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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
81

Identification and characterization of a Pseudomonas aeruginosa phospholipase C that contributes to lipid chemotaxis /

Barker, Adam Paul. January 2006 (has links)
Thesis (Ph.D. in Microbiology) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 145-167). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
82

NOVEL DOPAMINERGIC SIGNALING MODULATING HIPPOCAMPAL SYNAPTIC TRANSMISSION

Rizvi, Nisha 01 August 2015 (has links)
Dopaminergic systems regulate many brain functions and dysfunction of dopaminergic neurotransmission is thought to underlie numerous disorders, including schizophrenia, attention deficit hyperactivity disorder (ADHD), depression and Alzheimer’s disease. In the hippocampus, a dopaminergic projection from the ventral tegmental area (VTA) is proposed to be essential for controlling entry of sensory information into long-term memory through novelty and salience detection. However, the effects of the VTA-dopamine system on hippocampal synaptic transmission are largely under-explored and the underlying mechanisms are unclear. The goal of this project was to investigate mechanisms involved in dopaminergic modulation of hippocampal neurophysiology. Specifically, I (1) examined if dopamine modulates hippocampal synaptic transmission in a region- and input-specific manner, and (2) studied the signaling mechanisms underlying such modulation. In the first aim for the study, I tested whether SKF38393, a dopamine D1-like receptor agonist, differentially affects excitatory synaptic transmission in perforant path synapses onto dentate gyrus granule cells and whether such effects differ from those at area CA1 synapses. I found that SKF38393 produced a concentration-dependent increase in field excitatory postsynaptic potential (fEPSP) in both subregions, but that higher concentrations were needed in the dentate gyrus to produce comparable effects. This synaptic enhancement was long-lasting and largely irreversible which suggests it may be a form of long term enhancement (LTP). Also, the increase in synaptic transmission at medial perforant path synapses was larger than in the lateral perforant path. Importantly, effects in the dentate gyrus, unlike those in CA1, differed substantially along the dorsoventral axis, with effects being significantly larger at the dorsal compared to the ventral pole. In the second aim, various combinations of D1 and D2-like receptor agonists and antagonists as well as inhibitors of second messenger systems, demonstrated that differential mechanisms were required for initiation and maintenance of SKF38393-mediated early and late-phase enhancement and that a novel non-canonical phospholipase-C (PLC) dependent signaling pathway may be involved. Based on recent discoveries in other brain regions, we hypothesized that multiple subcellular signaling pathways may contribute to PLC activation which may include but are not limited to D1(5)-D2 heteromers and Gβγ complex. In conclusion, this work uncovers novel dopaminergic signaling pathways regulating hippocampal physiology, which will lead to development of better (functionally selective) therapeutic agents.
83

Propriedades eletrônicas, ópticas e vibracionais da região C- Terminal da fosfolipase A2 Lys 49 / Electronic, optical and vibrational properties of the C-terminal region of phospholipase A2 Lys 49

SANTOS, César Augusto Silva dos. 11 July 2018 (has links)
Submitted by Rosana Amâncio (rosana.amancio@ufcg.edu.br) on 2018-07-11T20:08:46Z No. of bitstreams: 1 propriedades eletronicas, ópticas e vibracionais da região C.pdf: 18345145 bytes, checksum: e90bc1985f0f75d2e3d83ecaa39d4a34 (MD5) / Made available in DSpace on 2018-07-11T20:08:46Z (GMT). No. of bitstreams: 1 propriedades eletronicas, ópticas e vibracionais da região C.pdf: 18345145 bytes, checksum: e90bc1985f0f75d2e3d83ecaa39d4a34 (MD5) Previous issue date: 2016-07-29 / Neste trabalho, apresentamos um estudo das propriedades eletrônicas, ópticas, vibracionais e termodinâmicas da região C-Terminal de Fosfolipases A2 Lisina 49 (PLA2s Lys 49). Este foi realizado por meio de cálculos quânticos através da Teoria do Funcional da Densidade (DFT), utilizando a aproximação da densidade local (Local Density Approximation - LDA) e a aproximação do gradiente generalizado (Generalized Gradient Approximation - GGA). Também foram realizados cálculos baseados no modelo tight binding. As PLA2 Lys 49 compõe um grupo de miotoxinas que apresentam pouca ou nenhuma atividade catalítica e ainda assim são capazes de atuarem na membrana celular por meio de um mecanismo alternativo propiciando a morte da célula. A região C-terminal destas proteínas, em particular a região compreendida entre os aminoácidos 115-129, é apontada como responsável pelo dano as membranas. Pouco se sabe sobre as características que propiciam a esta região tal capacidade e como acontece a sua interação com a membrana celular. Este trabalho realizou uma caracterização das propriedades físicas desta região. Buscando, portanto, estabelecer uma relação entre as propriedades físicas expressas por essa região e seu potencial de dano celular. Um resultado inicial obtido por este estudo foram as curvas corrente-voltagem (I-V ) para nove diferentes peptídeos que correspondem as regiões 115-129 de PLA2 de diferentes espécies de serpentes. As curvas I-V foram obtidas por meio do modelo tight binding. Elas demostraram que os peptídeos estudados apresentam características de semicondutores. Também apresentam semelhança com resultados experimentais obtidos por LOMONTE et al, 2003. Usando a DFT, foram realizados os cálculos da área acessível ao solvente, da densidade eletrônica, análise populacional de cargas, orbitais de fronteira, densidade de estados. Também foram realizados cálculos de propriedades vibracionais como espectro infravermelho, de propriedades ópticas e das propriedades termodinâmicas como capacidade térmica, entropia, entalpia e energia livre. As propriedades eletrônicas demostram que há a possibilidade de que a interação da região C-Terminal com a membrana seja predominantemente eletrostática. A análise populacional de carga demostrou que o aminoácido Lisina 122 possui carga igual a zero. Este fato indica que ele pode não possuir papel importante como é descrito na literatura. Os resultados obtidos para a área acessível ao solvente indicam que um peptídeo com maior área disponível para interagir com a membrana não causará maior dano. Os resultados ópticos apresentaram picos de absorção dentro da região visível. Estes resultados juntamente com os resultados vibracionais servem como uma "digital" para a identificação dos peptídeos estudados. Os resultados termodinâmicos apresentados neste trabalhos podem ser utilizados em futuras pesquisas envolvendo PLA2s Lisina 49. / In this work, we present a study of the electrical properties, optical, vibrational and thermodynamic of the C-terminal Phospholipase A2 Lysine region 49 (PLA2 Lys 49). This was done by means of quantum calculations by Density Functional Theory (DFT), using the approach of Local Density Approximation (LDA) and the approach of the Generalized Gradient Approximation (GGA). They were also made calculations based on the model tight binding. The PLA2 Lys 49 composes myotoxins group that presents a little or no catalytic activity and still are able to act on the cell membrane by providing an alternative mechanism of cell's death. The C-terminal region of these proteins, in particular the region between amino acids 115-129 is identi_ed as responsible for the damage the membranes. Little is known about the characteristics that propitiate to this region such capacity and how are their interaction with the cell membrane. This work constitutes a characterization of the physical properties of this region. Searching, therefore, establish a relationship between the physical properties expressed by this region and its potential for cell damage. An initial results obtained in this study were current-voltage curves (I-V) for nine di_erent peptides corresponding to regions 115-129 PLA2 from di_erent snake species. The (I-V) curves were obtained by the model tight binding. They demonstrate that the peptides studied have semiconductor characteristics. They also have similarity with experimental results obtained by LOMONTE et al., 2003. Using the DFT were performed the calculations of the area accessible to the solvent, the electron density, population analysis of load, orbital border, and density of states. They were also carried out calculations of vibrational properties as infrared spectrum, optical properties and thermodynamic properties such as heat capacity, entropy, enthalpy and free energy. The electronic properties show that there is a possibility the interaction of the C-terminal region with the membrane it's predominantly electrostatic. The load population analysis showed that the amino acid Lysine 122 has load zero. This indicates that it may not have important role as described in the literature. The consequences obtained for the solvent accessible area indicates that a peptide with the largest area available to interact with the membrane not cause greater damage. The optical results presented absorption peaks in the visible region. These results together with the results vibrational serve like a "digital"to identify the studied peptides. The Thermodynamic results presented in this work can be used in future research involving PLA2 Lysine 49.
84

Caracterização fisico-quimica e biologica de uma fosfolipase A2 isolada do veneno de Bothrops moojeni / Physicoquemical and biologic characterization of phospholipase A2 isolated from Bothrops moojeni venom

Calgarotto, Andrana Karla, 1983- 14 March 2008 (has links)
Orientador: Sergio Marangoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T15:45:07Z (GMT). No. of bitstreams: 1 Calgarotto_AndranaKarla_M.pdf: 1035943 bytes, checksum: f942243a78f40f0df6fd6b1150c07714 (MD5) Previous issue date: 2008 / Resumo: Bothrops moojeni é uma espécie de serpente de grande importância devido a sua ampla distribuição na América do Sul e Central além dos quadros clínicos que o veneno causa. Dentre as espécies peçonhentas brasileiras o gênero Bothrops é o mais numeroso e é o que apresenta os maiores índices de notificações relacionados a acidentes ofídicos. Os venenos de serpentes possuem uma mistura de substâncias biologicamente ativas sendo a maior parte composta por proteínas. Fosfolipases A2 (PLA2), proteínas presentes no veneno de serpentes, agem hidrolisando fosfolipídios de membrana na posição sn2 liberando lisofosfolipídios e ácidos graxos, além de exibir uma ampla variedade de efeitos farmacológicos. A isoforma de fosfolipase A2 (PLA2), denominada BmTX-I foi isolada através de um sistema de cromatografia em HPLC utilizando uma coluna de fase reversa µ-Bondapak C18. O alto grau de pureza foi confirmado através de eletroforese em SDS-PAGE Tricina (16,5%) e também através da determinação da massa molecular (14,238.71 Da) por espectrometria de massas (MALDI Tof). A caracterização cinética da BmTX-I PLA2 (Asp49) mostrou que tal isoforma é altamente estável e apresenta um pH ótimo de 8,0 e temperatura de 37º C. Frente a diferentes concentrações do substrato ácido 4-nitro-3-(octanoyloxy) benzóico a BmTX-I mostrou um comportamento com tendência alostérica. Na ausência de Ca2+ e na presença de alguns íons divalentes tais como Mg2+, Mn2+ e Cd2+ (na concentração de 10mM) a atividade BmTX-I foi significativamente diminuída, já na presença de Ca2+ (1mM) e com os mesmos íons divalentes citados apresentou uma discreta atividade. Também foi demonstrado o efeito inibitório de crotapotinas crotálicas sobre a atividade PLA2 da BmTX-I. A análise de composição de aminoácidos mostra que se trata de uma proteína de caráter básico pela alta presença de Lys, His e Arg. A presença de 14 resíduos de cisteína sugere a formação de 7 pontes dissulfeto. O estudo de homologia seqüencial da região N-terminal entre BmTX-I com outras PLA2 (Asp49) revelou um alto grau de homologia. O efeito neurotóxico in vitro do veneno total e da BmTX-I foi analisado na preparação biventer cervicis de pintainho. Nossos resultados mostraram que a ação do veneno de Bothrops moojeni na junção neuromuscular é menos potente quando comparado com venenos crotálicos, já que estes últimos levam a um bloqueio muito mais rápido e usando-se baixas concentrações. No entanto, não se pode negar que houve uma ação neurotóxica in vitro. O completo bloqueio tanto do veneno total quanto da BmTX-I não foi acompanhado pela inibição das respostas ao potássio (KCl) e a acetilcolina (ACh), exceto em altas concentrações de veneno (50 e 100 µg/ml) o que demonstra uma ação pré-sináptica primordial. Os testes in vivo do veneno total e da fração BmTX-I demonstraram o efeito miotóxico através da liberação de creatina quinase (CK) e o efeito inflamatório através de edema de pata e liberação de interleucina-6 (IL-6). O comportamento de liberação de CK foi semelhante tanto para o veneno total como para a PLA2 BmTX-I, os quais tiveram o maior liberação de CK uma hora após a injeção i.m. Oito horas após a injeção os níveis de CK estavam similares aos do controle. Ocorreu liberação de IL-6 bem como ação edematizante tanto do veneno total como da BmTX-I. A reprodutibilidade dos efeitos farmacológicos, só é possível com a utilização de frações quimicamente homogêneas que mantenham a integridade da função biológica. Essas frações são obtidas com metodologias de alta eficiência como HPLC, através do qual podemos isolar a BmTX-I em um único passo cromatográfico e o grau de pureza confirmado por espectrometria de massa. Estes resultados podem ser associados com a sua atividade biológica, eliminando a subjetividade causada por veneno total ou frações impuras. Essa abordagem pode ser aplicada nos estudos bioquímicos, estrutura-função, fisiológicos e farmacológicos, podendo revelar mecanismos ainda desconhecidos na relação estrutura-função das PLA2 procedentes de veneno de serpentes / Abstract: Bothrops moojeni is a very important snake species due to its great distribution in the South and Central America besides the clinic alterations caused by the venom. Among the Brazilian species the Bothrops genus is the most numerous and shows the highest registrations related to ophidian accidents. The snake venoms are source of biologically active substances which main components are proteins. Phospholipases A2 (PLA2), proteins present in the snake venom, hydrolyze the sn-2 acyl groups of phospholipids liberating fatty acids and lysophospholipds, besides exhibiting many pharmacological effects. The fosfolipase A2 (PLA2) isoform, named BmTX-I was isolated through RP-HPLC performed on a C18 column. The high degree of purity was confirmed by SDS-PAGE and also by determining the molecular mass (14238.71 Da) in a MALDI TOF mass spectrometry. The kinetic characterization of BmTX-I PLA2 (Asp49) showed that the isoform was highly stable and presented an optimum pH of 8.0 and temperature of 37° C. At different substrate acid 4-nitro-3-(octanoyloxy) benzoic concentrations the BmTX-I showed allosteric behavior. In the absence of Ca2+ and in the presence of some divalent ions such as Mg+2, Mn+2, Cd2+ (at the concentration of 10 mM) the BmTX-I activity significantly decreased. But in the presence of 1mM Ca+2, under the same divalent ions conditions, the isoforms showed a discreet activity. An inhibitory effect of crotalic crotapotins on the activity PLA2 was also demonstrated. The analysis of amino acids composition showed a high content of basic amino acids such as Lys, His and Arg indicating a basic character for the BmTX-I. The presence of 14 cysteine residues suggests the formation of 7 disulfide bridges. N-terminal amino acid sequence revealed a high level of homology between BmTX-I and other Asp49 PLA2s. The neurotoxic effect of the whole venom and of BmTX-I was analyzed at chick biventer cervicis muscle preparation. Our results showed that the blockage of the muscle contraction was lesser when compared with crotalic venoms, which blockage at lower concentrations. Nevertheless it is sure that there was an in vitro neurotoxic action. The complete blockage, as much the whole venom as the BmTX-I, was not accompanied by any significant inhibition of the responses to KCl and to Ach, excepting at higher concentrations of the venom (50 e 100 µg/ml) suggesting the primordial presynaptic action. The tests in vivo with the whole venom and BmTX-I fraction showed the miotoxic effect through the creatine kinase (CK) releases and the inflammatory effect through edema-forming activity and interleukin-6 release. The CK release was similar for both whole venom and BmTX-I, which showed higher liberation one hour after the i.m injection. After eight hours of injection the CK levels were similar to the controls. There was the IL-6 release as well as edema-forming activity both in to the whole venom and BmTX-I. The reprodubility of pharmacological effects, is just possible with the utilization of chemically homogenous fractions that maintain the integrity of biological function. These fractions were obtained with high efficient methodologies as HPLC, through which this we the BmTX-I could be isolated in only one chromatography step, with purity direct by mass spectrometry. These results may be associated with the biological activities, eliminating the subjectivity caused by total venom or impure fractions. This approximation may be applied to the biochemical, structure-function, physiological and pharmacological studies, and it may reveal still unknown mechanisms in the structure-function relationship of PLA2 from the serpents venom / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular
85

Caracterização farmacologica de uma fosfolipase A2 ASP49 isolada do veneno de Bothrops pauloensis / Pharmacological characterization of ASP49 PLA2 isolated from Bothrops pauloensis venom

Monzon, Georgina Sucasaca 12 August 2018 (has links)
Orientador: Lea Rodrigues Simioni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T23:41:00Z (GMT). No. of bitstreams: 1 Monzon_GeorginaSucasaca_M.pdf: 3056250 bytes, checksum: d246d55d0b29698ce7253fb6a9fc3f6a (MD5) Previous issue date: 2008 / Resumo: O veneno botrópico possui uma mistura de substâncias protéicas necessárias para a sobrevivência da serpente, dentre elas, as mais amplamente estudadas são as fosfolipases A2, que hidrolisam glicerofosfolipídios de membrana na posição sn-2, liberando isofosfolipídios e ácidos graxos, que além de desempenhar uma ação biológica no processo de digestão de lipídios, exibem também uma ampla variedade de efeitos farmacológicos. O presente trabalho teve como objetivo, caracterizar farmacologicamente uma nova fosfolipase A2 denominada Bp-13 do veneno de Bothrops pauloensis, serpente endêmica do cerrado brasileiro, avaliando sua ação sobre a junção neuromuscular, efeito miotóxico e atividade edematogênica. A Bp-13 foi purificada utilizando-se o sistema cromatográfico HPLC de fase reversa em coluna µ-Bondapack C18; possuindo uma massa molecular de 14 035 Da e atividade catalítica de 2,43 nmol/min/mg (30-35 ºC), dependente de Ca2+ porém inibida por Mg2+, Mn2+, Cd2+ e Sr2+. A atividade neuromuscular foi avaliada em preparações biventer cervicis (BC) de pintainho, extensor digitorum longus (EDL) e nervo frênico-diafragma (NFD) de camundongo, sob estimulação elétrica indireta durante 120 min a 37 ºC. A Bp-13 (3,56 µM) induziu bloqueio neuromuscular completo e irreversível, nas preparações de mamífero, que se mostraram mais sensíveis à ação da toxina do que as preparações de ave. Em preparação BC de pintainho o bloqueio neuromuscular foi de 28 ± 2 % após 120 min de incubação com a Bp-13 (7,12 µM) e a contratura evocada após a adição exógena de ACh foi parcialmente inibida. Concentrações menores (0,71 e 1,42 µM) foram testadas na preparação NFD de camundongo, induzindo o bloqueio da resposta contrátil em 29 ± 5 % e 55 ± 6 %, respectivamente. Em alguns experimentos, onde o Ca2+ foi substituído pelo Sr2+ na solução de Tyrode houve ausência do bloqueio neuromuscular característico da Bp-13 (3,56 µM); evento semelhante foi observado quando a temperatura do banho foi alterada de 37 ºC para 24 ºC, com bloqueio de 37,4 ± 6 % da resposta contrátil. A Bp-13 (3,56 µM) foi capaz de inibir a resposta contrátil a estímulo elétrico direto em preparações NFD de camundongo previamente curarizadas. Com o estudo eletrofisiológico realizado em preparação músculo hemidiafragma de camundongo, avaliaram-se os potenciais de membrana em repouso (PM). A Bp-13 (3,56 µM) causou uma despolarização progressiva do sarcolema, alcançando valores de -80 ± 1 mV (controle) até -37 ± 3 mV após 120 min de incubação com a toxina. Em preparações músculo diafragma pré-tratadas com d-Tubocurarina (10 µM), observou-se uma redução da despolarização induzida pela da Bp-13 (-37 ± 3 mV para -58 ± 2 mV). A miotoxicidade foi avaliada in vitro através de análise morfológica do músculo diafragma de camundongo e in vivo através da determinação da atividade de creatinoquinase (CK). Na análise morfológica considerou-se fibras normais as que mantiveram íntegro o sarcolema com o formato poligonal e fibras alteradas as que apresentavam hipercontração das miofibrilas, células com lesão tipo delta, edemaciadas e vacuolizadas. A porcentagem de fibras lesadas de preparações incubadas com o veneno de Bothrops pauloensis (100 e 50 µg/mL) foi de 14,4 ± 3 % e 8,7 ± 3 % respectivamente; e com a fração Bp-13 (20 e 50 µg/mL) foi de 8,3 ± 4 % e 30,6 ± 5 %, respectivamente. Quando as preparações foram mantidas em solução de Tyrode, onde o Ca2+ foi substituído pelo Sr2+, as alterações morfológicas foram de 36,7 ± 11 %, ou seja, semelhante aos valores observados com a Bp-13 na solução de Tyrode normal. O aumento nos valores de liberação de creatinoquinase, após 30 min da injeção da Bp-13, revelou o efeito miotóxico in vivo, valores que retornaram ao normal após 6 horas. A dose de 10 µg/pata de Bp-13 injetada em ratos via intraplantar induziram a formação de edema de pata após 15 min de injeção da toxina. Conclui-se, então, que a Bp-13 é uma miotoxina, que inibe a resposta neuromuscular dependente de temperatura e de Ca2+, em preparações isoladas EDL e NFD de camundongo, sugerindo que a atividade catalítica possivelmente contribua com o evento farmacológico, embora o efeito mionecrótico in vitro não seja afetado, indicando uma dissociação entre estes efeitos. / Abstract: Bothropic venom has several substances necessary to serpent survival, among them, one of the most widely studied are phospholipase A2 enzymes, that hydrolise sn-2 from membrane phospholipids, releasing lysophospholipids and fatty acids, which beyond playing a biological action in the lipid digestion process, also exhibit a wide variety of pharmacological effects. The present work had as objective to characterize pharmacologically a new phospholipase A2 denominated Bp-13 from the Bothrops pauloensis (endemic serpent to the Brazilian cerrado) venom, evaluating its action at neuromuscular junction, myotoxic effect and edematogenic activity. Bp-13 was purified through chromatographic system, a reverse phase HPLC on a µ-Bondapack C18 column; it has a molecular mass of 14 035 Da, 2,43 nmol/min/mg (30- 35 ºC) catalytic activity Ca2+ dependent, however it was inhibited by Mg2+, Mn2+, Cd2+ and Sr2+. Neuromuscular activity was evaluated in chick biventer cervicis (BC) preparations, extensor digitorum longus (EDL) and mouse phrenic nerve-diaphragm (PND), under indirect electrical stimulation during 120 minutes, at 37oC. Bp-13 (3,56 µM) induced complete and irreversible neuromuscular block in mammalian preparations, which showed to be more sensitive to toxin action than bird preparations. In chick BC preparation, neuromuscular block was 28 ± 2 % after 120 minutes incubation with Bp-13 (7,12 µM) and evoked contracture after exogenous ACh addiction was partially inhibited. Smaller concentrations (0,71 e 1,42 µM) were tested in mouse PND preparation, inducing block of the contractile response in 29 ± 5 % and 55 ± 6 %, respectively. In some experiments, where Ca2+ was substituted for Sr2+ in Tyrode's solution, there was absence of the Bp-13 characteristic neuromuscular block (3,56 µM); similar event was observed when the bath temperature was altered from 37 ºC to 24 ºC, with a block of 37,4 ± 6 % of the contractile response. Bp-13 (3,56 µM) was able to inhibit contractile response to direct electrical stimulus in previously curarized mouse PND preparations. Through electrophysiological study performed in mouse hemidiaphragm muscle preparation, membrane potentials at rest were evaluated (MP). Bp-13 (3,56 µM) caused a progressive sarcolemma depolarization, reaching values from -80 ± 1 mV (control) until - 37 ± 3 mV after 120 minutes in toxin incubation. In d-tubocurarine (10 µM) pre-treated hemidiaphragm muscle preparations, a depolarization reduction was observed, induced by Bp-13 (-37 ± 3 mV to -58 ± 2 mV). Myotoxicity was evaluated in vitro through morphological analysis of the mouse hemidiaphragm muscle and in vivo through determination of creatinkinase (CK) activity. Regarding morphological analysis, fibers which maintained intact sarcolemma with polygonal shape were considered normal, and fibers which presented hypercontraction of myofibrils, delta type lesion cells, edemaciated and vacuolized, were considered altered fibers. Lesioned fibers from Bothrops pauloensis (100 and 50 µg/mL) venom incubated preparations were 14,4 ± 3 % and 8,7 ± 3 % , respectively; and for Bp-13 fraction (20 e 50 µg/mL) they were 8,3 ± 4 % and 30,6 ± 5 %, respectively. When preparations were Ca2+ was substituted for Sr2+, morphological maintained in Tyrode's solution, where alterations were 36,7 ± 11 %, that is similar to the observed values with Bp-13 in normal Tyrode's solution. The increase in creatinkinase release values after 30 minutes of Bp-13 injection revealed in vivo myotoxic effect, values that returned to normal after 6 hours. Bp-13 concentration of 10 µg injected in rats via intraplantar induced paw edema formation after 15 minutes of toxin injection. In conclusion, Bp-13 is a myotoxin that inhibits temperature and Ca2+ dependent neuromuscular response in mouse EDL and PND isolated preparations, suggesting that its catalytic activity possibly contributes to the pharmacological event, although in vitro myotoxic activity was unaffected by these treatments, indicating a dissociation between these effects. / Mestrado / Mestre em Farmacologia
86

Aplicação de fosfolipase A2 de veneno de serpentes em biocatalise / Application of phospholipase A2 of serpents' poisons in biocatalysis

Pirolla, Renan Augusto Siqueira 13 August 2018 (has links)
Orientador: Jose Augusto Rosario Rodrigues / Dissertação ( mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-13T18:43:14Z (GMT). No. of bitstreams: 1 Pirolla_RenanAugustoSiqueira_M.pdf: 4621563 bytes, checksum: 4794daa19c9ceefe37205a1eed1b647b (MD5) Previous issue date: 2009 / Resumo: O projeto explora o potencial catalítico de fosfolipases A2, isoladas de venenos de serpentes brasileiras para efetuar resolução enzimática de substratos com relevância científica, visto que nenhum trabalho anterior foi feito analisando-se sua enantiosseletividade. Foram feitos estudos sobre a resolução do Binol, do a-tetralol, do 1-feniletanol, do para-nitro-1-feniletanol, do ácido 3-(2-bromo-hexanoiloxi)-4-nitrobenzóico e ácido 3-(2-metil-hexanoiloxi)-4-nitrobenzóico.Devido a dificuldade de obtenção e purificação das fosfolipases, a enzima foi imobilizada utilizando a formação de um agregado com ligações cruzadas (Cross-Linked Enzyme Aggregate . CLEA). Os agregados foram produzidos com quatro tipos de precipitantes (solução 55 % de sulfato de amônio, polietilenoglicol 600 Da, dimetoxietano e acetona) e dois adicionantes (TRITON-X100 e polietilenodiimina). Com os testes, observou-se que o CLEA formado com sulfato de amônio, sem adicionante apresentou os melhores resultados, sendo utilizado nas reações de biocatálise. A resolução dos substratos foi feita com a esterificação dos álcoois, formando-se ésteres (acetatos, propanoatos e hexanoatos), e posterior hidrólise com a enzima não-imobilizada e CLEA da fosfolipase A2, para comparação. Alíquotas das reações foram e analisadas por GC/FID com fase estacionária quiral para estudo dos excessos enantioméricos. As reações foram feitas a temperatura ambiente e a 45 °C. Os resultados indicam atividade enzimática sendo possível obter o tetralol com 16% de e.e. utilizando-se o CLEA e o p-nitro-1-feniletanol com 19% de ee usando-se a PLA2 livre. Os outros álcoois foram obtidos com baixos ee. O ácido 3-(2-bromo-hexanoiloxi)-4-nitrobenzóico não pode ser analisado por sofrer hidrólise química completa no meio reacional, e com a hidrólise do ácido 3-(2-metil-hexanoiloxi)-4-nitrobenzóico foi possível a obtenção do ácido 2-metil-hexanóico com 9 % utilizando-se CLEA e 7 % com a enzima livre. A baixa enantiosseletividade foi interpretada como decorrente da fraca interação dos substratos com o sítio ativo da enzima / Abstract: This project explores the catalytic potential of fosfolipases A2, isolated from poisons of brazilian serpents to effect enzymatic substrate resolution with scientific relevance, since no previous work was made analyzing its enantioselectivity. Studies on the resolution of several compounds had been made, including Binol, a-tetralol, 1-phenylethanol, para-nitro-1- phenylethanol, 3-(2-bromohexanoiloxy)-4-nitrobenzoic acid and 3-(2-methylhexanoiloxy)-4-nitrobenzoic acid.Due to difficulty of attainment and purification of the phospholipase, the enzyme was immobilized using the formation of an aggregate with cross-links (Cross-Linked Enzyme Aggregate ¿ CLEA). These aggregates had been produced with four types of precipitation agents (ammonium sulphate solution 55%, polietileneglycol 600 Da, dimethoxyethane and acetone) and two additives (TRITON-X100 and poliethylenediimine). With the tests, it was observed that the CLEA formed with ammonium sulphate, without additives presented the best results, being used in the reactions of biocatalysis.The resolution of substrates was made with the alcohol¿s esterification, forming different (acetates, propanoates and hexanoates) followed by hydrolysis with the free enzyme and CLEA, for comparison. Aliquots of the reactions had been made and analyzed with GC/FID with quiral stationary phase for study of the enantiomerics excesses. The reactions had been made at ambient temperature and 45 °C.The results indicate enzymatic activity and was possible to get tetralol with 16% of ee using CLEA and p-nitro-1-phenylethanol with 19% of ee. The other alcohols had been gotten with low ee. The 3- (2-bromohexanoiloxy) - 4-nitrobenzoic acid cannot be analyzed by suffering complete chemical hydrolysis during the reaction, and with hydrolysis of acid the 3- (2-metilhexanoiloxi) - 4-nitrobenzoic the attainment of the acid 2-metilhexanoic with 9% was possible using CLEA and 7% with the free enzyme. The low enantioselectivity was explained due to the weak interaction of substrates with the active site of enzyme / Mestrado / Quimica Organica / Mestre em Química
87

Molecular dynamics simulations on phospholipid membranes

Hyvönen, M. (Marja) 21 March 2001 (has links)
Abstract Phospholipids are the main components of cell membranes, lipoproteins and other membrane structures in living organisms. Properties of lipid molecules are important to the overall behaviour and interactions of membranes. Furthermore, characteristics of the biological membranes act as important regulators of membrane functions. Molecular dynamics (MD) simulations were applied in this thesis to study properties of biological membranes. A certain degree of acyl chain polyunsaturation is essential for the proper functioning of membranes, but earlier MD simulations had not addressed the effects of polyunsaturation. Therefore a solvated all-atom bilayer model consisting of diunsaturated 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine (PLPC) molecules was simulated. The analysis of the simulation data was focused on the effects of double bonds on a membrane structure. Self-organising neural networks were applied to the analysis of the conformational data from the 1-ns simulation of PLPC membrane. Mapping of 1.44 million molecular conformations to a two-dimensional array of neurons revealed, without human intervention or requirement of a priori knowledge, the main conformational features. This method provides a powerful tool for gaining insight into the main molecular conformations of any simulated molecular assembly. Furthermore, an application of MD simulations in the comparative analysis of the effects of lipid hydrolysis products on the membrane structure was introduced. The hydrolysis products of the phospholipase A2 (PLA2) enzyme are known to have a role in a variety of physiological processes and the membrane itself acts as an important regulator of this enzyme. The simulations revealed differences in the bilayer properties between the original and hydrolysed phospholipid membranes. This study provides further evidence that MD simulations on biomembranes are able to provide information on the properties of biologically and biochemically important lipid systems at the molecular level.
88

The Eosinophil Response in Mice Infected with Trichinella spiralis or Trichinella pseudospiralis as Indicated by Phospholipase B Activity

Hsu, Shing-Chien 12 1900 (has links)
The host eosinophil response was compared in mice infected with either T. spiralis or T. pseudospiralis by determination of levels of splenic and intestinal phospholipase B, a marker enzyme for eosinophils. Primary infection of naive mice and challenge infection of homologously sensitized mice with T. pseudospiralis resulted in significantly lower tissue phospholipase B activities than infection with T. spiralis. Mice homologously challenged with T. pseudospiralis did exhibit an anamnestic eosinophil response compared to mice given a primary T. pseudospiralis infection. This anamnestic response, however, was significantly lower than the eosinophil response seen in sensitized mice given a homologous T. spiralis challenge. Mice sensitized to T. spiralis or T. pseudospiralis and heterologous challenge demonstrated an elevated eosinophil response compared to mice given a primary infection with either parasite. The heterologous challenge response, however, was not as intense as found for sensitized mice given a homologous challenge.
89

The α<sub>1</sub>-Adrenoceptor Is Inactivated by Alterations in Membrane Phospholipids

Shreeve, S. M., Valliere, Julia E. 12 May 1992 (has links)
The influence of the membrane environment on the α1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the α1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic α1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the α1-adrenoceptor.
90

Lipoprotein-Associated Phospholipase a2 Predicts Progression of Cardiac Allograft Vasculopathy and Increased Risk of Cardiovascular Events in Heart Transplant Patients

Raichlin, Eugenia, McConnell, Joseph P., Bae, Jang Ho, Kremers, Walter K., Lerman, Amir, Frantz, Robert P. 01 April 2008 (has links)
BACKGROUND. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor for coronary artery disease (CAD) in nontransplant patients. We evaluated the association between Lp-PLA2, cardiac allograft vasculopathy (CAV) assessed by 3D intravascular ultrasound, and incidence of cardiac adverse events in heart transplant recipients. MATERIALS AND METHODS. Fasting blood samples were obtained and stored from a cross-section of 112 cardiac transplant recipients attending the Mayo cardiac transplant clinic in 2000 to 2001, mean of 4.7 years after transplant. Lp-PLA2 was measured in plasma aliquots using an enzyme-linked immunoassay. Fifty-six of these patients subsequently underwent two 3D intravascular ultrasound studies in 2004 to 2006 12 months apart. Cardiovascular (CV) events included percutaneous coronary intervention, coronary artery bypass grafting (CABG), reduction in left ventricular ejection fraction (LVEF) ≤45% secondary to CAV and CV death. RESULTS. High Lp-PLA2 level was associated with increase in plaque volume (r=0.43, P=0.0026) and percent plaque volume (r=0.45, P=0.0004). The association remained significant after adjusting for clinical and lipid variables. During follow-up of 5.1±1.6 years, 24 CV adverse events occurred in 15 of 112 (13%) heart transplant patients. Lp-PLA2 level>236 ng/mL (higher tertile) identified a subgroup of patients having a 2.4-fold increase of relative risk for combined endpoint of CV events (percutaneous coronary intervention, CABG, LVEF<45%, and CV death; 95% CI 1.16-5.19, P=0.012) compared with patients with Lp-PLA2≤236 ng/mL. CONCLUSIONS. Lp-PLA2 is independently associated with progression of CAV and predicts a higher incidence of CV events and CV death in transplant patients. This finding supports the concept that systemic inflammation is an important mediator of CAV. Lp-PLA2 may be a useful marker for risk of CAV and a therapeutic target in posttransplant patients.

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