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The crystal structure of malate synthase and mechanistic implications /Howard, Bruce Riley, January 1999 (has links)
Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-71). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9948022.
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Linking Human Genetic Variation to Mitochondrial MetabolismStrittmatter, Laura Anne January 2014 (has links)
Genetic variation has a powerful impact on human metabolism and disease. Traditionally, this relationship has either been studied at a high level using top-down descriptive studies of patients with genetically defined inborn errors of metabolism, or else from the bottom up, with molecular biology and biochemical studies of single proteins. Recent advances in genetic sequencing, metabolic profiling technology, and structural biology are rapidly enabling the integration of these approaches towards a more complete description of human metabolism.
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Two-state conformational behavior in protein active centersLohman, Jeremy R., 1981- 12 1900 (has links)
xiv, 82 p., ill. (some col.) / Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS).
GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide.
Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design.
This dissertation includes my unpublished co-authored materials. / Adviser: S. James Remington
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Prospecção de inibidores para a enzima malato sintase do Paracoccidioides brasiliensis: uma avaliação por triagem virtual e dinâmica molecular / Prospecting for inhibitors malate synthase ensyme the Paracoccidoides brasiliensis: an evaluation by virtual screening and molecular dynamicsCosta, Fausto Guimarães 16 April 2015 (has links)
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Previous issue date: 2015-04-16 / Paracoccidioidomycose / A Paracoccidioidomicose...
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Perfil transcricional e proteômico de Paracoccidioides em resposta à itraconazol e anfotericina B e identificação de compostos com potencial antifúngico / Proteomic and transcriptional profile of Paracoccidioides in response to itraconazole and identification of compounds with antifungal potentialSilva Neto, Benedito Rodrigues da 02 May 2013 (has links)
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Previous issue date: 2013-05-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The thermally dimorphic fungal pathogen Paracoccidioides is the agent of
paracoccidioidomycosis. This disease is characterized by a granulomatous inflammation
with clinical forms ranging from a benign localized infection to a disseminated one. The
triazole drugs are broad-spectrum antifungal agents and are currently used to treat
infections caused by various pathogenic yeast and molds. The mechanism of action of
azoles has been elucidated in some fungi, although little is known in Paracoccidioides.
Here we aim to investigate the mechanism of action of itraconazole on Paracoccidioides
by using Representational Difference Analysis from Paracoccidioides yeast cells grown
in the absence and presence of itraconazole for 1 and 2 h. Among the Paracoccidioides
genes up-regulated by itraconazole were those mainly involved in cellular transport,
metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6,
ERG3, ERG5 and ERG25 were up-regulated when evaluated in a timely manner. In vivo
infection experiment in mice corroborated in vitro results. The glyoxylate cycle and its
key enzymes isocitrate lyase and malate synthase (MLS) play a crucial role in the
pathogenicity and virulence of various fungi such as the human pathogens. Here, we
describe a study conducted to develop rational ligands as candidates to inhibit receptor
PbMLS. The important step in the search for ligands for this receptor based on structural
homology, molecular docking and molecular dynamics involving scanning virtual
(virtual screening) through the program AutoDock Vina. Identified from the database of
natural compounds (ZINC data bank) potential candidate ligands to inhibit the activity of
PbMLS when compared to the original binder. This process led us to monoterpene indole
alkaloids of the genus Palicourea (Rubiaceae) comprises about 230 species from shrubs
and small trees distributed mainly in tropical regions. From the molecular docking fifteen
compounds were tested as to its effectiveness in inhibiting the activity of PbMLS. The
specific activity of PbMLS was affected by the compounds. Four indol alkaloids showed
ability to reduce the enzyme activity. Since PbMLS is a linked surface protein that
behaves as an anchorless adhesin, and PbICL is here described as adhesin, we also
investigated if those compounds inhibit the adhesion of the protein to extracellular. Twodimensional
gel electrophoresis we used to investigate the proteins expressed
differentially during treatment with itraconazole and amphotericin B. Gels of three
independent biological replicates were digitalized and the images were analyzed using
the ImageMaster 2D Platinum 6.0 software (GE Healthcare). Spot intensities were
normalized and the statistics analyses were estimated by one-way ANOVA. The spots of
interest were excised, in-gel digested with trypsin, and the peptides were then analyzed
by MS and/or MS/MS and and sequenced. The results obtained here should assist in
understanding the mode of action of drugs in Paracoccidioides, and outline studies
identifying compounds with antifungal activity. / O fungo patógeno termodimórfico Paracoccidioides é o agente da
paracoccidioidomicose. Esta doença é caracterizada por uma inflamação granulomatosa
onde as formas clínicas vão da infecção localizada benigna a uma uma disseminada. As
drogas triazólicas são antifúngicos de amplo espectro e são usadas atualmente para tratar
infecções causadas por vários fungos patogênicos e fungos. O mecanismo de ação dos
azólicos foi elucidado em alguns fungos, embora pouco se sabe em Paracoccidioides.
Aqui, em primeiro lugar pretendemos investigar o mecanismo de ação do itraconazol em
Paracoccidioides usando análise de diferença representacional de Paracoccidioides
células de levedura crescidas na ausência e na presença de itraconazol por 1 e 2 horas.
Entre os genes Paracoccidioides up-regulados pelo itraconazol foram os envolvidos,
principalmente no transporte celular, metabolismo/energia, transcrição, defesa e
virulência. ERG11, ERG6, ERG3, ERG5 e ERG25 foram regulados quando avaliados de
forma temporal. Experimentos de infecção em camundongos corroborou resultados in
vitro. O ciclo do glioxilato e suas enzimas chave isocitrato liase (ICL) e malato sintetase
(MLS) desempenham um papel fundamental na patogenicidade e virulência de vários
fungos, assim como patogênese em humanos. Neste trabalho, descrevemos um estudo
realizado para desenvolver ligantes racionais como candidatos pra inibir o receptor
PbMLS. Apresentamos um passo importante na busca de ligantes para este receptor
baseando-se em homologia de estruturas, dinâmica molecular e acoplamento molecular
envolvendo varredura virtual (virtual screening) por meio do programa AutoDock Vina.
Identificamos a partir de banco de compostos naturais (data bank ZINC) potenciais
ligantes candidatos a inibir a atividade de PbMLS quando comparados ao ligante
original. Este processo nos conduziu aos alcalóides indólicos monoterpênicos do gênero
Palicourea (Rubiaceae) que compreende cerca de 230 espécies entre arbustos e pequenas
árvores distribuídas, principalmente, nas regiões tropicais.A partir da ancoragem
molecular quinze compostos foram testados quanto à sua eficácia na inibição da atividade
de PbMLS. A atividade específica de PbMLS foi afetado pelos compostos. Quatro
alcalóides indólico mostraram capacidade de reduzir a atividade da enzima. Desde que
PbMLS é uma proteína associada à superfície que se comporta como uma adesina
ancorada também foi investigado se os compostos inibem a adesão da proteína às
matrizes extracelulares. O processo de eletroforese em gel bidimensional foi utilizado
para investigar as proteínas diferencialmente expressas durante o tratamento com
itraconazol e anfotericina B. Gel de três réplicas biológicas independentes foram
digitalizadas e as imagens foram analisadas usando o software 6.0 Platinum 2D
ImageMaster (GE Healthcare). Intensidades dos spots foram normalizados e foram
estimadas as análises estatísticas por ANOVA one-way. Os spots de interesse foram
excisadas do gel digerida com tripsina e os péptidos foram analisados por MS e / ou MS /
MS e sequenciados. Os resultados obtidos aqui devem ajudar na compreensão do
mecanismo de ação de drogas em Paracoccidioides, e delinear estudos de identificação
de compostos com atividade antifúngica.
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Busca de peptídeos com potencial modulador da enzima malato sintase, a partir de estudos de interação proteínaproteína / Exploring peptides with modulation potential for malate synthase through protein-protein interaction studiesLima, Raisa Melo 03 October 2016 (has links)
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Previous issue date: 2016-10-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Brazil, where
are recorded about 80% of cases worldwide, and has Paracoccidioides sp. as the
etiologic agent. Malate synthase (MLS) is an important enzyme related to the
fungal metabolism, once it is essential in the glyoxylate cycle, a secondary
metabolic pathway of the citric acid cycle exclusive to microorganisms and plants.
Its absence in humans makes this enzyme an interesting subject to study, mainly
in rational drug design. From recent in vitro studies, several interacting proteins
of MLS (receiver) were classified, but the modes of interaction and key regions
involved in protein-protein interfaces (PPIs) have not yet been described. In this
work, six (6) binding proteins (BPs) were selected to describe the IP's of MLS.
Their tridimensonal structures, as well as MLS, were predicted by homology
modeling using I-TASSER server, and subsequent molecular dynamics
simulations (MD). The most common conformational modes of each protein were
obtained by cluster analysis of the trajectories generated by MD. Molecular
docking simulations using Gramm-X were then performed for the conformational
modes of MLS against the BP's, resulting in a total of 36 complexes. Based on
the higher frequency of some small fragments of proteins observed in the IPP's,
57 peptides with sizes between 5 and 20 residues, were initially selected from 5
regions of MLS which are considered more frequent in protein-protein interaction.
FlexPepDock simulations were performed to optimize the atomic coordinates of
the peptide complexed with MLS, and concomitantly, PepFOLD simulations were
performed to evaluate the stability of each peptide in solution. Based on the lower
energy score of peptides linked to MLS, and the stability of their structures in
solution (MLS-free), 6 peptides were selected as promising ligands to MLS mode
1. The stability and patterns of interactions of these peptides were evaluated in
detail. / A paracoccidioidomicose (PCM) é uma micose sistêmica endêmica no Brasil,
onde são registrados cerca de 80% dos casos mundiais, e possui como agente
etiológico o fungo Paracoccidioides spp. A Malato sintase (MLS) é uma
importante enzima relacionada ao metabolismo fúngico, uma vez que é essencial
no ciclo do glioxilato, uma via metabólica importante de produção de glicose para
parede celular, sendo exclusiva de micro-organismos e plantas. Sua ausência
em humanos a torna um alvo interessante de estudo, principalmente, no desenho
racional de fármacos. A partir de recentes estudos in vitro, várias proteínas que
interagem com a PbMLS (receptor) foram classificadas, porém os modos de
interação e as regiões chaves envolvidas nas interfaces proteína-proteína
(IPP’s), não foram ainda descritas. Neste trabalho, 6 (seis) proteínas ligantes
(PL) foram selecionadas para verificar suas interações com PbMLS. As
estruturas tridimensionais dessas proteínas, bem como de PbMLS, foram
preditas por homologia, usando o servidor I-TASSER. Simulações de dinâmica
molecular (DM) foram realizadas pelo programa GROMACS, e os modos das
conformações mais representativas de cada proteína foram determinados
baseando-se nas análises de agrupamentos a partir das trajetórias geradas por
DM. Simulações de ancoragem molecular com GRAMM-X foram então
realizadas entre os modos conformacionais de PbMLS contra os obtidos de PL’s,
resultando num total de 36 complexos. Baseado na frequência maior de alguns
pequenos fragmentos de proteínas, observados nas IPP’s, 57 peptídeos de
tamanhos entre 5 e 20 resíduos de aminoácidos, foram inicialmente
selecionados a partir de 5 regiões da PbMLS consideradas mais frequentes na
interação proteína-proteína. Simulações com FlexPepDock foram realizadas
para otimizar as coordenadas atômicas dos peptídeos complexados com MLS e,
concomitantemente, simulações com PepFOLD foram realizadas para avaliar a
estabilidade de cada peptídeo em solução. Com base nos mais baixos scores de
energia dos peptídeos ligados a MLS bem como na estabilidade de suas
estruturas não ligadas em solução , 5 peptídeos foram selecionados como
promissores ligantes ao modo 1 de MLS. A estabilidade e os padrões de
interações destes peptídeos são avaliados em detalhes.
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Influ?ncia do sistema anteoxidante da catalase no ciclo do glioxilato durante o estabelecimento p?s-germinativo de c?rtamo (Carthamus tinctorius L.) e de girassol (Helianthus annuus L.)Torres, Taffarel Melo 09 April 2013 (has links)
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Previous issue date: 2013-04-09 / Funda??o de Apoio ? Pesquisa do Estado do Rio Grande do Norte / Oilseeds are a high-value natural resource, due to its use as a substitute for
petroleum. However, the storage time can reduce seed viability and oil quality.
Therefore, scientific efforts have been made to provide a increment of storage time,
germination rates and plant establishment of high-value oilseeds. The seedling
establishment depends of the plant pass over the functional transition stage,
characterized by a metabolic change from heterotrophic condition to autotrophic one.
The storage oil mobilization is performed by β-oxidation process and the glyoxylate
cycle. Also, the functional transition involves acclimation to photosynthetic condition,
which generally includes the participation of antioxidant system and the reactive
oxygen species, the latter are produced in various reactions of primary and
secondary metabolism. In the present study, Catalase was inhibited during the
functional transition of sunflower and safflower, after were performed many analyzes
to elucidate the effects caused on the SOD and APX antioxidant systems. Also, were
checked the changes in expression pattern of the glyoxylate cycle enzymes markers,
ICL and MLS. It was observed that after CAT inhibition, the SOD and APX antioxidant
systems allow the seedling establishment. Besides, was verified that both oilseeds
can be accelerate the reverse mobilization and the photosynthetic establishment
when Catalase activity has dramatically decreased / As sementes oleaginosas armazenam ?leos, um recurso natural de alto valor
econ?mico devido a sua aplicabilidade como substituto aos derivados do petr?leo.
Por?m, o tempo de armazenamento pode inviabilizar a semente provocando a
redu??o da qualidade do ?leo e perda da viabilidade da semente. Por este motivo,
esfor?os cient?ficos t?m sido realizados para proporcionar um maior tempo de
armazenamento e incrementar taxas de germina??o e estabelecimento destas
plantas com alto valor econ?mico. O estabelecimento da pl?ntula depende da
capacidade do vegetal transpassar a etapa de transi??o funcional, caracterizada
pela mudan?a do estado heterotr?fico para o autotr?fico. O consumo das reservas
de ?leo depende das vias de β-oxida??o e do ciclo do glioxilato. No entanto, o
processo de transi??o envolve a aclimata??o do vegetal ? condi??o de organismo
fotossintetizante, que em geral conta com a participa??o fundamental do sistema
antioxidante do vegetal devido ? produ??o de esp?cies reativas de oxig?nio em
v?rias rea??es do metabolismo prim?rio e secund?rio. Neste estudo, a Catalase foi
inibida durante a transi??o funcional de girassol e c?rtamo para analisar os efeitos
provocados nos sistemas antioxidantes de APX e da SOD e verificar as
modifica??es no padr?o de express?o das enzimas marcadoras do ciclo do
glioxilato, ICL e MLS. Constatou-se que diante da inibi??o da Catalase o sistema
antioxidante da APX e da SOD permitem o estabelecimento da pl?ntula. Verificou-se
tamb?m que as duas oleaginoasas parecem acelerar a mobiliza??o de reservas e o
estabelecimento fotossint?tico quando a Catalase tem a atividade drasticamente
reduzida
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Malato sintase de Paracoccidioides brasiliensis é uma proteína ligada à superfície que se comporta como uma anchorless adesina / The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesinSILVA NETO, Benedito Rodrigues da 11 May 2009 (has links)
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Previous issue date: 2009-05-11 / The pathogenic fungus Paracoccidioides brasiliensis causative of
Paracoccidioidomycosis (PCM), a pulmonary mycose acquired by inhalation of fungal
airborne propagules, which may disseminate to several organs and tissues leading to a
severe form of the disease. Adhesion and invasion to host cells are essential steps
involved in the internalization and dissemination of pathogens. Inside host, P.
brasiliensis use the glyoxylate cycle for intracellular survival. Here, we provide
evidence that malate synthase of P. brasiliensis (PbMLS) is localized on the cell wall,
and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody
against this protein was obtained. By using Confocal Laser Scanning Microscopy and
Western blot analysis, PbMLS was detected in the cytoplasm and the cell wall of the
yeast phase of P. brasiliensis of mother and bud yeast cells. PbMLSr and the respective
polyclonal antibody produced against this protein inhibited the interaction of P.
brasiliensis to in vitro cultured epithelial cells A549. These observations indicated that
cell wall-associated MLS of P. brasiliensis could be mediating the binding of fungal
cells, thus contributing to the adhesion of fungus to host tissues and to the dissemination
of infection. / O fungo de patogênico Paracoccidioides brasiliensis agente causador da
Paracoccidioidomicose (PCM), uma micose pulmonar adquirida pela inalação de
propágulos aéreos do fungo que pode se disseminar a vários órgãos e tecidos levando a
uma forma severa da doença. Dentro do hospedeiro, P. brasiliensis usa o ciclo do
glioxalato (CG) para sobrevivência intracelular. Adesão e invasão das células do
hospedeiro são passos essenciais envolvidos na internalização e disseminação do
patógeno. Aqui, nós evidênciamos que malato sintase de P. brasiliensis (PbMLS) é
secretada, e é localizada na parede da célula. PbMLS foi superexpressa em Escherichia
coli, e o anticorpo policlonal contra esta proteína foi obtido. Usando Microscopia Laser
Confocal (CLSM) e análise de Western blot, PbMLS foi encontrada no citoplasma e na
parede da célula na fase leveduriforme de P. brasiliensis nas células mãe e broto.
PbMLSr e o respectivo anticorpo policlonal produzido contra esta proteína inibiram a
interação de P. brasiliensis com células epiteliais A549 cultivads in vitro. Estas
observações indicariam que MLS associada à parede da célula de P. brasiliensis pode
estar mediando a ligação do fungo às células, contribuindo assim com a adesão do
fungo aos tecidos hospedeiros e para a disseminação da infecção.
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Genom- und Transkriptionsanalyse von <i>Bacillus licheniformis</i> DSM13 - einem Organismus mit großem industriellem Potential / Genomic and transcriptional analyses of <i>Bacillus licheniformis</i> DSM13 - an organism of high industrial relevanceVeith, Birgit 25 January 2005 (has links)
No description available.
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