Mechanoelastic Properties Research Articles

Fibrillar, fibril-associated and basement membrane collagens of the arterial wall: architecture, elasticity and remodeling under stress.

The ability of a human artery to pass through 150 million liters of blood sustaining 2 billion pulsations of blood pressure with minor deterioration depends on unique construction of the arterial wall. Viscoelastic properties of this construction enable to re-seal the occuring damages apparently without direct immediate participance of the constituent cells. Collagen structures are considered to be the elements that determine the mechanoelastic properties of the wall in parallel with elastin responsible for elasticity and resilience. Collagen scaffold architecture is the function-dependent dynamic arrangement of a dozen different collagen types composing three distinct interacting forms inside the extracellular matrix of the wall. Tightly packed molecules of collagen types I, III, V provide high tensile strength along collagen fibrils but toughness of the collagen scaffold as a whole depends on molecular bonds between distinct fibrils. Apart of other macromolecules in the extracellular matrix (ECM), collagen-specific interlinks involve microfilaments of collagen type VI, meshwork-organized collagen type VIII, and FACIT collagen type XIV. Basement membrane collagen types IV, XV, XVIII and cell-associated collagen XIII enable transmission of mechanical signals between cells and whole artery matrix. Collagen scaffold undergoes continuous remodeling by decomposition promoted with MMPs and reconstitution from newly produced collagen molecules. Pulsatile stress-strain load modulates both collagen synthesis and MMP-dependent collagen degradation. In this way the ECM structure becomes adoptive to mechanical challenges. The mechanoelastic properties of the arterial wall are changed in atherosclerosis concomitantly with collagen turnover both type-specific and dependent on the structure. Improving the feedback could be another approach to restore sufficient blood circulation.

Stretching of red blood cells using an electro-optics trap

The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cells.

Characterization of unfolding mechanism of human lamin A Ig fold by single-molecule force spectroscopy-implications in EDMD.

A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath the nuclear envelope thereby conferring proper shape and mechanical rigidity to the nucleus. Lamin proteins are also shown to be related diversely to basic nuclear processes. More than 400 mutations in human lamin A protein alone have been reported to produce at least 11 different disease conditions jointly termed as laminopathies. These mutations in lamin A are scattered throughout its helical rod domain, as well as the C-terminal domain containing the conserved Ig-fold region. The commonality of phenotypes in all these diseases is characterized by misshapen nuclei of the affected tissues which might stem from altered rigidity of the supporting lamina hence lamins. Here we have focused on autosomal dominant Emery-Dreifuss Muscular Dystrophy, one such laminopathy where R453W is the causative mutation located in the Ig domain of lamin A. We have investigated by single-molecule force spectroscopy how a stretching mechanical perturbation senses the destabilizing effect of the mutation in the lamin A Ig domain and compared the mechanoelastic properties of the mutant R453W with that of the wild-type in conjunction with steered molecular dynamics. Furthermore, we have shown the interaction of Ig domain with emerin, another key player and interacting partner in the pathogenesis of EDMD, is disrupted in the R453W mutant. This altered mechanoresistance of Ig domain itself and consequent uncoupling of lamin A-emerin interaction might underlie the altered mechanotransduction properties of EDMD affected nuclei.

Detection and quantification of bacterial biofilms combining high-frequency acoustic microscopy and targeted lipid microparticles.

BackgroundImmuno-compromised patients such as those undergoing cancer chemotherapy are susceptible to bacterial infections leading to biofilm matrix formation. This surrounding biofilm matrix acts as a diffusion barrier that binds up antibiotics and antibodies, promoting resistance to treatment. Developing non-invasive imaging methods that detect biofilm matrix in the clinic are needed. The use of ultrasound in conjunction with targeted ultrasound contrast agents (UCAs) may provide detection of early stage biofilm matrix formation and facilitate optimal treatment.ResultsLigand-targeted UCAs were investigated as a novel method for pre-clinical non-invasive molecular imaging of early and late stage biofilms. These agents were used to target, image and detect Staphylococcus aureus biofilm matrix in vitro. Binding efficacy was assessed on biofilm matrices with respect to their increasing biomass ranging from 3.126 × 103 ± 427 UCAs per mm2 of biofilm surface area within 12 h to 21.985 × 103 ± 855 per mm2 of biofilm matrix surface area at 96 h. High-frequency acoustic microscopy was used to ultrasonically detect targeted UCAs bound to a biofilm matrix and to assess biofilm matrix mechanoelastic physical properties. Acoustic impedance data demonstrated that biofilm matrices exhibit impedance values (1.9 MRayl) close to human tissue (1.35 - 1.85 MRayl for soft tissues). Moreover, the acoustic signature of mature biofilm matrices were evaluated in terms of integrated backscatter (0.0278 - 0.0848 mm-1 × sr-1) and acoustic attenuation (3.9 Np/mm for bound UCAs; 6.58 Np/mm for biofilm alone).ConclusionsEarly diagnosis of biofilm matrix formation is a challenge in treating cancer patients with infection-associated biofilms. We report for the first time a combined optical and acoustic evaluation of infectious biofilm matrices. We demonstrate that acoustic impedance of biofilms is similar to the impedance of human tissues, making in vivo imaging and detection of biofilm matrices difficult. The combination of ultrasound and targeted UCAs can be used to enhance biofilm imaging and early detection. Our findings suggest that the combination of targeted UCAs and ultrasound is a novel molecular imaging technique for the detection of biofilms. We show that high-frequency acoustic microscopy provides sufficient spatial resolution for quantification of biofilm mechanoelastic properties.

Surface Interactions in Suspensions of Swimming Cells

Interactions between swimming cells and surfaces are essential to many microbiological processes, from bacterial biofilm formation to human fertilization. However, despite their fundamental importance, relatively little is known about the physical mechanisms that govern the scattering of flagellated or ciliated cells from solid surfaces. A more detailed understanding of these interactions promises not only new biological insights into structure and dynamics of flagella and cilia but may also lead to new microfluidic techniques for controlling cell motility and microbial locomotion, with potential applications ranging from diagnostic tools to therapeutic protein synthesis and photosynthetic biofuel production. Due to fundamental differences in physiology and swimming strategies, it is an open question of whether microfluidic transport and rectification schemes that have recently been demonstrated for pusher-type microswimmers such as bacteria and sperm cells, can be transferred to puller-type algae and other motile eukaryotes, because it is not known whether long-range hydrodynamic or short-range mechanical forces dominate the surface interactions of these microorganisms. Here, using high-speed microscopic imaging, we present direct experimental evidence that the surface scattering of both mammalian sperm cells and unicellular green algae is primarily governed by direct ciliary contact interactions. Building on this insight, we predict and experimentally verify the existence of optimal microfluidic ratchets that maximize rectification of initially uniform Chlamydomonas reinhardtii suspensions. Because mechano-elastic properties of cilia are conserved across eukaryotic species, we expect that our results apply to a wide range of swimming microorganisms.

Ciliary contact interactions dominate surface scattering of swimming eukaryotes

Interactions between swimming cells and surfaces are essential to many microbiological processes, from bacterial biofilm formation to human fertilization. However, despite their fundamental importance, relatively little is known about the physical mechanisms that govern the scattering of flagellated or ciliated cells from solid surfaces. A more detailed understanding of these interactions promises not only new biological insights into structure and dynamics of flagella and cilia but may also lead to new microfluidic techniques for controlling cell motility and microbial locomotion, with potential applications ranging from diagnostic tools to therapeutic protein synthesis and photosynthetic biofuel production. Due to fundamental differences in physiology and swimming strategies, it is an open question of whether microfluidic transport and rectification schemes that have recently been demonstrated for pusher-type microswimmers such as bacteria and sperm cells, can be transferred to puller-type algae and other motile eukaryotes, because it is not known whether long-range hydrodynamic or short-range mechanical forces dominate the surface interactions of these microorganisms. Here, using high-speed microscopic imaging, we present direct experimental evidence that the surface scattering of both mammalian sperm cells and unicellular green algae is primarily governed by direct ciliary contact interactions. Building on this insight, we predict and experimentally verify the existence of optimal microfluidic ratchets that maximize rectification of initially uniform Chlamydomonas reinhardtii suspensions. Because mechano-elastic properties of cilia are conserved across eukaryotic species, we expect that our results apply to a wide range of swimming microorganisms.

Resonance Condition in Microtubules Using Ultrasound Plane Waves

Microtubules (MTs) are protein filaments forming a major part of the cytoskeleton of all eukaryotic cells which directly contribute to the process of cell division by forming mitotic spindles and providing force for the segregation of chromosomes. MTs exhibit mechanoelastic properties of considerable interest in view of their functional roles in cell division and cell shape. In this work by introducing a new method, we have analytically solved equations of motion for the vibrational dynamics of an MT that is attached at its two ends. This is especially relevant for MTs during mitosis when they attach to chromosomes and centrosomes at the plus and minus ends, respectively. Our analysis applies to MTs present inside a viscous solution and when driven by an ultrasound plane wave. We have shown that with using ultrasound plane waves the resonance condition for the MT treated as a rigid rod cannot be provided, and in order to achieve resonance we should excite a single mode of the MT with a harmonic number larger than a threshold value introduced in this paper. Having a large enough amplitude for the resonant vibration effect is crucial in order to maximize the bending moment of an MT. Attaining a high quality factor for a resonance is crucial to have a better control of the energy transfer to the MT. Finally, single mode excitation not only helps to transfer the minimum amount of energy to the surrounding medium compared with multi-mode excitation but it also allows for a simultaneous high-amplitude and high-quality factor which is impossible when using plane waves.

Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes

Lamins are the major components of the nuclear lamina, a filamentous layer underlying the inner nuclear membrane and attached to the peripheral chromatin. Lamins are required for maintaining nuclear shape and are involved in most nuclear activities. Here, we studied the 3D organization of the nuclear lamina formed upon the expression of Caenorhabditis elegans lamin (Ce-lamin) within the nucleus of a Xenopus laevis oocyte. We show that Ce-lamin forms an intricate 3D meshwork of 5-6 nm lamin protofilaments. The diverse protofilament interactions and organization may shed light upon the unique mechano-elastic properties of the nuclear lamina scaffold supporting the nuclear envelope. The Q159K Hutchinson-Gilford Progeria Syndrome-linked mutation alters interactions between protofilaments within the lamina, leading to the formation of more bundled arrays of less isotropically-oriented protofilaments. Using this system, we show for the first time the organization of lamin proteins that were translated and assembled within the environment of a living cell.

Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.

A General Methodology for the Determination of 2D Bodies Elastic Deformation Invariants: Application to the Automatic Identification of Parasites

A novel methodology is introduced here that exploits 2D images of arbitrary elastic body deformation instances so as to quantify mechanoelastic characteristics that are deformation invariant. Determination of such characteristics allows for developing methods offering an image of the undeformed body. General assumptions about the mechanoelastic properties of the bodies are stated which lead to two different approaches for obtaining bodies' deformation invariants. One was developed to spot a deformed body's neutral line and its cross sections, while the other solves deformation PDEs by performing a set of equivalent image operations on the deformed body images. Both of these processes may furnish a body-undeformed version from its deformed image. This was confirmed by obtaining the undeformed shape of deformed parasites, cells (protozoa), fibers, and human lips. In addition, the method has been applied to the important problem of parasite automatic classification from their microscopic images. To achieve this, we first apply the previous method to straighten the highly deformed parasites, and then, apply a dedicated curve classification method to the straightened parasite contours. It is demonstrated that essentially different deformations of the same parasite give rise to practically the same undeformed shape, thus confirming the consistency of the introduced methodology. Finally, the developed pattern recognition method classifies the unwrapped parasites into six families, with an accuracy rate of 97.6 percent.

Molecular characterization of Xenopus lamin LIV reveals differences in the lamin composition of sperms in amphibians and mammals

Lamins are nuclear intermediate filament proteins. They are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. Somatic cells of vertebrates express lamins A, B1, and B2 while lamin LIII, a major component of the amphibian oocyte lamina is absent in mammals. The organization of the lamina of germ cells differs significantly from that of somatic cells. Mammalian spermatogenic cells express two short lamins, C2 and B3, that are splice isoforms of lamin A and B2, respectively. Here we identify the previously described Xenopus lamin LIV as splice variant of the lamin LIII gene. LIV contains 40 extra residues in coil 2A of the rod domain, which results in altered assembly properties. Xenopus lamin LIV and mammalian B3 assemble into short structures rather than into long IF-like filaments. Expression of lamin LIV is restricted to male germ cells suggesting that it might be the functional equivalent of mammalian lamin B3. We provide evidence that lamins C2 and B3 are restricted to the mammalian lineage and describe the lamin composition of Xenopus sperm. Our results show that the evolution of germ cell-specific lamins followed separate and distinctly different paths in amphibians and mammals.

Label-Free Cancer Cell Detection with Impedimetric Transducers

While cancer is still an implacable disease, many cancers can be cured if they are diagnosed in an early stage. Recently, it was reported that the transformation from normal cells to cancer cells can change their mechanoelastic properties to become softer and more deformable. If some cancer cells are more deformable, then a progressive increase of the volume of softer cancer cells should be induced as an abrupt change in osmolarity is applied. On the basis of this hypothesis, we developed a sensor that can electronically monitor the volume increase of cancer cells under hyposmotic pressure. By this methodology, K:Molv NIH 3T3 cells, 786-O human kidney carcinoma cells, and MPSC-1 ovarian cancer cells were successfully detected within 30 min using on the order of 10 cells. These cancer cells could be detected with the same sensitivity even in the presence of a vast excess of the respective noncancerous cells [NIH 3T3 cells, human embryonic kidney (HEK) 293 cells, ovarian surface epithelial (OSE) cells]. Since the proposed impedimetric sensor could be useful for detecting cancer cells fast and reliably, it could be further implemented in the screening of large populations of tissue samples and the detection of circulating tumor cells for point-of-care applications.

Membrane activity of a C-reactive protein

C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca 2+. We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein’s role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens.

The Supramolecular Organization of the C. elegans Nuclear Lamin Filament

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.

Single-Molecule Measurement of the Strength of a Siloxane Bond

Increasing the mechanical stability of artificial polymer materials is an important task in materials science, and for this a profound knowledge of the critical mechanoelastic properties of its constituents is vital. Here, we use AFM-based single-molecule force spectroscopy measurements to characterize the rupture of a single silicon-oxygen bond in the backbone of polydimethylsiloxane as well as the force-extension behavior of this polymer. PDMS is not only a polymer used in a large variety of products but also an important model system for highly flexible polymers. In our experiments, we probe the entire relevant force range from low forces dominated by entropy up to the rupture of the covalent Si-O bonds in the polymer backbone at high forces. The resulting rupture-force histograms are investigated with microscopic models of bond rupture under load and are compared to density functional theory calculations to characterize the free-energy landscape of the Si-O bond in the polymer backbone.

Co-Development of Intravital, Laser-Induced Experimental and Computational Models of Thrombogenesis.

We have developed modified protocols for confocal intravital monitoring of thrombus development in mouse mesenteric vessels following laser-induced injury. By including tetramethyrhodamine-labeled dextran (70,000 mw) in the blood, unlabeled cells (leukocytes and reticulocytes) form black silhouettes where the red fluorescent dye is excluded in the confocal image. In addition, platelets were labeled with rat monoclonal anti-mouse CD41/61 (Emfret Analytics, Germany) mixed with secondary goat anti-rat IgG antibody labeled with Alexa Fluor 488 and human fibrinogen was labeled with Alexa Fluor 350. Thus, high resolution confocal images of the developing thrombus included platelets in green, fibrin in blue, plasma in red and cellular components in black. Near real-time video recordings indicate the importance of flow parameters on the developing clot. In particular, turbulent flow patterns around the developing thrombus contribute to the incorporation of blood cells in the thrombus leading to the development of thrombi with a heterogeneous domain structure. Conceivably, different mechano-elastic properties of the heterogeneous domains might contribute to thrombus instability as the thrombus is subject to varying stresses and strains. In parallel with the modified experimental vascular injury model, we have begun development of a computational model of thrombus development. The modeling framework consists of a stochastic and discrete Cellular Potts Model (CPM) to describe platelet and cellular interactions and continuous submodels to describe hydrodynamic and biochemical reactions. The components of the computational model include the vessel wall, platelets (in resting and activated states), blood cells, coagulation reactions, fibrin formation, and hydrodynamic parameters. By varying input variables (such as flow rate, blood viscosity, coagulant activities) and monitoring properties of the developing thrombus, one can modify and validate the computational model. While our experimental systems enable us to determine essential components required for thrombus development, a validated model could help identify threshold levels of key components involved in thrombogenesis. This may be particularly useful in identifying hemostatic risk factors if these threshold levels are within the normal range found in the human population.

Delocalization of vaccinia virus components observed by atomic force and fluorescence microscopy

Better understanding of viral genomes is emerging as an urgent need as these genomes evolve and pandemic fears surface and for better understanding of viral infection processes. To address this need, we report a method to visualize intact, viral DNA and its interaction with viral proteins with the use of the atomic force microscope (AFM) in conjunction with fluorescence microscopy. Through a series of multifaceted experiments, we were able to visualize time-dependent progressive stages of proteolytic digestion and disassembly of extracellular enveloped vaccinia virus particles. After a 1-h treatment, the viral particles were partially digested and the viral cores showed slight disassociation in the AFM as evidenced by height analysis of individual virions. Most of the components of the virions were still intact. Further verification with florescence microscopy with nucleophilic and lipophilic stains demonstrated that viral DNA was, indeed still, co-localized within the viral core. However, with prolonged treatment with proteinase K and sodium dodecylsulfate, the AFM revealed that the viral core completely collapsed onto the substrate and had delocalized from the enclosed DNA. This process was again verified using fluorescence microscopy, the viral DNA was observed to be completely released from the viral core, in globular condensed form. These studies suggest that AFM imaging and fluorescence microscopy verification with stains specific for different constituents of viral particles is a valuable method to study the structural and mechano elastic properties of virus morphology and interactions of viral nucleoproteins with its DNA core.

High-temperature ferroelastic phase transitions in Li–Na niobate compounds

Measurements of mechanoelastic properties as a function of the temperature of (LixNa1−x)NbO3 (x⩽0.12) ceramics in the paraelectric phase show a sequence of anomalies in the complex Young’s modulus Y*. These anomalies are associated with structural phase transitions of the ferro–ferroelastic and ferro–paraelastic type, where strong elastic softening due to the order-parameter–spontaneous-strain coupling of linear quadratic character takes place. The existence of NbO6 octahedra tilts and the lack of significant volume change in the phase transition temperatures of the studied compositions allows considering the order parameters in the same way as that invoked in the case of the strontium titanate.

Micromechanical Studies of Mitotic Chromosomes

We review micromechanical experiments studying mechanoelastic properties of mitotic chromosomes. We discuss the history of this field, starting from the classic in vivo experiments of Nicklas (1983). We then focus on experiments where chromosomes were extracted from prometaphase cells and then studied by micromanipulation and microfluidic biochemical techniques. These experiments reveal that chromosomes have a well-behaved elastic response over a fivefold range of stretching, with an elastic modulus similar to that of a loosely tethered polymer network. Perturbation by microfluidic "spraying" of various ions reveals that the mitotic chromosome can be rapidly and reversibly decondensed or overcondensed, i.e., that the native state is not maximally compacted. We compare our results for chromosomes from cells to results of experiments by Houchmandzadeh and Dimitrov (1999) on chromatids reconstituted using Xenopus egg extracts. Remarkably, while the stretching elastic response of reconstituted chromosomes is similar to that observed for chromosomes from cells, reconstituted chromosomes are far more easily bent. This result suggests that reconstituted chromatids have a large-scale structure that is quite different from chromosomes in somatic cells. Finally, we discuss microspraying experiments of DNA-cutting enzymes, which reveal that the element that gives mitotic chromosomes their mechanical integrity is DNA itself. These experiments indicate that chromatin-condensing proteins are not organized into a mechanically contiguous "scaffold," but instead that the mitotic chromosome is best thought of as a cross-linked network of chromatin. Preliminary results from restriction enzyme digestion experiments indicate a spacing between chromatin "cross-links" of roughly 15 kb, a size similar to that inferred from classical chromatin loop isolation studies. These results suggest a general strategy for the use of micromanipulation methods for the study of chromosome structure.

Dielectric and mechanoelastic relaxations due to point defects inlayered bismuth titanate ceramics

Complex permittivity and Young's modulus provide relevant information on therole of point defects in the dielectric and mechano-elastic properties offerroelectric materials. Low-frequency measurements as a function of thetemperature performed on Bi4Ti3O12 (BIT) have shown that point anddipole defects are frozen close to domain walls. Low-temperature dipole defectrelaxation processes take place with characteristic times (τ0) of theorder of 10-11 s and 10-12 s and activation energies (Ea) of0.70 eV and 0.65 eV for dielectric and mechano-elastic relaxations,respectively. At higher temperatures new dielectric relaxation peaks appearthat can be attributed to jumps of de-iced oxygen vacancies(τ0≅10-11 s, Ea = 1.08 eV, T≅300 °C) and to vacancymigration (τ0≅10-15 s, Ea = 1.90 eV, T≅450 °C).Elastic relaxation peaks are also present close to 300 °C whoseactivation energy (1.50 eV) and characteristic time (10-15 s) suggest avacancy migration process. Close to 500 °C with Ea = 2.30 eV andτ0≅10-17 s another relaxation peak, which should correspond todomain wall viscous motion near the phase transition temperature, isobserved. The Young's modulus has a smooth step at T≅300 °C thatwe attribute to a change in the mobility of oxygen vacancies with respect tothe domain walls. Below 300 °C the vacancies are frozen in the domainwalls and they are de-iced and distributed throughout the material attemperatures above 300 °C. The experimental results show that thematerial is softer when the vacancies are linked to domain walls than whenthey are distributed throughout the material. The diffusion of vacancies backto the domain wall traps at room temperature takes a long time (days).