Cocaine (CA) exposure during pregnancy causes long-lasting negative effects on fetal brain development and eventually results in motor dysfunction or changes in learning and memory performance. £^-amino-butyric acid (GABA) is the primary inhibitory neurotransmitter in the adult brain and undergo a switch from excitatory to inhibitory during early postnatal period. The excitatory/inhibitory switch is resulted in the relative temporal expression of K+-Cl- cotransporter 2 (KCC2). GABA is the neurotransmitter in the rat was born from excitement to inhibition and until the growth of thirty days have completely inhibitory. Here we test the effect of CA prenatal exposure on the expression of KCC2 in prefrontal cortex (recognition), hippocampus (memory), VTA (reward) and nucleus accumbens (reward). Protein expression profile of control or prenatal CA treated groups were evaluated by western blot in 2 days interval from postnatal day (PND) 8 to 30. The expression of KCC2 was time-dependently enhanced from PND 8 and reaches its maximal expression around PND 18 in prenatal CA exposure groups. The time-dependent profile of KCC2 expression in prefrontal cortex and NAc was significantly delayed in prenatal CA exposure group. We then correlate the KCC2 expression and the cocaine sensitivity by locomotor activity assay. We found group A shows a higher sensitivity to cocaine than group B in control rats. Surprisingly, group A of prenatal cocaine reduce the sensitive to cocaine to a similar extend like group B in control rats, suggesting prenatal exposure of cocaine might enhance the KCC2 expression. Furthermore, age range of A group (PND 22~27) and B group (PND 29~34) to repeated cocaine exposure resulted in up-regulation of KCC2 expression in B group earlier than A group. We found that the KCC2 expressions of repeated cocaine exposure in B group were higher than A group. In other words, in the B group, the inhibitory effect of GABA was significant and the locomotor activity was relatively slow. Therefore, the A group was more easy be cocaine addiction than B group. We next explore the signaling mechanism underlying cocaine exposure-induced KCC2 expression inhibition. Brain slices were incubated with cocaine with or without dopamine receptor antagonists and KCC2 expression was evaluated by western blot. Either SCH23390 (dopamine D1-receptor inhibitor) or eticlopride (dopamine D2-receptor inhibitor) significantly hamper the inhibition of KCC2 expression by cocaine in normal slices. However, only D2 antagonist eticlopride but not SCH23390 is effective reverse cocaine-induced KCC2 expression inhibition. Overall, results from our current studies provide a further insight into the molecular mechanism of cocaine-induced synaptic modification.

This work has employed the Micro-wave Plasma enhanced Chemical Vapor Deposition (MPCVD) method to fabricate diamond PN junction device. The n+ <111> orientation single-crystal silicon has used as substrates. P-type diamond layer is doped with B(OCH3)3 and the N-type diamond layer is doped with ammonia. The surface structure of diamond film has been observed by scanning electron microscope; and the device rectification property of a PN junction has measured by current-voltage characteristic. The carrier density and mobility of diamond films have been analyzed by Hall measurement. Furthermore, the Cathodoluminescence (CL) spectroscopy showed the defect spectra in diamond PN junction. The N-type diamond film and P-type diamond film have deposited at temperature of 800 ¢J, for 30 minutes and 90 minutes, respectively. The process CVD has performed in the same chamber continually. A I-V curve of sample showed the set on positive voltage 0.5 V and the reverse breakdown voltage of 6 V. Further, CL results revealed a peak at 285 nm (4.4 eV), which represents the CVD diamond band and the other one is at 500 nm (2.5 eV), which stands for donor-acceptor recombination from defect in these diamond films.

Made available in DSpace on 2014-06-12T16:27:22Z (GMT). No. of bitstreams: 2 arquivo2084_1.pdf: 1052849 bytes, checksum: 5755ac115807355b687973e612634a6e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / A adoção da Política nacional de Medicamentos Genéricos pelo Governo Federal (Lei n° 9.787 de 10 de fevereiro de 1999) envolve a produção de medicamentos de melhor qualidade, mais seguros e eficazes, comprovados através da realização de testes de equivalência farmacêutica e bioequivalência, contribuindo para aumento do acesso aos medicamentos e fortalecendo a indústria nacional através do desenvolvimento tecnológico. Para isto, é necessário o desenvolvimento e a validação de metodologias analíticas capazes de determinar o fármaco em fluidos biológicos. No Brasil, ainda existe uma carência de recursos humanos qualificados para atender a crescente demanda do mercado. Como colaborador da implementação da política nacional de Medicamentos Genéricos, este trabalho tem por objetivo o desenvolvimento e validação de métodos bioanalíticos para aplicação em estudos de biodisponibilidade relativa de dois fármacos (cimetidina e hidroclorotiazida) em voluntários sadios. Aplicou-se a técnica de extração liquido-liquido para purificação das amostras e para quantificação foi utilizada a cromatografia líquida de alta eficiência com detecção por espectrometria de massas em tandem (CL-EM/EM). Após o desenvolvimento, validação e aplicação das metodologias; os resultados dos parâmetros farmacocinéticos foram obtidos através das curvas de concentração sangüínea do fármaco versus tempo, e analisados estatisticamente para determinação da bioequivalência. Os seguintes parâmetros farmacocinéticos devem ser determinados: ASC, Cmax e Tmax. Os métodos demonstraram-se econômicos, práticos, rápidos, sensíveis, precisos e exatos segundo as normas da ANVISA e da FDA. Portanto, os estudos apresentaram ferramentas adequadas para avaliação das formulações de cimetidina comprimido (teste e referência) e das formulações de hidroclorotiazida comprimido (teste e referência), ambas mostraram-se bioequivalentes

O presente estudo foi realizado com o objetivo de se desenvolver e validar uma metodologia analítica indicativa de estabilidade para separação e quantificação de anlodipino e seus produtos de degradação no medicamento besilato de anlodipino 5 mg por comprimido. Para tanto, realizou-se um estudo de degradação forçada, como forma de identificar os principais produtos de degradação, que podem vir a formar num estudo de estabilidade, bem como estabelecer possíveis rotas de degradação. Foi utilizada a técnica instrumental de separação, cromatografia a líquido (CL), com dois tipos de detectores: arranjo de diodos (DAD) e corona CAD (Detector de aerossol carregado), coluna cromatográfica Zorbax SB-Phenyl 1,8&#181;m 4,6 x 50 mm Agilent® e fase móvel constituída por tampão pH3,0, metanol e acetonitrila sob condições de gradiente (75% de tampão pH 3,0, 15% de metanol e 10% de acetonitrila; no tempo de 10 minutos tem-se 20% de tampão pH3,0, 60% de metanol e 20% de acetonitrila; no tempo de 13 minutos tem-se 75% de tampão pH3,0, 15% de metanol e 10% de acetonitrila; e no tempo de 15 minutos tem-se 75% de tampão pH3,0, 15% de metanol e 10% de acetonitrila); fluxo de 1,0mL/min até 10 minutos; de 10,1 a 13 minutos tem-se fluxo de 0,5 mL/min; e de 13,1 a 15 minutos volta ao fluxo de 1,0 mL/min; e detecção em 238 nm. O método apresentou-se linear, preciso, exato, robusto e seletivo, e proporcionou resultados confiáveis, sem interferência de degradantes e impurezas. / This study was conducted in order to develop an analytical methodology indicative of stability for separation and quantification of the antihypertensive amlodipine and its degradation products in the drug amlodipine besylate 5 mg per tablet. For this development was performed a forced degradation study, in order to identify the main degradation products that may form in a stability study, as well as establish possible degradation routes. It was used the instrumental separation technique, liquid chromatography (LC) with two types of detectors: diode array (DAD) and corona CAD (Charged Aerosol Detector), chromatographic column Zorbax SB-Phenyl 4.6 x 50 mm 1,8&#181;m Agilent® and mobile phase of buffer pH3,0, methanol and acetonitrile under gradient conditions (75% buffer pH3,0, 15% methanol and 10% acetonitrile; 10 minutes has 20% buffer pH3,0, 60% methanol and 20% acetonitrile; 13 minutes has become 75% of buffer pH3,0, 15% methanol and 10% acetonitrile; and 15 minutes has become 75% of buffer pH3,0, 15% methanol and 10% acetonitrile); flow 1.0 mL/ min up to 10 minutes; a flow 0,5 mL/min is obtained from 10,1 to 13 minutes; and from 13,1 minutes the flows returns to 1,0 mL/min; and detection in 238 nm. The method is linear, precise, accurate, robust and selective, and provided reliable results without interference from degradation products and impurities.

The Salmonella enterica subsp. enterica serovar Typhimurium PhoPQ two-component system is activated within the intracellular phagosome environment, where it promotes remodeling of the outer membrane and resistance to innate immune antimicrobial peptides. Maintenance of the PhoPQ-regulated outer membrane barrier requires PbgA, an inner membrane protein with a transmembrane domain essential for growth, and a periplasmic domain required for PhoPQ-activated increases in outer membrane cardiolipin. Here, we report the crystal structure of cardiolipin-bound PbgA, adopting a novel transmembrane fold that features a cardiolipin binding site in close proximity to a long and deep cleft spanning the lipid bilayer. The end of the cleft extends into the periplasmic domain of the protein, which is structurally coupled to the transmembrane domain via a functionally critical C-terminal helix. In conjunction with a conserved putative catalytic dyad situated at the middle of the cleft, our structural and mutational analyses suggest that PbgA is a multifunction membrane protein that mediates cardiolipin transport, a function essential for growth, and perhaps catalysis of an unknown enzymatic reaction. IMPORTANCE Gram-negative bacteria cause many types of infections and have become increasingly resistant to available antibiotic drugs. The outer membrane serves as an important barrier that protects bacteria against antibiotics and other toxic compounds. This outer membrane barrier function is regulated when bacteria are in host environments, and the protein PbgA contributes significantly to this increased barrier function by transporting cardiolipin to the outer membrane. We determined the crystal structure of PbgA in complex with cardiolipin and propose a model for its function. Knowledge of the mechanisms of outer membrane assembly and integrity can greatly contribute to the development of new and effective antibiotics, and this structural information may be useful in this regard.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18181号 / 医博第3901号 / 新制||医||1004(附属図書館) / 31039 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 松田 道行, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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