Spelling suggestions: "subject:"diynamics simulation"" "subject:"bdynamics simulation""
121 |
Structure and Dynamics of the Copper-binding Octapeptide Region in the Human Prion ProteinRiihimäki, Eva-Stina January 2005 (has links)
<p>The copper-binding ability of the prion protein may be closely connected to its function. Identifying the exact function of the prion protein can clarify the underlying mechanism in prion diseases. In this work, the copper-binding octapeptide region in the human prion protein has been studied. The structural characteristics of the binding site are examined by quantum chemical structural optimization. The calculations aim at identifying a substitute for copper(II) to be used in NMR-spectroscopic studies of the copper-binding region. The dynamical and structural features of the peptide region are investigated in molecular dynamics simulations. Aspects of importance in the development of model systems in molecular dynamics simulation are addressed.</p>
|
122 |
Molecular Dynamics Simulation of the Effect of the Crystal Environment on Protein Conformational Dynamics and Functional MotionsAhlstrom, Logan Sommers January 2012 (has links)
Proteins are dynamic and interconvert between different conformations to perform their biological functions. Simulation methodology drawing upon principles from classical mechanics - molecular dynamics (MD) simulation - can be used to simulate protein dynamics and reconstruct the conformational ensemble at a level of atomic detail that is inaccessible to experiment. We use the dynamic insight achieved through simulation to enhance our understanding of protein structures solved by X-ray crystallography. Protein X-ray structures provide the most important information for structural biology, yet they depict just a single snapshot of the solution ensemble, which is under the influence of the confined crystal medium. Thus, we ask a fundamental question - how well do static X-ray structures represent the dynamic solution state of a protein? To understand how the crystal environment affects both global and local protein conformational dynamics, we consider two model systems. We first examine the variation in global conformation observed in several solved X-ray structures of the λ Cro dimer by reconstructing the solution ensemble using the replica exchange enhanced sampling method, and show that one X-ray conformation is unstable in solution. Subsequent simulation of Cro in the crystal environment quantitatively assesses the strength of packing interfaces and reveals that mutation in the lattice affects the stability of crystal forms. We also evaluate the Cro models solved by nuclear magnetic resonance spectroscopy and demonstrate that they represent unstable solution states. In addition to our studies of the Cro dimer, we investigate the effect of crystal packing on side-chain conformational dynamics through solution and crystal MD simulation of the HIV microbicide Cyanovirin-N. We find that long, polar surface side-chains can undergo a strong reduction in conformational entropy upon incorporation into crystal contacts, which supports the application of surface engineering to facilitate protein crystallization. Finally, we outline a general framework for using network visualization to aid in the functional interpretation of conformational ensembles generated from MD simulation. Our results will enhance the understanding of X-ray data in establishing protein structure-function-dynamics relationships.
|
123 |
Smart hydrogels as storage elements with dispensing functionality in discontinuous microfluidic systemsHaefner, Sebastian, Frank, Philipp, Elstner, Martin, Nowak, Johannes, Odenbach, Stefan, Richter, Andreas 07 April 2017 (has links) (PDF)
Smart hydrogels are useful elements in microfluidic systems because they respond to environmental stimuli and are capable of storing reagents. We present here a concept of using hydrogels (poly(N-isopropylacrylamide)) as an interface between continuous and discontinuous microfluidics. Their swelling and shrinking capabilities allow them to act as storage elements for reagents absorbed in the swelling process. When the swollen hydrogel collapses in an oil-filled channel, the incorporated water and molecules are expelled from the hydrogel and form a water reservoir. Water-in-oil droplets can be released from the reservoir generating different sized droplets depending on the flow regime at various oil flow rates (dispensing functionality). Different hydrogel sizes and microfluidic structures are discussed in terms of their storage and droplet formation capabilities. The time behaviour of the hydrogel element is investigated by dynamic swelling experiments and computational fluid dynamics simulations. By precise temperature control, the device acts as an active droplet generator and converts continuous to discontinuous flows.
|
124 |
Metodologia de aperfeiçoamento de suspensões veiculares através de modelo virtual em ambiente multicorpos / Improvement methodology of vehicle suspensions through model in virtual environment multibodyVieira Neto, Alaor Jose 19 April 2011 (has links)
Entre as etapas do desenvolvimento de automóveis pode-se apontar a definição das características de suas suspensões. A fase de definição da suspensão pode ser dividida dentro do seguinte cenário: a escolha de um determinado tipo de suspensão, os pontos (geometria) e quais os valores de rigidez/amortecimento para todo o sistema irá resultar em um comportamento dinâmico desejado para o veículo, bem como a viabilidade de produção. Além disso, o entendimento da interação entre os parâmetros de suspensão, é crucial para a otimização do desempenho. Este trabalho pretende propor um método para aperfeiçoar a fase de \"tuning\" da suspensão, com foco principal no conforto. O veículo considerado é um caminhão comercial, e entre os seus parâmetros considerados estão rigidezes de molas da cabine e suspensão, amortecimento da suspensão de cabine e curvas do amortecedor da suspensão primária. O modelo virtual do veículo foi desenvolvido em ambiente ADAMS, o qual, previamente à otimização, foi validado contra dados experimentais. Métricas foram especialmente desenvolvidas levando em consideração aspectos subjetivos de conforto veicular, para dessa forma eliminar a variabilidade entre as avaliações subjetivas e análises das simulações. Os resultados mostraram expressivas melhorias no conforto e através de dados experimentais essas melhorias foram confirmadas. / Among the development phases of an automotive vehicle one can point out the definition of the characteristics of its suspensions. Suspension definition phase can be understood as the following scenario: given a suspension type, which hard points (geometric) and what values of stiffness/damping for the whole system will result in a desired dynamic behavior for the vehicle as well as production feasibility. Moreover, understanding the iteration among the suspension parameters, even considering just the tuning ones, is crucial for performance optimization. This work intends to propose a method for vehicle tuning characteristics optimization, having as a target the ride comfort. The vehicle considered here is a commercial truck, and among its parameters one considers cabin and suspension springs, cabin dampers and suspension damper curves. A vehicle model was developed in ADAMS environment and prior to the optimization the vehicle was validated against experimental data. Metrics were specially developed to take into account subjective aspects of ride, and, in this way, eliminating the gap between subjective evaluations and simulations analysis. Results showed improvements in ride comfort. The resulting setup was measured and the improvements were confirmed with experimental data.
|
125 |
Computer simulations exploring conformational preferences of short peptides and developing a bacterial chromosome modelLi, Shuxiang 15 December 2017 (has links)
Computer simulations provide a potentially powerful complement to conventional experimental techniques in elucidating the structures, dynamics and interactions of macromolecules. In this thesis, I present three applications of computer simulations to investigate important biomolecules with sizes ranging from two-residue peptides, to proteins, and to whole chromosome structures.
First, I describe the results of 441 independent explicit-solvent molecular dynamics (MD) simulations of all possible two-residue peptides that contain the 20 standard amino acids with neutral and protonated histidine. 3JHNHα coupling constants and δHα chemical shifts calculated from the MD simulations correlated quite well with recently published experimental measurements for a corresponding set of two-residue peptides. Neighboring residue effects (NREs) on the average 3JHNHα and δHα values of adjacent residues were also reasonably well reproduced. The intrinsic conformational preferences of each residue, and their NREs on the conformational preferences of adjacent residues, were analyzed. Finally, these NREs were compared with corresponding effects observed in a coil library and the average β-turn preferences of all residue types were determined.
Second, I compare the abilities of three derivatives of the Amber ff99SB force field to reproduce a recent report of 3JHNHα scalar coupling constants for hundreds of two-residue peptides. All-atom MD simulations of 256 two-residue peptides were performed and the results showed that a recently-developed force field (RSFF2) produced a dramatic improvement in the agreement with experimental 3JHNHα coupling constants. I further show that RSFF2 also improved modestly agreement with experimental 3JHNHα coupling constants of five model proteins. However, an analysis of NREs on the 3JHNHα coupling constants of the two-residue peptides indicated little difference between the force fields’ abilities to reproduce experimental NREs. I speculate that this might indicate limitations in the force fields’ descriptions of nonbonded interactions between adjacent side chains or with terminal capping groups.
Finally, coarse-grained (CG) models and multi-scale modeling methods are used to develop structural models of entire E. coli chromosomes confined within the experimentally-determined volume of the nucleoid. The final resolution of the chromosome structures built here was one-nucleotide-per-bead (1 NTB), which represents a significant increase in resolution relative to previously published CG chromosome models, in which one bead corresponds to hundreds or even thousands of basepairs. Based on the high-resolution final 1 NTB structures, important physical properties such as major and minor groove widths, distributions of local DNA bending angles, and topological parameters (Linking Number (Lk), Twist (Tw) and Writhe (Wr)) were accurately computed and compared with experimental measurements or predictions from a worm-like chain (WLC) model. All these analyses indicated that the chromosome models built in this study are reasonable at a microscopic level. This chromosome model provides a significant step toward the goal of building a whole-cell model of a bacterial cell.
|
126 |
Flexible multibody dynamics approach for tire dynamics simulationYamashita, Hiroki 01 December 2016 (has links)
The objective of this study is to develop a high-fidelity physics-based flexible tire model that can be fully integrated into multibody dynamics computer algorithms for use in on-road and off-road vehicle dynamics simulation without ad-hoc co-simulation techniques. Despite the fact detailed finite element tire models using explicit finite element software have been widely utilized for structural design of tires by tire manufactures, it is recognized in the tire industry that existing state-of-the-art explicit finite element tire models are not capable of predicting the transient tire force characteristics accurately under severe vehicle maneuvering conditions due to the numerical instability that is essentially inevitable for explicit finite element procedures for severe loading scenarios and the lack of transient (dynamic) tire friction model suited for FE tire models. Furthermore, to integrate the deformable tire models into multibody full vehicle simulation, co-simulation technique could be an option for commercial software. However, there exist various challenges in co-simulation for the transient vehicle maneuvering simulation in terms of numerical stability and computational efficiency. The transient tire dynamics involves rapid changes in contact forces due to the abrupt braking and steering input, thus use of co-simulation requires very small step size to ensure the numerical stability and energy balance between two separate simulation using different solvers.
In order to address these essential and challenging issues on the high-fidelity flexible tire model suited for multibody vehicle dynamics simulation, a physics-based tire model using the flexible multibody dynamics approach is proposed in this study. To this end, a continuum mechanics based shear deformable laminated composite shell element is developed based on the finite element absolute nodal coordinate formulation for modeling the complex fiber reinforced rubber tire structure. The assumed natural strain (ANS) and enhanced assumed strain (EAS) approaches are introduced for alleviating element lockings exhibited in the element. Use of the concept of the absolute nodal coordinate formulation leads to various advantages for tire dynamics simulation in that (1) constant mass matrix can be obtained for fully nonlinear dynamics simulation; (2) exact modeling of rigid body motion is ensured when strains are zero; and (3) non-incremental solution procedure utilized in the general multibody dynamics computer algorithm can be directly applied without specialized updating schemes for finite rotations. Using the proposed shear deformable laminated composite shell element, a physics-based flexible tire model is developed. To account for the transient tire friction characteristics including the friction-induced hysteresis that appears in severe maneuvering conditions, the distributed parameter LuGre tire friction model is integrated into the flexible tire model. To this end, the contact patch predicted by the structural tire model is discretized into small strips across the tire width, and then each strip is further discretized into small elements to convert the partial differential equations of the LuGre tire friction model to the set of first-order ordinary differential equations. By doing so, the structural deformation of the flexible tire model and the LuGre tire friction force model are dynamically coupled in the final form of the equations, and these equations are integrated simultaneously forward in time at every time step.
Furthermore, a systematic and automated procedure for parameter identification of LuGre tire friction model is developed. Since several fitting parameters are introduced to account for the nonlinear friction characteristics, the correlation of the model parameters with physical quantities are not clear, making the parameter identification of the LuGre tire friction model difficult. In the procedure developed in this study, friction parameters in terms of slip-dependent friction characteristics and adhesion parameter are estimated separately, and then all the parameters are identified using the nonlinear least squares fitting. Furthermore, the modified friction characteristic curve function is proposed for wet road conditions, in which the linear decay in friction is exhibited in the large slip velocity range. It is shown that use of the proposed numerical procedure leads to an accurate prediction of the LuGre model parameters for measured tire force characteristics under various loading and speed conditions. Furthermore, the fundamental tire properties including the load-deflection curve, the contact patch lengths, contact pressure distributions, and natural frequencies are validated against the test data. Several numerical examples for hard braking and cornering simulation are presented to demonstrate capabilities of the physics-based flexible tire model developed in this study.
Finally, the physics-based flexible tire model is further extended for application to off-road mobility simulation. To this end, a locking-free 9-node brick element with the curvature coordinates at the center node is developed and justified for use in modeling a continuum soil with the capped Drucker-Prager failure criterion. Multiplicative finite strain plasticity theory is utilized to consider the large soil deformation exhibited in the tire/soil interaction simulation. In order to identify soil parameters including cohesion and friction angle, the triaxial soil test is conducted. Using the soil parameters identified including the plastic hardening parameters by the compression soil test, the continuum soil model developed is validated against the test data. Use of the high-fidelity physics-based tire/soil simulation model in off-road mobility simulation, however, leads to a very large computational model to consider a wide area of terrains. Thus, the computational cost dramatically increases as the size of the soil model increases. To address this issue, the component soil model is proposed such that soil elements far behind the tire can be removed from the equations of motion sequentially, and then new soil elements are added to the portion that the tire is heading to. That is, the soil behavior only in the vicinity of the rolling tire is solved in order to reduce the overall model dimensionality associated with the finite element soil model. It is shown that use of the component soil model leads to a significant reduction in computational time while ensuring the accuracy, making the use of the physics-based deformable tire/soil simulation capability feasible in off-road mobility simulation.
|
127 |
Folding and aggregation of amyloid peptidesKittner, Madeleine January 2011 (has links)
Aggregation of the Amyloid β (Aβ) peptide to amyloid fibrils is associated with the outbreak of Alzheimer’s disease. Early aggregation intermediates in form of soluble oligomers are of special interest as they are believed to be the major toxic components in the process. These oligomers are of disordered and transient nature. Therefore, their detailed molecular structure is difficult to access experimentally and often remains unknown. In the present work extensive, fully atomistic replica exchange molecular dynamics simulations were performed to study the preaggregated, monomer states and early aggregation intermediates (dimers, trimers) of Aβ(25-35) and Aβ(10-35)-NH2 in aqueous solution.
The folding and aggregation of Aβ(25-35) were studied at neutral pH and 293 K. Aβ(25-35) monomers mainly adopt β-hairpin conformations characterized by a β-turn formed by residues G29 and A30, and a β-sheet between residues N27–K28 and I31–I32 in equilibrium with coiled conformations. The β-hairpin conformations served as initial configurations to model spontaneous aggregation of Aβ(25-35).
As expected, within the Aβ(25-35) dimer and trimer ensembles many different poorly populated conformations appear. Nevertheless, we were able to distinguish between disordered and fibril-like oligomers. Whereas disordered oligomers are rather compact with few intermolecular hydrogen bonds (HBs), fibril-like oligomers are characterized by the formation of large intermolecular β-sheets. In most of the fibril-like dimers and trimers individual peptides are fully extended forming in- or out-of-register antiparallel β-sheets. A small amount of fibril-like trimers contained V-shaped peptides forming parallel β-sheets. The dimensions of extended and V-shaped oligomers correspond well to the diameters of two distinct morphologies found for Aβ(25-35) fibrils.
The transition from disordered to fibril-like Aβ(25-35) dimers is unfavorable but driven by energy. The lower energy of fibril-like dimers arises from favorable intermolecular HBs and other electrostatic interactions which compete with a loss in entropy. Approximately 25 % of the entropic cost correspond to configurational entropy. The rest relates to solvent entropy, presumably caused by hydrophobic and electrostatic effects.
In contrast to the transition towards fibril-like dimers the first step of aggregation is driven by entropy. Here, we compared structural and thermodynamic properties of the individual monomer, dimer and trimer ensembles to gain qualitative information about the aggregation process. The β-hairpin conformation observed for monomers is successively dissolved in dimer and trimer ensembles while instead intermolecular β-sheets are formed. As expected upon aggregation the configurational entropy decreases. Additionally, the solvent accessible surface area (SASA), especially the hydrophobic SASA, decreases yielding a favorable solvation free energy which overcompensates the loss in configurational entropy. In summary, the hydrophobic effect, possibly combined with electrostatic effects, yields an increase in solvent entropy which is believed to be one major driving force towards aggregation.
Spontaneous folding of the Aβ(10-35)-NH2 monomer was modeled using two force fields, GROMOS96 43a1 and OPLS/AA, and compared to primary NMR data collected at pH 5.6 and 283 K taken from the literature. Unexpectedly, the two force fields yielded significantly different main conformations. Comparison between experimental and calculated nuclear Overhauser effect (NOE) distances is not sufficient to distinguish between the different force fields. Additionally, the comparison with scalar coupling constants suggest that the chosen protonation in both simulations corresponds to a pH lower than in the experiment. Based on this analysis we were unable to determine which force field yields a better description of this system.
Dimerization of Aβ(10-35)-NH2 was studied at neutral pH and 300 K. Dimer conformations arrange in many distinct, poorly populated and rather complex alignments or interlocking patterns which are rather stabilized by side chain interactions than by specific intermolecular hydrogen bonds. Similar to Aβ(25-35) dimers, transition towards β-sheet-rich, fibril-like Aβ(10-35) dimers is driven by energy competing with a loss in entropy. Here, transition is mediated by favorable peptide-solvent and solvent-solvent interactions mainly arising from electrostatic interactions. / Die Aggregation des Amyloid β (Aβ) Peptids zu Amyloidfibrillen wird mit dem Ausbruch der Alzheimer Krankheit in Verbindung gebracht. Die toxische Wirkung auf Zellen wird vor allem den zeitigen Intermediaten in Form von löslichen Oligomeren zugeschrieben. Aufgrund deren ungeordneter und flüchtiger Natur kann die molekulare Struktur solcher zeitigen Oligomere oft experimentell nicht aufgelöst werden. In der vorliegenden Arbeit wurden aufwendige atomistische Replica-Exchange-Molekulardynamik-Simulationen durchgeführt, um die molekulare Struktur von Monomeren und Oligomeren der Fragmente Aβ(25-35) und Aβ(10-35)-NH2 in Wasser zu untersuchen.
Die Faltung und Aggregation von Aβ(25-35) wurde bei neutralem pH und 293 K untersucht. Monomere dieses Fragments bilden hauptsächlich β-Haarnadelkonformationen im Gleichgewicht mit Knäulstrukturen. Innerhalb der β-Haarnadelkonformationen bilden die Residuen G29 und A30 einen β-turn, während N27–K28 and I31–I32 ein β-Faltblatt bilden. Diese β-Haarnadelkonformationen bildeten den Ausgangspunkt zur Modellierung spontaner Aggregation.
Wie zu erwarten, bilden sich eine Vielzahl verschiedener, gering besetzter Dimer- und Trimerkonformationen. Mit Hilfe einer gröberen Einteilung können diese in ungeordnete und fibrillähnliche Oligomere unterteilt werden. Ungeordnete Oligomere bilden kompakte Strukturen, die nur durch wenige intermolekulare Wasserstoffbrückenbindungen (HBB) stabilisiert sind. Typisch für fibrillähnliche Oligomere ist hingegen die Ausbildung großer intermolekularer β-Faltblätter. In vielen dieser Oligomere finden wir antiparallele, in- oder out-of-register β-Faltblätter gebildet durch vollständig ausgestreckte Peptide. Ein kleiner Teil der fibrillähnlichen Trimere bildet parallele, V-förmige β-Faltblätter. Die Ausdehnungen ausgestreckter und V-förmiger Oligomere entspricht in etwa den Durchmessern von zwei verschiedenen, experimentell gefundenen Fibrillmorphologien für Aβ(25-35).
Die Umwandlung von ungeordneten zu fibrillähnlichen Aβ(25-35) Dimeren ist energetisch begünstigt, läuft aber nicht freiwillig ab. Fibrillähnliche Dimere haben eine geringere Energie aufgrund günstiger Peptidwechselwirkungen (HBB, Salzbrücken), welche durch den Verlust an Entropie kompensiert wird. Etwa 25 % entsprechen dem Verlust an Konfigurationsentropie. Der restliche Anteil wird einem Verlust an Lösungsmittelentropie aufgrund von hydrophoben und elektrostatischen Effekten zugesprochen.
Im Gegensatz zur Umwandlung in fibrillähnliche Dimere, ist die Assoziation von Monomeren oder Oligomeren entropisch begünstigt. Beim Vergleich thermodynamischer Eigenschaften der Monomer-, Dimer- und Trimersysteme zeigt sich im Verlauf der Aggregation, wie erwartet, eine Abnahme der Konfigurationsentropie. Zusätzlich nimmt die dem Lösungsmittel zugängliche Oberfläche (SASA), insbesondere die hydrophobe SASA, ab. In Verbindung damit beobachten wir eine Abnahme der freien Solvatisierungsenergie, welche den Verlust an Konfigurationsentropie kompensiert. Mit anderen Worten, der hydrophobe Effekt in Kombination mit elektrostatischen Wechselwirkungen führt zu einem Ansteigen der Lösungsmittelentropie und begünstigt damit die Aggegation.
Die spontane Faltung des Aβ(10-35)-NH2 Monomers wurde für zwei verschiedene Proteinkraftfelder, GROMOS96 43a1 und OPLS/AA, untersucht und mit primären NMR-Daten aus der Literatur, gemessen bei pH 5.6 und 283 K, verglichen. Beide Kraftfelder generieren unterschiedliche Hauptkonformationen. Der Vergleich zwischen experimentellen und berechneten Kern-Overhauser-Effekt (NOE) Abständen ist nicht ausreichend, um zwischen beiden Kraftfeldern zu unterscheiden. Der Vergleich mit Kopplungskonstanten aus Experiment und Simulation zeigt, dass beide Simulationen einem pH-Wert geringer als 5.6 ensprechen. Basierend auf den bisherigen Ergebnissen können wir nicht entscheiden, welches Kraftfeld eine bessere Beschreibung für dieses System liefert.
Die Dimerisierung von Aβ(10-35)-NH2 wurde bei neutralem pH und 300 K untersucht. Wir finden eine Vielzahl verschiedener, gering besetzter Dimerstrukturen, welche eher durch Seitenkettenkontakte als durch spezifische HBB stabilisiert sind. Wie bei den Aβ(25-35) Dimeren, ist die Umwandlung zu β-Faltblattreichen, fibrillähnlichen Aβ(10-35) Dimeren energetisch begünstigt, konkurriert aber mit einem Entropieverlust. Die Umwandlung wird in diesem Fall durch elektrostatische Wechselwirkungen zwischen Peptid und Lösungsmittel und innerhalb des Lösungsmittels bestimmt.
|
128 |
Investigation of Structural Behaviors of Methyl Methacrylate Oligomers within Confinement Space by Coarse-grained Configurational-bias Monte Carlo SimulationChang, Chun-Yi 16 August 2010 (has links)
The coarse-grained configurational-bias Monte Carlo (CG-CBMC) simulation was employed to study the structural behaviors of methyl methacrylate (MMA) oligomers adsorbed on grooved substrate due to molecular dynamics (MD) simulation is probably trapped at some local energy minima and difficult to carry out over a long enough time to allow relaxation of chain motion for an enormous polymeric system. Therefore, the CG-CBMC simulation was adopted in the present study. In this study, three types of chains are classified according to their positions relative to the groove. Type 1, Type 2, and Type 3 represent the entire MMA-oligomer within the groove, the MMA-oligomer partially within the groove, and the oligomer outside the groove, respectively. The orientational order parameters of Type 1 and Type 2 oligomers decrease with the increase of groove width, but the orientational order parameter of Type 3 oligomers is approximately equal to 0.1. In addition, observation of the orientational order parameters of Type 1 oligomers interacting with the grooved substrate at different interaction strengths decrease with increasing the groove width. Furthermore, the orientational order parameters of Type 1 oligomers within the narrowest (20 Å) and the widest (35 Å) groove with different depths were determined. For the narrowest groove, the arrangement of Type 1 oligomers will be influenced by the groove width. However, in the case of the widest groove, the orientational order parameter of Type 1 oligomers is approximately equal to 0.2. This study can help engineers clarify the characteristics and phenomena of physical adsorption of the molecules, as well as contributing to the application of recent technology.
|
129 |
Simulation of Fluorescence Spectroscopy Experiments / Simulation fluoreszenzspektroskopischer ExperimenteSchröder, Gunnar 06 October 2004 (has links)
No description available.
|
130 |
Collective Dynamics Underlying Allosteric Transitions: A Molecular Dynamics Study / Kollektive Dynamiken in allosterischen Übergängen: Eine MolekulardynamikstudieVesper, Martin David 18 December 2012 (has links)
No description available.
|
Page generated in 0.154 seconds