flexibles et compacts afin de moduler les propriétés biologiques et la pharmacocinétique des traceurs peptidiques. Le but de mon projet de maîtrise est de développer une série de chélateurs acycliques dérivés de l'histidine et de l'acide glutamique et fonctionalisés avec des groupements hydroxamates pour identifier un CBF offrant un complexe stable in vivo avec le [indice supérieur 64]Cu(II). Les CBFs ont été préparés en solution pour faciliter l’optimisation de chaque étape réactionnelle. Les groupements chélatants hydroxamates ont été sélectionnés pour leur habilité à former des complexes stables avec différents métaux et ils ont été liés en position N-terminale et sur la chaîne latérale des acides aminés grâce à des réactions de substitution nucléophile. Les groupements para-methoxy-benzyles ont été judicieusement sélectionnés pour la protection des groupements hydroxamates afin de faciliter, au besoin, une déprotection sélective sous des conditions très douces. L’optimisation du marquage a été effectuée avec l’isotope stable du cuivre et ensuite avec le [indice supérieur 64]Cu en faisant varier le contre ion métallique, le pH, la concentration, et la température. Le CBF offrant la plus grande stabilité, soit celui dérivé de l’histidine, a été conjugué à un peptide, le H[indice inférieur 2]N-PEG-[D-Tyr[indice supérieur 6],βAla[indice supérieur 11],Thi[indice supérieur 13],Nle[indice supérieur 14]]bombesin(6-14) (BBN), se liant fortement aux récepteurs de la relâche de la gastrine surexprimés dans les cancers du sein et de la prostate. La stabilité et l’activité spécifique du CBF-histidine et du radiotraceur marqués au [indice supérieur 64]Cu s’est avérée faible in vitro. Il est connu que l’activité antibactérienne de ligands hydroxamates est associée à leur capacité à complexer le fer. En perspective, comme nos chélateurs complexent très fortement le Fe(III), une alternative pour ces composés serait d’évaluer leur capacité à inhiber la croissance et la prolifération des bactéries. || Abstract : Thanks to its particular physical characteristics, [superscript 64]Cu (T[subscript ½= 12.7 h; β[superscript +], 0.65MeV [17.8 %]; β[superscript −], 0.58MeV [38.4 %]) is an ideal candidate for PET imaging and targeted cancer radiotherapy. Currently, its use is limited by the availability of bi-functional chelators (BFCs) which give high resistance to in vivo transmetallation reactions. Recently, we developed two new cyclic BFCs, DOTHA[subscript 2] and NOTHA[subscript 2], bearing hydroxamate pendant arms for the complexation with [superscript 64]Cu. Those BFCs have fast labeling kinetics under very mild conditions, a high in vivo stability and a biodistribution profile which is favorable with a fast clearance. Now, we propose to expand our approach to the preparation of acyclic BFCs, which are more flexible and compact, in order to better modulate biological properties and the pharmacokinetics of the peptidic tracers. The goal of my Master’s degree project is to develop a series of acyclic chelators derived from histidine and glutamic acid and functionalized with hydroxamate pendant arms to identify a BFC that shows highly stable in vivo complexes with [superscript 64]Cu(II). BFCs have been prepared in solution to facilitate the optimization of each reactive step. Hydroxamate chelating groups have been selected for their ability to form stable complexes with different metals and they have been conjugated in N-terminal position and on the lateral chain of amino acids via nucleophilic substitution reactions. Para-methoxy-benzyl groups have been judiciously selected for the protection of the hydroxamate groups to facilitate, if needed, a selective deprotection under mild conditions. The labeling optimization has been performed with a stable copper isotope, and then with [superscript 64]Cu varying the metallic counter-ion, pH, concentration and temperature. The BFC having the highest stability, the one derived from histidine, was conjugated to a peptide, H[subscript 2]N-PEG-[D-Tyr[superscript 6],βAla[superscript 11],Thi[superscript 13],Nle[superscript 14]]bombesin(6-14) (BBN), strongly bounding the gastrin releasing peptide receptor, which is overexpressed in breast and prostate cancers. Both the stability and specific activity of BFC-histidine of the radiotracer labeled with [superscript 64]Cu were low in vitro. It is known that the antibacterial activity of hydroxamate ligands is associated with their ability to complex iron. In perspective, because our hydroxamate ligands strongly complex Fe(III), an alternative for these compounds would be to assess their ability to inhibit the growth and proliferation of bacteria.

Des nanoparticules multifonctionnelles polymères, préparées par auto-assemblage de plusieurs dérivés du poly (L-glutamate de gamma-benzyle) (PBLG), ont été conçues afin d’assurer le ciblage des tissus osseux et la libération contrôlée de molécules actives. Des propriétés d'attachement aux tissus osseux leur ont été conférées par la présentation en surface de différents ligands ostéotropes, l'alendronate et l' acide poly(glutamique), seuls ou en combinaison. Leur affinité pour les tissus osseux a été évaluée in vivo ainsi que leur distribution fine dans ces tissus. Par ailleurs, des propriétés anticancéreux ont été conférées aux nanoparticules grâce à un mécanisme originale d’association du cisplatin par complexation. Le procédé mis en œuvre permet d’obtenir des cinétiques de libération très progressives de dérivés actifs du platine et déclenchée par la présence des ions chlorure. Enfin, leur cytotoxicité a été mesurée. Cette stratégie constitue donc une approche prometteuse en vue d’améliorer le traitement des métastases osseuses. / Multifunctional bone targeted polymeric nanoparticles prepared by self-assembly of several poly(gamma-benzyl-L-glutamate) (PBLG) derivates have been developed. Their bone binding properties were provided by two different osteotropic moieties, alendronate or/and poly(glutamic acid) exposed on the nanoparticle surface. Their affinity for bone tissues has been evaluated in vitro, ex vivo and in vivo, including their detailed distribution in bone tissues structures. Further, in view of bone cancer therapeutics, nanoparticles were provided with anticancer properties thanks to the complexation of cisplatin, which leaded to very well controlled release properties. Finally, cytotoxicity were studied. Therefore, this strategy constitute a promising approach for the improvement of bone cancer therapeutics.

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

Metallic nanoparticles are unique materials for optical, electronic, catalytic, and sensing applications. Due to the vast flexibility in controlling the surface chemistry of these particles through functionalization there is a great deal of interest in using metallic nanoparticles as building blocks in the development of more complex nanostructures through the use of a 'bottom-up' approach. Using self assembly techniques, one can exploit spontaneous chemical interactions to build complex constructs on the nanometer scale. Towards this end, gold nanorods have been synthesized and modified with various polymers, inorganic oxides and organic ligands to establish principles for self-assembly of these unique nanomaterials. Gold nanorods are of great interest due to their strong optical absorption in the visible and near infrared regions, which can be tuned through material preparation and modification of the surrounding environment. This thesis focuses on investigating approaches for both irreversible and reversible self-assembly of gold nanorods. Techniques such as dynamic light scattering (DLS), ultraviolet-visible (UV) spectroscopy, transmission electron microscopy (TEM), and polarization modulation infrared reflection absorbance spectroscopy (PM-IRRAS) were used to characterize the colloidal particles and gold surfaces. A novel contribution of this work is the successful demonstration of end-to-end linking of gold nanorods in a rapid and reversible manner using a pH responsive polypeptide.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009.

Στην παρούσα διατριβή μελετήθηκε το ντοπαμινεργικό και γλουταμινεργικό σύστημα των βασικών γαγγλίων, χρησιμοποιώντας δύο διαφορετικά μοντέλα ζώων. Ο πρώτος στόχος ήταν να εξεταστεί εάν η μεταφορά του γονιδίου TrkA σε νευρώνες της μέλαινας ουσίας (SN) ενήλικων επίμυων επιδρά στις νευροχημικές τους ιδιότητες, απουσία ή παρουσία εξωγενούς νευροαυξητικού παράγοντα (NGF) στο ραβδωτό σώμα. Η εκτοπική έκφραση του TrkA στην SN οδήγησε σε σημαντική μείωση του mRNA της υδροξυλάσης της τυροσίνης (TH), της TH ανοσοδραστικότητας και του mRNA του DAT στη δεξιά SN σε σύγκριση με την ετερόπλευρη, ενώ δεν βρέθηκε καμία διαφορά στο mRNA των υποδοχέων ντοπαμίνης D2 και της ειδικής δέσμευσης του [3Η]raclopride στην SN. Δεν παρατηρήθηκαν μεταβολές στις θέσεις δέσμευσης του [3Η]WIN35428 και της ανοσοδραστικότητας του DAT στο ομόπλευρο ραβδωτό σώμα, καθώς και στις θέσεις δέσμευσης των μετασυναπτικών υποδοχέων ντοπαμίνης D1 και D2, όπως καθορίζεται από τους ιχνηθέτες [3H]SCH23390 και [3Η] raclopride, αντίστοιχα. Επιπλέον, δεν βρέθηκαν σημαντικές μεταβολές στους υποδοχείς NMDA και AMPA. Τα αποτελέσματα αυτά δείχνουν ότι η εκτοπική έκφραση του TrkA στην SN ρυθμίζει αρνητικά την ΤΗ και οδηγεί σε ανεξάρτητες από τον NGF αποκρίσεις. O δεύτερος στόχος της διατριβής ήταν η μελέτη του ντοπαμινεργικού συστήματος σε ένα μοντέλο DYT1 δυστονίας μυός. Σε διαγονιδιακούς μυς με υπερκινητική και μη υπερκινητική συμπεριφορά παρατηρήθηκε μείωση των υποδοχέων ντοπαμίνης D2 στο ραβδωτό σώμα, όπως προσδιορίστηκε από τη δέσμευση του [3H]raclopride, και τoυ mRNA των D2 στην SNpc, σε σχέση με μη διαγονιδιακούς μυς. Δεν παρατηρήθηκε διαφορά στη δέσμευση του [3H]SCH23390 ή του [3H]WIN35428 στο ραβδωτό σώμα διαγονιδιακών μυών. Τα δεδομένα προτείνουν μία πιθανή εμπλοκή της ντοπαμινεργικής νευροδιαβίβασης στην παθοφυσιολογία της DY1 δυστονίας. / In the present thesis we examined the dopaminergic and glutamatergic neurotransmission systems of basal ganglia, using two different animal models. The first goal was to investigate whether TrkA gene transfer into substantia nigra (SN) neurons of adult rats influence some of their neurochemical properties, in the absence or presence of exogenous nerve growth factor (NGF) delivery in the striatum. Ectopic expression of TrkA in SN resulted in a significant decrease of tyrosine hydroxylase (TH) immunoreactivity, TH mRNA and DAT mRNA expression in the right SN compared to the contralateral side, while no difference was found in the mRNA expression of D2 DA receptors and [3H]raclopride binding in SN. No significant changes were seen in the density of DAT by measuring [3H]WIN35428 binding sites and DAT immunoreactivity in the ipsilateral striatum, as well as in the number of postsynaptic striatal D1 and D2 receptor binding sites, as determined by [3H]SCH23390 and [3H]raclopride, respectively. Furthermore, no significant changes were found in NMDA and AMPA receptors. These data suggest that ectopic TrkA expression in SN downregulates TH in nigral dopaminergic neurons and elicits NGF-independent responses. The second goal of the present thesis was to examine the dopaminergic system of basal ganglia in a mouse model of DYT1 dystonia. A decrease in striatal D2 binding sites, measured by [3H]raclopride binding, and D2 mRNA expression in substantia nigra pars compacta (SNpc) was revealed in affected and unaffected transgenic mice when compared with non-transgenic. No difference in D1 receptor binding and DAT binding, measured by [3H]SCH23390 and [3H]WIN35428 binding, respectively, was found in striatum of transgenic animals. These data suggest a possible involvement of dopamine neurotransmission in the pathophysiology of DYT1 dystonia.

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.

O ácido y-poliglutâmico (y-PGA) é um biopolímero solúvel em água, aniônico, atóxico, biodegradável e biocompatível, produzido por Bacillus e que possui aplicações nas áreas química, médica, ambiental e de alimentos. Este trabalho tem como objetivo estudar o processo de produção do y-PGA a partir do cultivo de uma nova linhagem de Bacillus isolada de ambiente amazônico, o Bacillus subtilis BL53. Este estudo inclui a seleção de culturas, a otimização de condições de cultivo e a prospecção de substratos de produção alternativos. A habilidade de produção de y-PGA por linhagens de Bacillus isoladas na região amazônica foi investigada. A linhagem BL53 foi a linhagem selecionada, sendo identificada como Bacillus subtilis, através da análise da seqüência do gene 16S DNAr e de suas características bioquímicas. A avaliação das condições de cultivo submerso foi conduzida em agitador orbital mediante delineamento composto central rotacional (DCCR) que apontou como ponto ótimo de produção de y-PGA, a temperatura de 37 oC, o pH inicial de 6,9 e a concentração de 1,22 mM de Zn2+ suplementada ao Caldo E. Nas condições otimizadas, a produção de y-PGA foi igual a 10,4 g/L, cerca de três vezes maior que a obtida em condições convencionais de cultivo em Caldo E, utilizando a mesma linhagem. A influência da disponibilidade de oxigênio sobre a produção de y-PGA por B. subtilis BL53 foi avaliada em biorreator agitado de 5 L, com o emprego das velocidades de agitação de 500, 750 e 1000 rpm, sendo mantida fixa a taxa de aeração de 2 vvm. A produção de y-PGA mostrou-se altamente dependente da transferência de oxigênio, sendo que o teor de oxigênio dissolvido decaiu rapidamente nas primeiras 15 horas de cultivo, como resultado da elevada demanda de oxigênio pelas bactérias. O aumento na velocidade de agitação no biorreator possibilitou uma maior transferência de oxigênio e induziu o aumento na taxa de consumo de oxigênio pelas bactérias, conduzindo a maior produção e maior produtividade de y-PGA. A intensificação da agitação também influenciou os parâmetros cinéticos de crescimento do Bacillus subtilis BL53, provocando um aumento na velocidade específica de crescimento na fase logarítmica (max) sem provocar a perda da viabilidade celular. Com a utilização da velocidade de agitação de 1000 rpm em biorreator, o tempo de cultivo pode ser reduzido para menos de 48 h, cerca de 50% do tempo necessário para operação a 500 rpm. A investigação de substratos alternativos para produção de y-PGA mostrou o glicerol residual de biodiesel e o resíduo fibroso de soja como substratos promissores, apontando para a possibilidade de investigação em trabalhos futuros. / The poly-gamma-glutamic acid (y-PGA) is a water-soluble biopolymer, anionic, non toxic, biodegradable and biocompatible, it is produced by Bacillus and it has applications in chemical, medical, environmental and food industries. This work aims to study the process of production of y-PGA through cultivation of a new strain of Bacillus isolated from the Amazonian environment, Bacillus subtilis BL53. This study includes the screening of strains, the optimization of culture conditions and the investigation of alternative substrates. The ability of y-PGA production by Bacillus strains isolated from Amazonian environment was investigated. The BL53 strain was selected and identified as Bacillus subtilis, through analysis of 16S rDNA gene sequence and its biochemical characteristics. Evaluation of culture conditions in submerged cultivation was conducted in shaker using central composite design (CCD), which showed the temperature of 37 oC, the initial pH 6.9 and concentration of 1.22 mM Zn2+ in Medium E as the optimal conditions to y-PGA production. Under optimized conditions, the production of y-PGA was 10.4 g/L, about threefold what was obtained using this strain under conventional cultivation in Medium E. The influence of oxygen availability on the production of y-PGA by B. subitlis BL53 was evaluated in a 5 l stirred bioreactor with the use of stirring rates of 500, 750 and 1000 rpm, using a fixed aeration rate of 2 vvm. Production of y-PGA was highly dependent on oxygen transfer, and the dissolved oxygen content decreased rapidly in the first 15 hours of culture as a result of high oxygen demand by bacteria. The increase in stirring rate in the bioreactor allowed a better oxygen transfer and induced a rise in the oxygen uptake rate by bacteria, leading to higher production and higher productivity of y-PGA. The intensification of the stirring also influenced the kinetics growth parameters of Bacillus subtilis BL53, producing an increase in specific growth rate in the logarithmic phase (max) without causing reduction of cell viability. Using the stirring rate of 1000 rpm in a bioreactor, the cultivation time can be reduced to less than 48 h, about 50% of the time required for operation at 500 rpm. The investigation of alternative substrates for the production of y-PGA showed crude glycerol from biodiesel and soybean industrial fibrous residue as promising ones, pointing to research possibilities in future work.

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.

LIMA, Cleânio da Luz. Estudo espectroscópico de materiais: a mútipla aplicabilidade da espectroscopia Raman na caracterização de aminoácidos, molibidato e tungstato de sódio, e óxidos nanomoldados. 2011. 143 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2014-11-13T20:27:15Z No. of bitstreams: 1 2011_tese_cllima.pdf: 6232743 bytes, checksum: c0525f4a943562b9a02c7976bc0aa766 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2014-11-13T20:37:41Z (GMT) No. of bitstreams: 1 2011_tese_cllima.pdf: 6232743 bytes, checksum: c0525f4a943562b9a02c7976bc0aa766 (MD5) / Made available in DSpace on 2014-11-13T20:37:41Z (GMT). No. of bitstreams: 1 2011_tese_cllima.pdf: 6232743 bytes, checksum: c0525f4a943562b9a02c7976bc0aa766 (MD5) Previous issue date: 2011 / Structure determination of aminoacid (C5H9NO4), tungstate (Na2WO4) and molybdate (Na2MoO4), and MOx nanostructureed oxides was performed through Raman spectroscopy. L-glutamic acid was investigated by Raman spectroscopy with the aim of evaluating the effect of the hydrostatic pressure on their structural and vibrational properties. The nanocrystalline samples were obtained by slow evaporation method; and two polymorphic phases were produced. To identify the abovementioned phases, X-ray diffraction (XRD), thermal analysis (DSC), infrared spectra (FT-IR) and Raman spectroscopy experiments were performed. The latter was decisive to distinguish the two phases formed, and was complementary to XRD. When high pressures were applied, modifications in the L-glutamic acid structure were observed. Such a modification was due to vibrational and changes in the wavenumber versus pressure curves. These modifications evidenced that the β-phase of the crystal had four structural phase transitions from room pressure to 21.5 GPa; for α-phase crystal, there structural phase transitions from room pressure to 7.5 GPa. Decompression to room pressure provided the restored original spectra, which indicating that the phase transitions were reversible to the aforesaid phases. Variation of the temperature of the polycrystalline Na2MoO4 and Na2WO4 was performed to obtain information about the structural change occurred the solids. Cubic phase stability of the Na2MoO4 and Na2WO4 was verified and these studies indicated that the crystals maintained the cubic phase in the 8- 823 K and 15 – 773 K range for Na2MoO4 and Na2WO4 crystals, respectively; a first order transition phase was shown through the Raman modes behavior: the splitting of the new modes appeared below to 100 cm-1. These transitions were due to the disorders of MoO4 and WO4 sites from Na2MoO4 and Na2WO4 structure, respectively. The structure determination of nanostructured oxides (monoxides and binary oxides) was investigated by Raman spectroscopy. Nanoparticles mode of CeO2, ZrO2 and TiO2 as well as that of CeO2-ZrO2 and Ce0.08Zr0.2O2 were obtained. XRD analysis, textural and surface properties and Raman spectroscopy, being the latter responsible for a deeper understanding of the structural properties of the nanoparticles and nanostructure when applied as catalysts for glycerol dehydration to acrolein and 1-hydroxyacetone the solids were actives, and actively was related to the solids structure. / A elucidação da estrutura do aminoácido (C5H9NO4), molibidato (Na2MoO4) e tungstato (Na2WO4) de sódio, e óxidos nanomoldados (CeO2, ZrO2, TiO2 e Ce0,8Zr0,2O2) foi realizada através da espectroscopia Raman. O ácido L-glutâmico (C5H9NO4) foi investigado através de experimentos de espectroscopia Raman, visando avaliar os efeitos da aplicação da pressão hidrostática em suas propriedades estruturais e vibracionais. As amostras monocristalinas foram obtidas através do método de evaporação lenta, gerando duas fases. Para a identificação das referidas fases, foram realizados experimentos de difração de raios-X (DRX), análise térmica (DSC), espectroscopia na região do infravermelho (FT-IR) e Raman. Esta última técnica foi decisiva para a distinção das duas fases, de modo a complementar o DRX. Quando o ácido L-glutâmico foi submetido a altas pressões, foram observado modificações em sua estrutura; tais modificações ocorridas foram no número de modos vibracionais e descontinuidade nas curvas número de onda vs pressão; as modificações evidenciaram que o cristal da fase β sofreu quatro transições de fase estruturais desde a pressão ambiente até 21,5 GPa. Para o cristal da fase α, observou-se três transições de fase estruturais, desde a pressão ambiente até 7,5 GPa. Na descompressão para a pressão ambiente, os espectros originais foram recuperados, indicando que as transições de fase são reversíveis, para as referidas fases α e β. Os experimentos de espectroscopia Raman com variação de temperatura dos materiais policristalinos Na2WO4 e Na2MoO4, foram realizados para se obter informações sobre a estabilidade estrutural destes sólidos. A estabilidade da fase cúbica dos sistemas Na2WO4 e Na2MoO4 foram verificadas e estes estudos indicaram que os cristais permanecem com tal fase no intervalo de 8-823 K e 15-773 K para Na2WO4 e Na2MoO4, respectivamente. Destaca-se, que sobre o cristal de Na2WO4 houve uma transição de fase em 898 K aproximadamente. O cristal de Na2MoO4, por outro lado, exibiu três transições de fase em altas temperaturas, 783-803 K, 823-913 K e 943-950 K. Em ambos os cristais as transições de fase são de primeira ordem, e evidênciadas pelo comportamento dos modos Raman: o splitting de novos modos abaixo de 100 cm-1. Estas transições foram relacionadas à inclinação e/ou rotações dos tetraedros WO4 e MoO4 que provocam uma desordem nos sítios WO4 e MoO4, os quais são constituintes da estrutura Na2WO4 e Na2MoO4, respectivamente. A elucidação da estrutura de óxidos nanoestruturados (monóxidos e óxidos binários) foi realizada por espectroscopia Raman. Nanopartículas constituídas de CeO2, ZrO2 e TiO2, bem como nanoestruturas constituídas por CeO2-ZrO2 e Ce0,8Zr0,2O2 foram obtidos. As análises de DRX, propriedades texturais e superficiais foram comparadas através da espectroscopia Raman, sendo que esta técnica possibilitou a compreensão das propriedades estruturais das nanopartículas e nanoestruturas. Os referidos sólidos mostram ser ativos quando aplicados como catalisadores na desidratação do glicerol a acroleina e 1-hidroxicetona, sendo a atividade catalítica relacionada à estrutura dos sólidos.