This article investigates the material behavior within multiple-component systems. Specifically, a structural concrete element strengthened to flexure with externally-bonded fiber-reinforced polymer (FRP) material is considered. Enhancements of mechanical performances of the composite structural element resulting from synergies in the framework of the multiple-component system are studied. The research work comprises the determination of the constitutive relations for the materials considered separately as well as the investigation of materials’ response within a complex system such as the composite structural element. The definition of the material models involves a calibration of the model constants based on characterization tests. The constitutive relations are integrated into the finite element model to study the material behavior within the multiple-component system. Results obtained by finite element analysis are compared with experimental results from the literature. The finite element analysis provides valuable information about the evolution of some internal variables, such as mechanical damage accumulation. The material synergies find expression in the load-carrying capacity enhancement and the delay in the damage accumulation in concrete. This article investigates the structural response of materials considered either separately or as constituencies of a multiple-component composite system. The prediction of the behavior of a complex system is not always a straightforward task and requires the implementation of sophisticated techniques.For the identification of multiple-component systems, one of the available approaches is the study of the mechanical wave propagation. The velocities of the compressional and shear waves depend on the elastic moduli of the continuum. Therefore, the estimation of these velocities is a basis of evaluation and testing of engineered materials  . SpecifiFor soft elastic materials, the study of wave propagation also has a number of practical applications. Among others, these are non-destructive defect detection ,6 and acGiven the complexity of the problem, reliable characterization procedures are necessary for the definition of the material models and the constitutive laws. The behavior of the multiple-component system can be characterized by a remarkable synergy of the constituent materials. This hypothesis should be verified for different cases. On the one hand, the material synergies enhance the performances of an FRP-strengthened reinforced concrete beam in terms of increased load-carrying capacity and stiffness. One the other hand, negative effects due to material interaction might be also present in some cases. A well-known example is the so-called premature failure of FRP-strengthened structural elements ,23,24,25Adding external FRP reinforcement  leads to an increase in the load-carrying capacity. The flexural performance enhancement of concrete/reinforced concrete beams strengthened with externally bonded FRP plates pioneered by , has beeThe complex mechanical behavior of a composite structural element, specifically, a reinforced concrete beam strengthened to flexure with adhesively-bonded composite material, is the research object of this study. Assuming that the behavior of the composite structural element essentially depends on failure mechanisms in concrete, the investigation focuses on the strain-softening concrete response against a background defined by the other materials’  responses.Finite element simulation can provide valuable insight into the evolution of some internal variables , which are difficult to measure experimentally. Material models, needed as an input for the finite element analysis, are generally defined based on experimental data. Model constants are identified in numerical procedures designed to search for the set of constants providing the best fit with experimental data. The following materials characterization tests are required to build a numerical model of an FRP-strengthened structural element: (a) compression tests on concrete cylindrical specimens; (b) tension by flexure tests on concrete prismatic specimens; (c) tension tests on steel specimens ; (d) tension tests on specimens made of the adhesive; (e) tension tests on specimens of the composite material plate. The identification of the response of the composite material/concrete interface requires additional tests, such as the single-lap shear test ,40,41.Complex systems may include “unknown” parameters . In the A numerical model is built to predict the complex behavior of the composite structural element. The model assumes experimentally identified constitutive laws to simulate constituent materials. The model is verified through a comparison of numerical results with experimental data. For an illustrative example, the experimentally-obtained data on the evolution of the mid-span deflection is compared with predictions on the evolution of the same parameter obtained by finite element analysis. In this context, the finite element analysis sheds light on the interaction between different components of the system . A constitutive relation defined based on damage mechanics fundamentals is chosen to model the behavior of concrete and to investigate the synergy effect improving structural performance. This approach provides an insight into the evolution of some internal (or non-observable) variables, such as the damage accumulated in the representative volume element. On the basis of the distribution of this variable obtained (for example) by finite element analysis, an accurate assessment of the resources remaining in the material subjected to a specified loading history can be assessed.The behavior of materials is defined through the stress-strain relationship: In Equation (1), The formulation of the employed material models, as well as the identification tests used as a basis for their definition, are discussed in this section.The FRP materials can be used as external reinforcement  or as internal reinforcement . It is known from experiments that the mechanical response of the FRP materials is linear and elastic until failure. However, there might be some nuances in the modeling of the FRP material’s behavior in the function of the material symmetries. For example, the unidirectional FRP sheets should be modeled by employing transverse isotropy. Thus, for a material in which the y–z plane is a plane of symmetry .The components of the compliance matrix are identified by performing tension tests on specimens made of composite material. Loading in different material directions is simulated by varying the angle between the applied load direction and the orientation of the fiber. Two approaches are possible to model the inelastic concrete response through the assessment of the accumulated damage. The elasticity tensor can be directly related to the current damage state, or the compliance matrix can be considered as a variable characterizing of the state of damage .Within the first of the above-mentioned approaches, the inelastic mechanical response of concrete is modeled based on the stress-strain relationship of a linear, elastic material:variable ,48:(6)σiE and tD) and compression (cD) is defined as follows [In Equations (5) and (6),  follows : (7)Dt=1As can be seen, the evolution of either of the components of the damage variable is governed by the so-called equivalent strain:The equivalent strain retains only the positive components of the principal strains This feature of the model is in line with the phenomenological observation that cracking in concrete takes place in regions where tensile strains (stresses) are present. From a physical standpoint, the equivalent strain is related to the crack opening mode I . In otheThe behavior of concrete in compression was identified by testing standard cylindrical specimens (80 mm in radius and 320 mm in height) whereas the behavior of concrete in tension—by testing prismatic concrete specimens .cA, and cB are needed for the evaluation of the compression component of the damage variable (see Equation (8) and tA and tB are employed to define the evolution of the tensile component of the damage variable (Equation (7) and The identification tests are used to characterize the concrete response not only by determining compression strength, tensile strength, and maximum strain, but also to identify the model constants in Equations (7) and (8). These constants are identified through the curve fitting procedure. To this end, finite element models designed to simulate identification tests, specifically compression tests on cylindrical concrete specimens and tension by flexure tests on prismatic concrete specimens a, are buSteel response is characterized by an initial elastic phase as long as stress remains within the elastic domain. Upon reaching the yield stress, plastic flow is initiated. For common grades of steel reinforcement, it is followed by a hardening phase. A standard model based on the plasticity theory fundamentals is used ,52. In tThe strain tensor increment can be obtained as a sum of elastic and plastic increments:The yield criterion is a scalar function of the stress  and internal  variables .Q is the plastic potential, and Equation (13) defines the yield surface in the considered stress space. The evolution of the plastic strain is determined by the flow rule:DX pointing to the origin of the yield surface in the stress space; see In Equation (15), The material constitutive relations, formulated based on characterization tests, are implemented into the numerical model of a multiple-component system. In the present study, this is a reinforced concrete beam strengthened to flexure by adhesively bonded composite material. The behavior of the multiple-component system is also studied experimentally. The model reproduces the geometry and the reinforcement arrangement  of the test specimen. It is validated based on a comparison between numerical and experimental results on the evolution of the mid-span deflection.Taking into account the existing symmetries of the experimentally tested specimens, a quarter-model space is considered. Appropriate symmetry boundary conditions are applied to nodes located in the symmetry planes. In the finite element (FE) model, boundary conditions are applied to lines . Nodes attached to the line of the  contact between the roller support and the beam, are constrained to move in the global y-direction. One of these nodes is also constrained in global x-direction. Forces in the global y-direction are applied to nodes attached to lines defined with respect to the application point of the concentrated load . The sum of the magnitudes of these forces equals the current value of the applied load.A perfect bond is assumed for all the interfaces (steel/concrete and concrete/CFRP).In the 3-D FE model, the concrete, the steel bars, steel stirrups, and the adhesive layer, are meshed with Solid 95 . Shell 186  is used to model the external CFRP reinforcement. The simulation of damage accumulation should take into account the effect of localization . A possible option is to remesh damaged regions with a finer mesh. With the implementation of higher-order elements containing mid-side nodes, the need for mesh refinement during analysis can be avoided. On the other hand, higher-order finite elements are a favorable option if the solution implies a refinement of the initial finite element mesh in specified regions. Additionally, the use of finite elements with mid-side nodes is a prerequisite for more accurate analysis results because of the improved shape function.With the hypothesis that the global response of the composite structural element is influenced mainly by the failure behavior of concrete, a finer mesh is generated for concrete. Additionally, the finite element size should comply with the requirements of the representative volume element size derived based on the energy balance in the framework of the continuum damage mechanics . The sizThe quasi-static loading is modeled by incrementally increasing the applied load. For each step of the applied load, a nonlinear static solution is performed. The damage-based constitutive relation for concrete is applied in the post-processing phase of each step. The damage variable is calculated based on the stress and strain distributions obtained in the finite element solution for the current value of the applied load. Before the next step of the solution, the material properties of the finite elements affected by damage are modified. Moreover, the finite elements in which the critical damage is reached are deactivated. In the next step, they don’t contribute to the overall rigidity. Thus, with the simulation of the propagating cracks, the finite element model becomes eventually insufficiently constrained. It is no more possible to perform nonlinear static analysis, and the numerical routine stops.Finite element simulations reproduce force-controlled tests. From an experimental standpoint, displacement-controlled tests generally provide more accurate and more reliable results. However, the focus here is on the accuracy of the results obtained by finite element simulation, assessed through a comparison with the experimental data.FRPt = 0.2 mm), the cross-section of the FRP plate would be:n is the number of FRP layers and FRPb stands for the thickness of the FRP plate. This cross-section can be further used in the design of the FRP-strengthened concrete element. In the present study, finite elements with the corresponding number of layers and material properties model the CFRP reinforcement.By assuming a specified value for the thickness of the FRP fabric f=, “f corresponds to a better fit with the experimental data. The comparison between experimental and numerical results shows that for the beam strengthened with three layers of CFRP, the value of f is 75%, and for the beam, strengthened with five layers of CFRP—93%. A higher value of The good agreement between experimental data and FE results  shows thThe analysis based on the effective moments of inertia is discussed here with the only purpose to provide an intuitive classification of the different phases in the response of a composite structural element subjected to a quasi-static loading. The analysis of both experimental and numerical results  leads tomax,nF = 0.9 × max,expF where max,nF is the failure load predicted by the numerical simulation and max,expF is the failure load obtained in the experiment. In the numerical analyses, the failure load and the maximum displacement are less than those obtained in the experimental study. This underestimation is referred to as a “premature numerical failure.” It can be attributed to excessive distortions in the initially generated finite element mesh. One possible solution to this problem could be the generation of new finite element mesh based on the current position of nodes, choosing one of the steps preceding the step at which the routine stops. The development of such an algorithm is the subject of ongoing research. Possibly, after testing, it will be possibly integrated into the analyses of structures’ responses within the large displacement domain. On the other hand, a modification of the solution options (number of steps and substeps) might increase the accuracy of the predictive results in the last phase of the loading history. A detailed and in-depth explanation of the premature, numerical failure also requires the extension of the database available. A conclusion made based on a large number of comparisons between numerical and experimental results would be more relevant.As can be seen in F1 < F2 < F3), finite elements of a particular degree of mechanical damage are not displayed. Damage is initiated in the vicinity of the mid-span of the strengthened beam and propagates towards the supports.The strategy chosen to simulate the strain-softening concrete response, see Equations (6)–(8), involves the calculation of damage variable for each increment of the driving force parameter and for each finite element used to model concrete. Technically, the calculated damage variables are stored in predefined arrays. This algorithm makes possible the simulation of the initiation and propagation of the damage in an initially undamaged, homogeneous, and isotropic medium. In With the increase of the applied load, mechanical damage accumulates, and the damaged zone propagates in concrete. When the damage variable, calculated for a given finite element, reaches a predefined critical value, the procedure deactivates this finite element in the next step of the incremental solution. As a result of the application of this algorithm, zones of zero rigidity nucleate and propagate in concrete. In the finite element simulation, the behavior of the flexural steel reinforcement and the externally bonded CFRP reinforcement intersecting a new-formed zone of zero rigidity  is analogous to their responses within an experimental study. Strains in CFRP and steel, within the regions of the intersection with cracks, are higher to resist the tensile force.The finite element analysis sheds light on the debonding failure mechanism. Analysis of previous  research leads to a classification of the debonding failure modes into two types . DebondiThe mechanical behavior of structural materials considered as elements of a multiple-component system has been investigated. Material models have been defined based on empirical data obtained in material characterization tests. A finite element model has been built to shed light on the unknowns related to the composite action: synergies in the materials’ response and material failure behavior within the structural element. The numerical model has been validated through a comparison with experimental data. The comparisons showed that the numerical model predicts with an adequate accuracy the experimentally-obtained evolution of the observable variable. The verification results demonstrate the capability of the numerical model to reproduce accurately the strain-softening concrete response taking into account the behavior of the other materials . The discussion focuses on concrete behavior since it is estimated that the global failure of the studied structural element results from a local failure in concrete.A specific feature of the proposed approach is the possibility to track the evolution of the damage accumulation in concrete, which is directly related to the initiation and propagation of cracks on the macroscopic scale. Based on this monitoring, the load-carrying capacity and stiffness of the composite structural element remaining after a specified period of exploitation can be rationally estimated.In the reported study, the enhancement of the mechanical performances of the composite structure demonstrates the synergy of the constituent materials. The external adhesively bonded CFRP reinforcement is a factor that enhances the load-carrying capacity of the strengthened beams. Also, it results in the delay in the failure mechanisms taking place in concrete.


In this study, the effects of shrinkage reduction agent (SRA) content and filler type on the deformability characteristics of unsaturated polyester (UP) resin-based polymer concrete were experimentally investigated. Specifically, the setting shrinkage, thermal expansion, maximum compressive strain and the modulus of elasticity of UP polymer concrete were all analyzed. Setting shrinkage was found to be influenced by the UP resin, the SRA and filler. The thermal expansion, maximum compressive strain and modulus of elasticity were also affected by the aggregate. The effect of SRA content on deformability was found to be greater than that of the filler type. To put UP polymer concrete to efficient use, it is essential to secure proper deformability according to the intended purpose. At that time, it is desirable that the deformation characteristics resulting from the SRA content and filler type sufficiently reflect when the mix proportion is determined. The effects of filler type on the deformability of UP polymer concrete are such that: A uniform dispersion of filler particles impacts the setting shrinkage; the thermal expansion is influenced by the filler’s various thermal expansion properties; the compressive strain is related to the nature of the small spherical particles that tend to fill porosity, producing better packing of the aggregate materials; and the modulus of elasticity is influenced by the density, which is related to the strength of the filler. However, additional in-depth studies are required on all of these elements. Polymer concrete (PC) is a composite material produced with polymeric binders instead of the cementitious binders found in ordinary Portland cement concrete . Hence, PC is employed in a wide range of civil and structural applications, such as bridge decking, pavement overlays, hazardous waste containers, sewer pipes and decorative construction panels . PC is tVarious polymer binders have been employed with aggregates to produce PC, such as epoxy (EP), unsaturated polyester (UP) and vinyl ester (VE) resins, as well as methyl methacrylate monomer (MMA). Among these binders, the most common polymeric binder for PC is UP resin ,8. UP reDespite these advantages, UP resin-based polymer concrete (UP polymer concrete) shows substantial shrinkage during the curing process, as compared to what is seen with ordinary Portland cement concrete , resultiThus, UP polymer concrete is not desirable in terms of dimensional stability. In order to reduce the setting shrinkage, a shrinkage reduction agent (SRA) can be incorporated as an additive when the PC is produced. In particular, the addition of fillers improves the dimensional stability of the PC and minimizes the temperature dependence of the mechanical properties . Based oPrevious studies on UP polymer concrete have focused on research associated with the material’s physical and mechanical properties ,15,16,17In particular, the novelty of the present study is that the setting shrinkage, thermal expansion and the stress–strain relationship were all investigated. There are currently no studies on the concentration of SRAs and fillers, which are important parameters for the deformation of UP polymer concrete. In the use of fillers, some research has employed fly ash ,6,7,16, Thus, this study identifies whether the uses of SRAs and certain fillers effectively improve the dimensional stability of UP polymer concrete.Two component systems  of UP binders were used to produce the PC. The UP resin employed in this study was of a pre-accelerated, orthophthalic type; the catalyst and its properties are shown in The initiator applied with the UP resin was methyl ethyl ketone peroxide , containing 45% dimethyl phthalate ; its properties are listed in About 6% to 12% volumetric shrinkage occurs when pure UP resin is cured without a filler. However, the amount of shrinkage is reduced when the material is employed to produce PC . Though When in a paste form, fillers fill voids between the surfaces of the aggregate. UP binders are liquid. A moisture content of less than 0.5%, no impurities, and low absorption are all required components of a filler .In this study, four types of fillers were employed. These fillers can be categorized into either crystalloid or blast and amorphous fillers. Ground calcium carbonate (GCC) and silica flour (SF) were used as crystalloid fillers. Fly ash (FA) and blast furnace slag (BFS) were employed as amorphous fillers. Density was tested via ASTM D 7481-18: Standard Test Methods for Determining Loose and Tapped Bulk Densities of Powders using a Graduated Cylinder . ParticlRiver sand, gravel, crushed sand and stone can all be used as aggregates. The requirements for aggregates include suitable particle shape, gradation, good soundness, high strength, a moisture content of less than 0.5%, low absorption of binders, etc. . For thiDetermining the effective mix proportions is essential to reduce the production cost of PC and ensure its structural performance . To determine the most effective mix proportions, the basic properties of the PC, such as workability, bleeding or segregation, and strength, should be evaluated. The mix proportions of PC depend upon many parameters, such as binder type, shape and gradation of the aggregate, temperature at the worksite, etc. Standards for mix design exist for cement concrete mixtures. However, such standards do not exist for PC. Thus, the mix proportions of PC are generally determined by trial and error. Hence, uniform mix proportions do not exist, and the mix proportions employed in every study are different. The mix proportions applied in the present study are shown in A forced mixing-type mixer was used to produce the specimens of PC because of the fast hardening reaction and high viscosity of the binder. In the mix order, after their weights were measured, the aggregates and fillers were dry-mixed using the mixer. Next, a binder was produced by mixing a resin, SRA, and the initiator, with prepared aggregates and fillers. At that time, the rotational speed of the mixer was set to 60 ± 5 rpm. To evenly compact the concrete specimens, hand rodding was carried out for each section. The total height of the cylindrical specimens (Φ 50 × 100 mm) was divided into three sections. The total height of the prismatic specimens (40 × 40 × 160 mm) was evenly divided into two sections. After that, each specimen was put onto a vibrating table (3000 rpm) for three minutes for additional compaction. The specimens were cured at a temperature of 25 ± 2 °C and a humidity of 60 ± 5%. The areas where the load would be applied to the cylindrical specimens were ground for planeness before the compressive stress–strain tests were carried out. The shape of the specimens used in this study is shown in Setting shrinkage tests were carried out based on ASTM C596-18: Standard Test Method for Drying Shrinkage of Mortar Containing Hydraulic Cement . The sizThermal expansion tests were carried out per ASTM C531–18: Standard Test Method for Linear Shrinkage and Coefficient of Thermal Expansion of Chemical Resistance Mortars, Grouts, Monolithic Surfacings, and Polymer Concretes . The sizThe modulus of elasticity was measured according to ASTM C 469M–14: Standard Test Method for Static Modulus of Elasticity and Poisson’s Ratio of Concrete in Compression . A wire UP polymer concrete offers many advantages, as mentioned above. However, the material also has certain disadvantages , as compared to conventional Portland cement concrete. Nevertheless, studies of UP polymer concrete are rarely carried out to resolve these disadvantages. Further research on the substantial setting shrinkage and thermal expansion of UP polymer concrete should be conducted because these significant strains influence the dimensional stability of a structure. If the setting shrinkage and thermal expansion are repeated as the temperature changes, this could cause interface cohesion failure or shear failure in the substrate, or even loss of broadcast aggregates in the polymer ,26. ThusAs with the substantial dry shrinkage that is seen in ordinary Portland cement concrete, the setting shrinkage of PC should be considered significant when the material is applied in real structures. Significant setting shrinkage can affect the accuracy of molding or placing, the design of the formwork, and adhesion to the reinforcement or substrate . The set−6 to 6849 × 10−6 strain units when the SRA content was 0%, was from 1799 × 10−6 to 2445 × 10−6 strain units when the SRA content was 10%, and it was from 1020 × 10−6 to 1560 × 10−6 strain units when the SRA content was 15%. It was identified that the setting shrinkage tended to decrease when the SRA content increased. These test results are similar to what was found by Mani et al. [According to the test results of the setting shrinkage by the SRA contents, the range of the maximum setting shrinkage was from 6333 × 10i et al. , which i−6 strain units when the SRA content was 0%, was 646 × 10−6 strain units when the SRA content was 10%, and finally it was 540 × 10−6 strain units when the SRA content was 15%. The differences in setting shrinkage by filler type were not apparent, but a smaller difference in setting shrinkage was identifiable because the particle size mode was smaller.According to the test results of the setting shrinkage by filler types, the amount of the setting shrinkage of FA was the smallest, and the amount of the setting shrinkage of SF was the largest. The changes of the setting shrinkage were 516 × 10−6 to 1200 × 10−6 strain units [−6 to 6000 × 10−6 strain units [Conversely, according to the previous study, although the dry shrinkage of ordinary Portland cement mortar and concrete depends upon the aggregate/cement ratio, it ranged from approximately 200 × 10in units . The setThe coefficient of thermal expansion is defined as the change per unit length when the temperature changes. The thermal shrinkage strain is determined by considering the magnitude of the temperature drop of the concrete and the linear coefficient of thermal expansion at the same time . The coeThe thermal strain of PC is caused by two parameters: the internal temperature caused by the polymerization reaction of the polymer binder and the external temperature of the atmosphere . If the −6 to 19.5 × 10−6 °C−1 at an SRA content of 0%, from 13.1 × 10−6 to 16.8 × 10−6 °C−1 at the SRA content of 10%, and from 14.2 × 10−6 to 16.6 × 10−6 °C−1 when there was an SRA content of 15%. It shows that the coefficient of thermal expansion is decreased when the SRA contents increased. This result shows that the coefficient of thermal expansion also changes depending on the composition and quantity of the SRA added to the resin.The test results determined in this study regarding the coefficient of the thermal expansion of the UP polymer concrete are shown in −6 °C−1 when the SRA content was 0%, 3.7 × 10−6 when the SRA content was 10% and finally 2.4 × 10−6 °C−1 when the SRA content was 15%. This result shows that the change of the coefficient of the thermal expansion by filler types was decreased when these SRA contents were increased. The cause of these differences by filler type is closely related to the thermal expansion properties of the chemical composition of the filler employed.The change in the coefficient of thermal expansion by filler type indicates that the smallest thermal expansion occurred when FA was employed, and the largest when SF was used. The differences of the coefficient of the thermal expansion were 8.0 × 10−6 °C−1 for flexible PC and 13.9 × 10−6 °C−1 for rigid PC [−6 °C−1 [Previous studies related to the coefficient of thermal expansion of PC found 23.9 × 10rigid PC . These v[−6 °C−1 .−6 °C−1 is usually employed for bridge deck overlays. This means that the coefficient of thermal expansion of this PC for the bridge deck overlay was approximately twice as large as that of Portland cement concrete [The stiffness of the polyester–styrene matrix depends upon the chemical composition of the resin. Hence, the more flexible resin with a coefficient of thermal expansion of approximately 23.4 × 10concrete ,33.−6 °C−1; mortar, 12 × 10−6 °C−1; and concrete, ranging from 6 × 10−6 to 12 × 10−6 °C−1 [−6 and 13 × 10−6 °C−1 [The (approximate) coefficients of linear thermal expansion of cementitious mixtures were: cement paste, 18 × 100−6 °C−1 . Also, t0−6 °C−1 . These lStress–strain curves are the basis for determining the modulus of elasticity. Since concrete is an incomplete elastic material, there are limitations to determining the elastic modulus with this curve. Therefore, the secant modulus of elasticity, also known as the chord modulus, is applied to determine the elastic modulus when the linear elastic section of a material is not clear, such as with concrete . The mod−6 to 5558 × 10−6 at 0%, 3765 × 10−6 to 4798 × 10−6 at 10% and 3595 × 10−6 to 4004 × 10−6 at 15%. It shows that the compressive strain decreased with increasing SRA content. Here, the compressive strain decreased when the size of the spherical particles employed in the UP polymer concrete was decreased because the small size of the particles contributed to a better packing density and reduced porosity [The maximum compressive strain is a critical parameter when materials are being selected for a structure’s design. In the present research, the maximum compressive strain was determined based on It was determined that the maximum compressive strain of UP polymer concrete was approximately two to three times higher than that of Portland cement concrete (about 0.002), with the stress at approximately 30 MPa; that of cement paste (about 0.0027) was about 34 MPa . ChandraAlso, Many studies have investigated the compressive strength of PC. For example, according to the work of Chandra et al. , the comThe modulus of elasticity is an important parameter for measuring deformability, not only in PC, but also in other structural materials. The PC employed for overlays of concrete pavement should have a low modulus of elasticity to reduce the shear stresses caused by temperature changes that occur between newly applied overlays and existing concrete substrate . However3 to 30.6 × 103 at 0% of SRA, from 25.8 × 103 to 29.5 × 103 at 10% of SRA and from 22.0 × 103 to 28.9 × 103 at 15% of SRA. It showed that the modulus of elasticity was decreased when the SRA content was increased. This shows that the compressive strengths and the moduli of elasticity were influenced by the applied resin (a binder of UP polymer concrete) and the SRA contents. In terms of the change in the elastic modulus by the filler types, the elastic modulus was the highest when SF was used, and the lowest elastic modulus was when FA was employed. The differences of elastic moduli were 2.5 × 103 at SRA of 0%, 3.7 × 103 at SRA of 10% and 6.9 × 103 MPa at SRA of 15%. As the SRA content increases, the modulus of elasticity tended to increase. In this study, the cause of the difference in elastic modulus by filler type was found to potentially be related to the mode of particle size. However, more research is needed to identify the exact reasons.The elastic moduli obtained through this study are shown in 3 MPa for polyester–styrene polymer concrete and 25.1 × 103 MPa for ordinary Portland cement concrete. Chandra et al. [3 to 35 × 103 MPa, and the modulus of Portland cement concrete ranged from 20 × 103 to 40 × 103 MPa. Compared to these previous results, it was determined in this research that the elastic modulus of the UP-polymer concrete was similar to the elastic modulus values presented in previous studies. However, the elastic modulus of PC is lower than the elastic modulus of Portland cement concrete, because the elastic modulus of polymer is lower than the elastic modulus of the aggregates. The elastic modulus of PC depends on the elastic modulus of the polymer [There have been many studies on the elastic moduli of polymer and ordinary Portland cement concrete. Among these previous studies, O’Connor  found th polymer .The relationship between the compressive strength and modulus of elasticity of all the obtained data regarding UP polymer concrete is shown in cE is modulus of elasticity of the UP polymer concrete in × 103 MPa and cf’ is the compressive strength in MPa.Where, 1)The setting shrinkage was generally stabilized from 12 to 24 h after casting was completed. As the SRA content was increased, the setting shrinkage tended to decrease significantly, and there was no significant difference in setting shrinkage based on filler type.2)Thermal expansion tended to decrease as the amount of SRA was increased, and the difference in thermal expansion was relatively substantial. Changes in filler produced significant differences depending upon the type used.3)The maximum compressive strain also tended to decrease as the SRA content was increased, but the difference in compressive strain by the amount of SRA was not significant. There was no significant difference in the compressive strain based on changes in the filler type. As a result, it was determined that the effects of SRA content on setting and thermal shrinkage were not significant.4)The modulus of elasticity decreased as the SRA content increased. In addition, there were significant differences in the decrease of the moduli of elasticity as the SRA content increased. However, there was no significant difference in the modulus of elasticity by filler types. Also, in this case, the effect of SRA contents on the elastic modulus was greater than the effect of filler types on that.5)Setting shrinkage was affected by resin, SRA and filler. Additionally, the thermal expansion, maximum compressive strain and modulus of elasticity were affected not only by the resin, SRA, and filler, but also the aggregate. In this study, the effects of SRA content and filler type on setting shrinkage were investigated. As a result, the effect of SRA content on the deformability of UP polymer concrete was found to be more substantial than the effect of filler type.6)It is desirable to determine the mix proportions, considering both the SRA content and the deformation properties by filler type as determined in this study, in order to secure the proper deformability of UP polymer concrete when it is applied to repair endeavors, structural or nonstructural precast products, etc.7)The effects of filler type on deformability indicate that: setting shrinkage is influenced by evenly dispersed filler particles; the thermal expansion characteristics of fillers affect thermal expansion; small spherical particles tend to fill porosity, leading to better packing of the aggregate materials and influencing compressive strain; and the modulus of elasticity is influenced by density, which is related to the strength of the filler. Additional research is required to gather greater detail on these topics.This study experimentally examined the effects of SRA content and filler type on the deformability of UP polymer concrete in terms of aspects, such as setting shrinkage, thermal expansion, maximum compressive strain, modulus of elasticity, etc.


Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks both effective patient stratification strategies and therapeutic targets. Whilst elevated levels of the MET receptor tyrosine kinase are associated with TNBCs and predict poor clinical outcome, the functional role of MET in TNBC is still poorly understood. In this study, we utilise an established Met-dependent transgenic mouse model of TNBC, human cell lines and patient-derived xenografts to investigate the role of MET in TNBC tumorigenesis. We find that in TNBCs with mesenchymal signatures, MET participates in a compensatory interplay with FGFR1 to regulate tumour-initiating cells (TICs). We demonstrate a requirement for the scaffold protein FRS2 downstream from both Met and FGFR1 and find that dual inhibition of MET and FGFR1 signalling results in TIC depletion, hindering tumour progression. Importantly, basal breast cancers that display elevated MET and FGFR1 signatures are associated with poor relapse-free survival. Our results support a role for MET and FGFR1 as potential co-targets for anti-TIC therapies in TNBC. Clinically, patients with TNBC have fewer therapeutic options due to the lack of HER2 amplification and negative oestrogen and progesterone receptor  status. Patients with TNBC are at high risk of local and metastatic recurrence within the first 5 years post treatment and have poor overall survival2. The main challenge in treating TNBC is the paucity of patient stratification strategies capable of informing therapeutic decisions. Conventionally, TNBC is largely divided into the basal-like and claudin-low molecular subtypes of breast cancer4. While more recent efforts to further stratify patients have identified four to seven subtypes based on gene expression and mutational spectra, with the exception of a subtype enriched for the androgen receptor, the clinical relevance of each of these subtypes remains to be fully established8. Consequently, treatment for TNBC still typically involves combinations of anthracycline and taxane-based chemotherapies along with surgery and in some cases radiotherapy, with no clear consensus on new therapeutic regimens2.Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of aggressive breast cancers, accounting for ~15% of all invasive breast cancers12. TICs are capable of self-renewal and regenerate the heterogeneity of the original tumour following transplantation13, display resistance to conventional therapies16 and are postulated to seed local recurrence and distant metastases20. As a result, therapies that debulk tumours by indiscriminately eradicating highly proliferating cells, but do not target TICs, are now recognised as unlikely to cure patients22. Signalling pathways that promote TICs, as well as inhibitory compounds that target them, have therefore been the subject of intense research focus24. Highly tumourigenic and stem-like properties observed in TIC populations can be enhanced by the process of epithelial to mesenchymal transition (EMT)26, a cellular programme whereby epithelial cells decrease cell–cell junctions, lose apical–basal polarity, and display increased migratory and invasive capacities associated with mesenchymal cells. EMT is orchestrated by a restricted number of transcription factors namely those in the SNAI, TWIST, and ZEB families28. Indeed, breast TICs possess a mesenchymal gene expression signature supportive of mesenchymal and stem-like properties observed in breast cancer cells undergoing EMT such as elevated expression of ALDH and CD44 and low expression of CD2429. The level of expression of these markers is associated with poor clinical outcome in TNBC patients11.A growing body of evidence suggests that disease relapse in breast cancer is attributable to the survival of a subpopulation of tumour cells with stem-like properties, termed tumour-initiating cells (TICs) or cancer stem cells30. MET also regulates a variety of distinct biological processes characteristically exploited by tumours, including cell scattering, epithelial remodelling, cell proliferation and cell survival31. Elevated levels of MET protein are detected in 15–20% of all breast cancers and are associated with poor outcome across subtypes and within the TNBC subclass itself35. Despite these correlations, the functional role of MET in TNBC remains poorly understood. We have previously developed a murine model in which mammary gland expression of Met (MMTV-Metmt)34 and loss of the tumour suppressor gene Trp53  synergise to promote tumours displaying a spindloid, mesenchymal pathology associated with the molecular and pathological features of claudin-low breast cancer36. Cross-species gene expression analysis revealed that spindloid tumours were characterised by mesenchymal properties and high expression of EMT and stem cell gene signatures, reminiscent of the basal B molecular subtype of breast cancer37. In this murine model of basal breast cancer, the endogenous MET locus becomes amplified and Met kinase activity is required to sustain both the EMT phenotype and proliferation of spindloid tumour cells36.MET is a receptor tyrosine kinase (RTK) that, when bound by its ligand hepatocyte growth factor (HGF), coordinates a programme of invasive growth that broadly overlaps with the process of EMT, both in cancer and during the physiological process in development38. Amplification of the genomic locus of FGFR1 at chromosomal region 8p11-12 occurs in ∼10% of breast cancers39. While in ER + breast cancers, FGFR1 amplification is associated with poor prognosis40 and resistance to endocrine therapy39, the role of FGFR1 in TNBCs remains poorly understood.Fibroblast growth factor receptors (FGFRs) are a family of four RTKs (FGFR1-4) that are activated by fibroblast growth factors (FGFs) and regulate predominantly cell survival, proliferation and differentiation, and are implicated in many cancersHGF and FGFR1, and that co-expression predicts poor prognosis among basal breast cancer patients. These studies identify MET and FGFR1 as co-regulators of TIC properties in basal breast cancer and provide a new understanding of treatment modalities for highly mesenchymal TNBC tumours.In this study, we assay TIC properties to directly investigate the role of Met in tumour initiation and identify FGFR1 signalling as a key convergent pathway with Met for the maintenance of TICs. We find that simultaneous inhibition of Met and FGFR1 activity abrogates properties of stemness and reduces TICs, hindering tumour progression. Importantly, we show that both human TNBC cell lines and TNBC patient-derived xenografts (PDXs) with co-expression of MET and FGFR1 are highly sensitive to dual-MET-FGFR1 inhibition in TIC assays. Finally, we find that human TNBCs with mesenchymal characteristics are significantly enriched for Metmt;Trp53fl/+;Cre murine basal-like mammary tumour model are associated with increased mesenchymal status and tumourigenicity36. Unsupervised clustering of gene expression data from three independent MMTV-Metmt;Trp53fl/+;Cre-derived spindloid and non-spindloid tumours revealed that spindloid tumours displayed a gene expression profiling distinct from the non-spindloid tumours , and cell junction gene signatures were enhanced in non-spindloid tumours  were elevated in the spindloid tumours, while higher levels of cell junction markers  and differentiation genes (Gdpd2 and Ogn)29 are elevated in non-spindloid tumours  formed a higher number of and larger tumourspheres when compared to non-spindloid cell lines (A1221 and A1222), hence supporting the idea that spindloid cell lines possess higher proportions of TICs that form tumourspheres with greater proliferative capacity   were nearly three times more proliferative than cell lines derived from non-spindloid tumours (A1221 and A1222) Fig. . To funcity Fig. . Since l23) Fig. , providiMetmt;Trp53fl/+;Cre spindloid cells on both plastic and in soft agar are dependent on Met kinase activity36. To determine whether active Met signalling was also required to maintain TIC populations in these cells, tumourspheres derived from A1005 and A1129 cells were cultured in the presence of the small-molecule Met inhibitor Crizotinib for 5 days. Met inhibition significantly decreased but did not completely abrogate tumoursphere formation , basic fibroblast growth factor (bFGF) and insulin43. To test this, tumoursphere assays were performed with sequential removal of individual growth factors. Strikingly, in the absence of bFGF, treatment with Crizotinib abrogated tumoursphere formation of both A1005 and A1129 cell lines , showed a significant reduction in sphere proliferation when treated with both inhibitors when compared to either inhibitor alone  over multiple rounds of passage. Since spindloid tumourspheres fail to form in the presence of Met and FGFR inhibitors in combination, A1005 and A1129 tumourspheres were allowed to establish over 2 days before inhibitors were added for a further 3 days. On day 5, tumoursphere numbers in each condition were determined, tumourspheres were enzymatically and mechanically dissociated into single cells, re-plated, and the treatment with inhibitors was repeated. Strikingly, Met and FGFR inhibitor combination-treated tumourspheres exhibited a decrease in SFE by the tertiary passage that was not observed in any other conditions Fig. . Under tFGFR1 transcript to be selectively elevated in cells from spindloid tumours when compared to all other tumour types and normal mammary gland, while FGFR2 and FGFR3 were not modulated . The increase of FGFR1 expression in spindloid tumours was confirmed at the protein level  following treatment with Met or FGFR inhibitor alone , or in combination for 24 h. All three cell lines treated with inhibitor combinations displayed a gene expression signature distinct from that of cells treated with either inhibitor alone  and stemness  were confirmed independently by qPCR  on tumourspheres subjected to dual-MET-FGFR inhibition and found negative enrichment indicating loss of gene signatures associated with EMT, stemness, breast cancer progenitors, and Met signalling when compared to DMSO-treated control tumourspheres Fig. . ReducedPCR Fig. . Interesx46 Fig. . An incrx46 Fig. , with siPCR Fig. . In accoPCR Fig. , a signived Fig. . Notablyres Fig. . OverallTo investigate whether co-targeting Met and FGFR in vivo could suppress tumour-initiation, female athymic mice were injected with A1129 tumour cells and randomised into four groups, receiving either vehicle control, Crizotinib, the orally available FGFR inhibitor BGJ398, or both agents in combination by daily gavage Fig. . Mice re3 , we found that basal B cell lines exhibited the highest expression of FGFR1 when compared to luminal, HER2, and basal A cell lines . In contrast, FGFR2 and FGFR3 were not significantly correlated with any subgroup of breast cancer cell lines, whereas FGFR4 expression was elevated in HER2 and luminal cell lines  were cultured as tumourspheres in the presence of DMSO, Crizotinib, PD173074, or both inhibitors in combination. In addition to bFGF, a regular component of tumoursphere medium, tumourspheres were also cultured in the presence of HGF to ensure MET activation. Measurement of tumoursphere proliferation revealed that tumourspheres from all four basal B cell lines consistently exhibited a greater decrease in proliferation under conditions of MET and FGFR co-inhibition when compared to all other conditions, while in contrast, basal A tumourspheres varied in their sensitivity Fig. .+CD24−/low. Reflecting their strong overlap with mesenchymal/stem gene expression signatures, basal B cell lines typically have high percentages of CD44+CD24−/low cells23. To investigate whether the CD44+CD24−/low phenotype is regulated by MET or FGFR signalling, basal B cell lines Hs578t and BT-549 were cultured in the presence of HGF and/or bFGF and treated with Crizotinib and/or PD173074 for 3 days. In both cell lines, FGFR inhibition led to TIC depletion, which was rescued upon activation of MET by HGF. However, MET inhibition alone did not affect the CD44+CD24−/low population  are primary patient tumour fragments that are serially transplanted in immunocompromised mice that are considered to recapitulate properties of the original patient tumoursTo assess the role of MET and FGFR signalling in putative TICs derived from PDXs, the GCRC1915 PDX tumour was collected, digested into single cells and cultured as tumourspheres in media containing DMSO, Crizotinib, PD173074, or both inhibitors in combination. A reduction in SFE was observed in both Crizotinib and combination-treated tumourspheres, with the latter exhibiting a larger decrease Fig. . Serial HGF gene in the murine locus (NSG-hHGFki), ensuring that both FGFR and MET signalling pathways can be activated. Combination treatment with both Crizotinib and BGJ398 by oral gavage resulted in a moderate but significant reduction in tumour burden compared to all other treatment arms , TNBC patients from The Cancer Genome Atlas (TCGA)s41 Fig. . Accordient Fig. . DespiteFGFR1 and HGF, we assessed whether these two genes in combination could predict outcome in breast cancer patients. Analysis of publicly available breast cancer expression profiles revealed that high expression of both FGFR1 and HGF were associated with poor relapse-free survival (RFS) specifically in basal tumours  is a heterogeneous disease that lacks both effective patient stratification and therapeutic targets, and where strategies to target TICs are still poorly defined. Here, we identify a co-dependency between the MET and FGFR RTKs for self-renewal of TICs and tumourigenesis of mesenchymal TNBC. This was established by functional studies using transgenic mouse models of TNBC, human TNBC cell lines and PDXs from patients with TNBC. These multiple complementary preclinical and clinical models, together with quantitative biochemical data, and gene expression and TCGA data analyses, support a model whereby MET cooperates with FGFR to regulate TICs, and where dual inhibition of MET and FGFR1-FRS2 signalling results in TIC depletion, hindering tumour progression in TNBC.Increasing evidence supports that TICs can promote intratumoural heterogeneity given their capacity to give rise to multiple different cell types within a single tumour66, where MET acts as a functional marker of TICs, facilitating their survival under stress such as hypoxia, as well as conferring therapeutic resistance and metastatic potential68. This is consistent with our observation that MMTV-Metmt;Trp53fl/+;Cre mouse mammary tumours with a spindloid pathology are highly enriched in TIC populations that are dependent on Met signalling for survival, proliferation and self-renewal. FGFR signalling is essential for normal mammary gland development and promotes the growth of TNBC cell lines47. FGFR1-3 inhibition with AZD4547 has been reported to selectively target mammary stem cells and TIC populations in an ErbB2-overexpressing breast cancer mouse model69. However, while FGFR1 is an independent prognostic marker of survival in TNBC patients70, the contribution of FGFR1 signalling in TIC renewal in these tumours remains to established.A role for MET signalling in TIC biology has been documented in several cancers including glioblastoma, prostate, pancreatic and breast cancers74 and conversely, FGFR1-dependent resistance to MET inhibitors is observed in leukaemia, kidney and prostate cancers77. Resistance is typically associated with amplification of MET or FGFR1, supporting the ability of MET and FGFR to activate similar key signalling pathways required for tumour progression. Despite extensive research into the therapeutic applications of MET and FGFR inhibitors, studies exploring the potential benefit of targeting both receptors in TNBC are lacking.The ability of MET and FGFR1 signalling to compensate for one another in support of tumour growth has been identified in various human cancers. In FGFR1-amplified lung cancer cell lines, MET-amplification is a mechanism of resistance to FGFR-targeting drugs80. This unexpected crosstalk between these RTKs for FRS2 phosphorylation and TNBC TICs is further supported by in vivo studies where tumour initiation was inhibited only upon combination therapy with Met and FGFR inhibitors. Accumulating evidence highlights the strict requirement of ERK1/2 signalling in the maintenance of TIC properties83. As both Met and FGFR are robust activators of ERK1/2 signalling, it is improbable that there are unique pathways downstream of ERK1/2 that regulate TIC/stemness properties that cannot be activated by either MET or FGFR alone. Yet transcriptional changes in response to MET and FGFR inhibition are distinct, implying that alternate pathways can be activated and/or the amplitude of the signal may alter transcriptional responses. Whereas Met inhibition alone exerted a limited effect on FRS2 phosphorylation in tumourspheres, Crizotinib was sufficient to reduce FRS2 phosphorylation in tumours to a level similar to FGFR inhibitor.Here, we report that MET or FGFR-dependent TIC renewal in TNBC requires tyrosine phosphorylation of the downstream scaffold protein FRS2. FRS2 phosphorylation generates multiple binding motifs for tyrosine phosphatase Shp2 and adaptor protein Grb2, recruiting additional scaffold proteins such as GAB1, and is required for maintenance of ERK1/2 signalling+CD24−/low TIC populations, a process that can be prevented by HGF stimulation, further highlighting the role of MET activity in the maintenance of TICs. Notably, in these TNBC cells, tyrosine phosphorylation of FRS2 can occur via HGF-dependent activation of MET. Although FRS2 tyrosine phosphorylation has been identified in a MET-amplified gastric cancer cell line84, this is the first study to establish FRS2 as a common downstream signal in response to ligand activation of non-amplified MET. FRS2 contains a PTB domain for binding to RTKs, including FGFR, RET, ALK and the neurotrophin receptor86. MET can form complexes with multiple RTKs87 leading to crosstalk downstream from RTKs. We may envision that proximity of MET and FGFR complexes is enriched in highly mesenchymal and invasive cells, subsequently facilitating FRS2 phosphorylation downstream of MET.The specific sensitivity of mesenchymal TNBCs to MET and FGFR inhibitors reflects the dual expression of these RTKs. Although MET is elevated in most basal breast cancer cell lines, we found that both FGFR1 protein and mRNA levels are preferentially enriched in basal B cell lines. Consistent with MET and FGFR1 signalling crosstalk in TNBCs, following FGFR inhibition, basal B cell lines underwent reduction of CD44FGFR1 and HGF. Importantly, we found that co-expression of HGF and FGFR1 predicts poor relapse-free survival specifically among basal breast cancers, providing further support that FGFR1 and MET pathways contribute to the tumourigenicity of highly mesenchymal breast cancers. Accordingly, we found that combined inhibition of MET and FGFR in a basal B PDX resulted in a decrease in sphere-forming and CD44+CD24−/low populations, indicative of a loss of TICs and reduction in overall tumour burden. The finding that co-inhibition of MET and FGFR signalling reduces but does not completely abrogate sphere formation and tumour growth in human cell lines and PDXs may suggest the involvement of other compensatory pathways, such as EGFR, which has been associated with mesenchymal TNBCs88. We can also envision that MET and FGFR1 inhibition targets a specific subgroup of TICs. This is consistent with data where MET was specifically correlated with ALDH1A3 and CD133 in breast cancers66. In addition, phenotypically and functionally distinct TIC populations can exist within a single tumour. Several studies have also documented the spontaneous conversion between TIC and non-TIC states, demonstrating a robust capacity for plasticity89. These complexities have implications for TIC-targeting therapies and warrant further study. Taken together, our data support that MET and FGFR1-FRS2 pathways cooperate to promote a population of mesenchymal-like TICs in TNBCs and, consistent with other potential therapies targeting mesenchymal-like TIC populations91, the clinical benefits of inhibiting these receptors are likely maximised if paired with tumour de-bulking by a chemotherapeutic agent.We found that TIC/EMT-enriched breast cancers were elevated in gene signatures associated with both MET and FGFR1 signalling pathway activation. Consistent with basal B PDXs, the TIC/EMT high patient group was enriched for co-expression of HGF and FGFR1 co-expression in basal breast cancers offers a new strategy for stratifying patients, as well as potential anti-TIC therapeutic targets that warrant further investigation.In summary, we have established that MET and FGFR1 are co-expressed in highly mesenchymal TNBCs and that they co-regulate TICs therein through a compensatory interplay. We demonstrate for the first time to our knowledge, the ability of non-amplified MET to independently phosphorylate FRS2. Finally, the prognostic power of Metmt, Trp53fl/+;Cre, and MMTV-Metmt;Trp53fl/+;Cre mammary tumours as previously described36. Cells were cultured in DMEM (Gibco) supplemented with 5% serum, epidermal growth factor (5 ng/ml), insulin (5 μg/ml), bovine pituitary extract (35 μg/ml) and hydrocortisone (1 μg/ml). BT-20, HCC70, HCC1937, HCC1954, HCC1395, MDA-MB-468, MDA-MB-436, MDA-MB-157, MDA-MB-231, BT-549 and Hs578T were from ATCC and cultured according to ATCC recommendations. All cells were grown at 37 °C and 5% CO2.Primary mouse cell lines were established by dissociation of MMTV-53. Briefly, all human participants provided informed consent for this study, and tissue was collected at McGill University Health Center in accordance with the protocols approved by the research ethics board (SUR-99-780). All animal studies linked to this protocol were approved by the McGill University Animal Care Committee (2014–7514). The Biobank protocol (05-006) and the protocol to generate PDX from biobank tissues (14–168) were both approved by the Jewish General Hospital ethics committee.PDXs have been previously describedCommercial antibodies used include Met (R&D System), Claudin-1 (Thermo Fisher), FRS2 (Santa Cruz) and β-actin (Sigma). Antibodies against phospho-Met Y1234/1235), FGFR1, Akt, phospho-Akt (S473), ERK1/2, phospho-ERK1/2 (T202/Y204), phospho-Frs2 (Y196), phospho-Frs2 (Y436), β-tubulin and cleaved-Caspase-3 were from Cell Signaling. CD24-PE and CD44-FITC for flow cytometry were from BD Biosciences. Antibodies dilutions are provided in Supplementary Table /1235, FGFGFR1 and Frs2 in A1005 and A1129 cells were carried out using shRNAs cloned into pLKO.1 lentiviral vectors shRNAs (Sigma-Aldrich). The following clones were used for FGFR1: TRCN0000023295 (Sequence: CCGGCCTGGAGCATCATA ATGGATTCTCGAGAATCCATTATGATGCTCCAGGTTTTT) and Frs2: TRCN0000097281 (sequence: CCGGCCGACAGTCTTTAACTTTGATCTCGAGATCAAAGTTAAAGACTGTCGGTTTTTG) HEK293T cells were transfected using FuGENE HD (Promega) to produce lentivirus. Packaging vectors used were pRSV-Rev, pHCMV-VSVg and pMDLg/pRRE. Media containing viral particles was collected and passed through a 0.45-μm filter. Infected A1005 and A1129 cells with stable knockdown were selected under Puromycin (2 μg/ml) and the drug efflux inhibitor Cyclosporin A (2.5 μM).Knockdown of Single cells were seeded in six-well ultra-low attachments plates (Corning) in 2 ml serum-free DMEM/F12 supplemented with 1× B27, 10 μg/ml insulin (Gibco), 20 ng/ml EGF (BP Bioscience), 20 ng/ml bFGF (StemRD), 10 μg/ml heparin (StemCell Technologies) and 0.5 μg/ml hydrocortisone (Wisent). Tumoursphere number and size were determined after 5–7 days of culture using the software AxioVision (Carl Zeiss). To serially passage, tumourspheres were enzymatically and mechanically dissociated in 0.05% Trypsin-EDTA (Gibco), passed through a 25-G needle, and re-seeded as single cells. All experiments were performed in triplicate.The proliferation of tumourspheres was determined using the CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s instructions with some modifications. Briefly, single cells were seeded in 96-well ultra-low-attachment plates (Corning) and cultured for 5–7 days. Plates were collected on the first and last days of culture and centrifuged at 2000 rpm for 20 min to pellet the tumourspheres. Plates were then inverted to remove media, frozen at −80 °C for a minimum of 24 h, and thawed at room temperature when ready to quantify. The CyQUANT dye/lysis buffer solution was added to all wells, and each plate was analysed using a VarioSkan plate reader (Thermo Scientific). Proliferation was expressed as a ratio of fluorescence on the last day to the first day of culture. All experiments were performed in triplicate.23. Whole-cell lysates were resolved by SDS/PAGE and transferred to PVDF membranes. Membranes were blocked with Li-COR Blocking Buffer (Li-COR Biosciences) and probed with primary antibodies overnight at 4 °C. After TBS-Tween washes, membranes were incubated with infrared-conjugated (Li-COR Biosciences) or HRP-conjugated  secondary antibodies for 1 h at room temperature for signal detection by Odyssey IR Imaging System (Li-COR Biosciences) or enhanced chemiluminescence (Amersham Biosciences) respectively. All blots derive from the same experiment and were processed in parallel.Tumour-derived cell lines were lysed in 1% Triton lysis buffer , and snap-frozen mammary tumours were lysed as previously describedHprt and Gapdh genes for the FGFR transcript quantification and RNA-Seq gene expression validation. For RNA-Seq gene expression validation, the data presented represent the merge of three different cell lines for the DMSO, PD173074 and Crizotinib-treated tumourspheres and two cell lines for the combination-treated tumourspheres.RNA was isolated using the RNAeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesised using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. qPCR reactions were performed using SYBR Green I Master on a LightCycler480 (Roche). Primers sequences (Integrated DNA Technologies) are provided in Supplementary Table 36, and sections were cut at 4 μm. Sections were deparaffinized xylene and re-hydrated in ethanol, followed by antigen retrieval in Tris-EDTA at boiling temperature. Slides were cooled and blocked with Power Block (BioGenex) and incubated overnight with primary antibody at 4 °C. Rinse slides in distilled water then incubated in 3% H2O2 for 30 min. SignalStain Boost  was used as the secondary antibody, and the SignalStain DAB substrate kit  was used to detect signal prior to counterstaining with Harris’ hematoxylin. Finally, slides were dehydrated, mounted, and scanned using Aperio-XT slide scanner (Aperio).Tumour tissues were formalin-fixed and paraffin-embedded in a previous study92 and Lima. After unpaired analysis, only genes with False Discovery Rate (FDR) < 0.05, and log2 fold change ≥1.5 were considered. Hierarchical clustering of differentially expressed genes was used to represent the results (R package ggplot2). The gene set enrichment analysis was performed using the Molecular Signature-DB 7.1 and the GSEA software version 4.1093 with the complete set of normalised input values, using the Hallmark, canonical pathway gene sets , and Gene ontology gene sets . Heatmaps were constructed using Partek software. For all statistical analysis, differences were considered statistically significant if the adjusted P values calculated by Student’s t test with Bonferroni correction were <0.05. RNA-Sequencing of the tumoursphere (GSE162272). The microarray of the MMTV-Metmt;Trp53fl/+;Cre tumours (GSE41601) were previously published36. RNA-Sequencing of the PDXs (GSE142767) were previously published94.A1005, A1129 and A1471 tumourspheres treated with inhibitors as indicated were collected, and the total RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. High RNA quality was verified using the Bioanalyzer RNA 6000 Nano assay (Agilent). The samples were sequenced using a NextSeq500 sequencer (Illumina) by the Genomics Core Facility of the Institute for Research in Immunology and Cancer, Université de Montréal. Reads were mapped to mouse genome version mm10 using Spliced Transcripts Alignment to a Reference (STAR). Reads counts were normalised using mean-centred and log-transformed. Differentially expressed genes among groups were identified using the R packages DESeq2http://bioinf.wehi.edu.au/software/elda/.Mice were housed in accordance with McGill University Animal Ethics Committee guidelines. A1129 and A1221 cells were resuspended in sterile PBS and injected at decreasing numbers  into the 4th mammary fat pad of female athymic mice aged between 6 and 8 weeks (Taconic Farms). The study was conducted over 12.5 weeks, during which mice were palpated every 2–3 days monitor for tumour outgrowth. TIC frequency was calculated using the Extreme Limiting Dilution Analysis (ELDA) online software available at n = 10), with each group receiving vehicle control, Crizotinib , BGJ398 , or both drugs in combination. For the tumour progression study, mice were injected with MMTV-Metmt;Trp53fl/+;Cre cells (A1129) as above. Once tumours reached 50–100 mm3, mice were randomised into four treatment groups (n = 10) and gavaged daily as above. Tumour diameters were measured every 2 days with callipers, and tumour volume (mm3) was calculated by the following formula: (length × 4.18879)/2 × (width/2)2. PDX GCRC1915 tumours were transplanted as fragments into the 4th mammary fat pad of female NSG mice with HGFtm1.1(HGF)Aveo “humanised” knock-in allele (NSG-hHGFki) (https://www.jax.org/strain/014553). Once tumours reached 50–100 mm3, mice were randomised into four treatment groups (n = 5) and gavaged daily as above. Tumour growth was monitored and volumes calculated as above.For the tumour-initiation study, 100,000 A1129 cells were resuspended in sterile PBS and injected into the 4th mammary fat pad of female athymic mice . On the day of injection, mice were randomised into four groups  was added to each sample. Multi-colour cell sorting was performed on a FACS CantoII (BD Biosciences) and data analysis was performed using FlowJo (Tree Star Inc.). An example of gating is presented in Supplementary Fig. 2O2 for 30 min, incubated with secondary antibody for 45 min at room temperature, and counterstained with 0.5 ng/ml DAPI. Tyramide signal amplification (Thermo Fisher) was used according to the manufacturer’s instructions. Finally, slides were imaged with an LSM800 confocal microscope and analysed using Zen software (Zeiss).Tumour tissues were formalin-fixed and paraffin-embedded, and sections were cut at 4 μm. Sections were deparaffinized in xylene and re-hydrated in ethanol, followed by citrate buffer (pH 6) at boiling temperature. Slides were cooled and blocked with 2% BSA and incubated overnight with primary antibody at 4 °C. Slides were rinsed in distilled water, treated with 3% HTumours were minced using razor blades and transferred to a 50-ml conical tube containing tissue digestion media (DMEM + 2.5 FBS with collagenase IV). Tubes were placed in rotating ovens and to dissociate over 2–3 h at 37 °C. The suspension was centrifuged to pellet the epithelial and fibroblast cell content. The pellet was then resuspended in warm PBS, and the epithelial organoids/cells were allowed to gravity sediment for ~5 min. The stromal cell-rich supernatant was removed, and the epithelial content washed in warm PBS then pelleted. The pellet was then trypsinized in 0.05% Trypsin-EDTA and passed through a 40-μm cell strainer to generate a single-cell suspension. Murine stromal cells were further removed using a Mouse Cell Depletion Kit (Miltenyi) according to the manufacturer’s instructions.95 was used to analyse data from TNBC patients of the TCGA dataset41. In order to enrich for patients with high expression of TIC or EMT gene signatures, ssGSEA was applied using the indicated published gene signatures. Welch two-sample t test was used to compare the expression of single genes, as well as MET and FGFR1 activation signatures between TIC/EMT high and TIC/EMT low patient cohorts.The R package GSVAhttp://co.bmc.lu.se/gobo/) was used to determine the association of HGF, FGFR1, EGF and EGFR expression with relapse-free survival across a large set of breast cancers analysed by Affymetrix U133A arrays96.The gene expression-based outcome for breast cancer online (GOBO) tool ; ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001. Data distribution was assumed to be normal, but this was not formally tested. P values and the number of experiments used for quantification and statistical analysis are indicated in the corresponding figure legends.Quantitative data are presented as means ± SEM. Statistical significance was assessed using a two-tailed Student’s Further information on research design is available in the Supplementary InformationReporting Summary Checklist


Both rosiglitazone and metformin have effects on blood glucose regulation and the proliferation of liver cancer cells. Combination therapy with these two drugs is common and effective for the treatment of diabetes in the clinic, however, the application of these two drugs is influenced by the poor dissolution of rosiglitazone and the gastrointestinal side-effect of metformin resulting from a high solubility. The formation of a multidrug crystal form (Rsg-Met) by a solvent evaporation method can solve the solubility issue. Crystal structure data and intramolecular hydrogen bonds were detected by X-ray diffraction and infrared spectroscopy. Surprisingly, Rsg-Met shortens the time spent in solubility equilibrium and multiplies the dissolution rate of Rsg. Finally, we found that a low concentration of Rsg-Met enhanced the proliferation inhibition effect on liver cancer cells  compared with rosiglitazone, without affecting the human normal cell line LO2. Among drugs for type 2 diabetes mellitus (T2DM) used in its clinical treatment, rosiglitazone (Rsg) and metformin (Met) are the first line agents . Rsg, a Recently, extensive research has shown that Rsg and Met can decrease the occurrence rate of some cancers and improve cancer prognosis in T2DM patients, especially for liver cancer ,10,11,12However, on the basis of the Biopharmaceutical Classification System (BCS), Rsg is ranked as a class II molecule with low solubility and high permeability . It is nIn this context, medicine modification through crystal engineering and supramolecular techniques offers a route. We could synthesize a multidrug crystal aimed at T2DM patients with liver cancer. Co-crystal synthesis is a relatively simple approach to modify an existing active pharmaceutical ingredient (API) to achieve desirable properties, such as improved dissolution . MultidrAdditionally, from the structural point of view, Rsg and Met possess multiple sites which have potential hydrogen bonding capacities . The guaIn this article, we aimed to synthesize a novel multicomponent crystal which we have named Rsg-Met. We also performed structural analysis and dissolution rate of this multidrug crystal. Finally, its cytotoxicity effect was detected by the MTT assay, which indicated that Rsg-Met had a better inhibitory effect on some liver cancer cell lines  compared with the mixture and Rsg alone.Powder x-ray diffraction (PXRD) was used for the preliminary detection. Clearly, the patterns of Rsg-Met  are different from those of the Met  and Rsg , as shown in Following the disappearance of the characteristic peaks of two pure ingredients, the appearance of novel characteristic peaks in the Rsg-Met spectra was recorded. Moreover, the experimental PXRD patterns of Rsg-Met are similar to the simulated patterns of the corresponding single crystal. The similarity with the simulated powder pattern shows that the prepared Rsg-Met powder is a pure phase. The crystallographic data of Rsg-Met, listed in Rsg-Met is characterized by complex 2D hydrogen-bonded layers parallel to the (1 0 0) plane, as illustrated in The most surprising aspect of the data is the molecules of Rsg and Met were connected not only by hydrogen bonds, but also by an ionic bond . More spFourier-transform infrared (FT-IR) spectroscopic analysis is a standard detection method. In the first instance, the Rsg-Met IR spectrum should be a juxtaposition of the IR spectra of the single ingredients. There were slight shifts in absorption as the corresponding groups involved in strong hydrogen bonding and ionic bonding. This indeed was the case, as shown in −1) of Rsg moved to high-frequency and showed a novel absorption peak at 3446 cm−1, which was influenced by the H atom of N between lactam and the thiolactone from Rsg moving to a biguanide bond from the Met molecules. The thiolactone C=O vibration (1693 cm−1) and the lactam C=O vibration (1609 cm−1) of Rsg moved to low-frequency and showed a novel absorption peak at 1675 cm−1 and 1559 cm−1, which were influenced by producing a slight electron pair effect due to the formation of N anions. Additionally, the N-H vibration (3371 cm−1), -NH2 vibration (3173 cm−1) and C=NH vibration (1625 cm−1) from Met moved to high/low-frequency compared with the corresponding groups in the pure Met material, respectively. Overall, these results indicate that Rsg-Met formed hydrogen bonds and ionic bonds between Rsg and Met.In the infrared spectra of the Rsg-Met, the N-H vibration . Therefore, we conducted this experiment in phosphate buffer (pH = 6.8). As can be seen from the Notably, Met powder was directly soluble when it contact with an alkaline medium (pH = 6.8) ,22, so iThe results of the MTT assay are shown in Rsg and Met are classical oral anti-diabetic drugs used for T2DM patients . The comAdditionally, recent epidemiologic studies have suggested that both Rsg and Met have an anti-hepatoma effect ,36. As tIn this context, Rsg-Met was created with the basis of supermolecular chemistry and crystal engineering. A simple and cheap process was applied to prepare this novel multidrug crystal. The single most striking observation to emerge from the data analysis was the enhancement of the dissolution and cytotoxicity of Rsg.2O molecules because most of the hydrogen bond donor and acceptor moieties in Met actually formed hydrogen bonds between Met and Rsg molecules. Correspondingly, with the introduction of the highly soluble Met molecule, the dissolution of Rsg-Met was enhanced.The SCXRD data showed that the two pure ingredients were integrated by novel intramolecular hydrogen-bonds  and ionic-bonds. One molecule of Rsg-Met consisted of one molecule of Rsg and one of Met. From the perspective of hydrogen bonding analysis, Rsg-Met reduces the opportunity to form hydrogen bonds between Met and HFurthermore, we found that Rsg-Met had higher efficacy in inhibiting the proliferation of HepG2 and SK-hep1 liver cancer cells at lower concentrations than the mixture and Rsg and it has no effect of the human normal cell line LO2. This may be as the result of the improved dissolution of Rsg while a complementary effect between Rsg and Met cannot be excluded .The results of the experiments enhance our knowledge about these classical hypoglycemic agents, especially for Rsg. They provide an alternative method to address the problems related to poor solubility and the traditional fixed high-dose combinations. It also offers a potential application in the design and development of relevant and similar clinical anti-liver cancer drugs for T2MD patients.Rsg was purchased from Adamas Reagent Company  and used as received. Met was prepared by adding metformin hydrochloride  and sodium hydroxide  into 70 mL of ethanol and filtering the suspension after stirring at 25 °C for 12 h, followed by solvent removal with a rotary evaporator. The obtained free base of Met was freshly used in the next experiments. Other chemicals were purchased from Adamas Reagent Company, and used without any further purification.Boiling ethanol (35 mL) containing Rsg  was added to the same volume of ethanol containing Met  and vigorously stirred. The resulting solution was covered by parafilm perforated with a few small holes, and allowed to evaporate slowly under a surrounding temperature of 20 °C. After three days, this process yields colorless triclinic-shaped crystals that are suitable for single crystal x-ray diffraction. The obtained solids were filtered and dried under room conditions for further characterization.PXRD data for the crystalline products were collected using a Bruker D8 Advance x-ray diffractometer , operating in transmission geometry with Cu Kα radiation (λ = 1.5406 Å), 40 kV/100 mA, and fitted with a LynxEye linear detector . The samples were prepared on silicon single crystal sample holders with a 20 mm depth. Data for each sample were collected from 2θ = 5° to 50° at 25 °C with step and scan speed of 5 °/min.SCXRD data were collected on a SMART CCD diffractometer  using Cu-Kα radiation λ = 1.54184 Å) with a graphite monochromator at 293 K. The integrated and scaled data were empirically corrected for absorption effects with spherical harmonics, implemented with the SCALE3 ABSPACK scaling algorithm. Using Olex2 , the str84 Å with−1.Mortars and pestles were previously washed and placed in a dryer for 30 min. Then, each compound (1 mg) was ground into powder with dried KBr (50 mg) in a mortar. The mixture was pressed into a piece of slice and recorded on a Spectrum FT-IR spectrometer  in the range of 4000–400 cmw/v) of sodium lauryl sulfate as a dissolution medium under constant temperature 37 °C and 75 rpm. The powder equivalent to 100 mg of Rsg was weighed and added into dissolution medium. Ten mL samples were withdrawn at 10, 20, 30, 45, 60, 80, 100, 120, 160, 180 min, and fresh medium added at the same volume and temperature at the same time to maintain the volume constant. Three mL of the secondary filtrate was taken by filtering with 0.45 μm filter  and discarding the primary filtrate. The UV-Visible detector  was set at a wavelength of 318 nm. Every procedure describe above was performed three times.The dissolution rate of Rsg and Rsg-Met in powder form was studied by using a US Pharmacopoeia tablet dissolution test apparatus  in 900 mL of phosphate buffer (pH 6.8) containing 0.25% (2 = 0.9995), which valid from 50 to 90 μg/mL. Then, the dissolution rate can be measured by the equation w = CV/m × 100%. In this formula, w means the dissolution rate, C means concentration, V means 10 mL which were withdrawn from samples and m means 100 mg Rsg.The Lambert-Beer law was used to measure concentration of samples, and the equation is C=El/A. In this formula, A means absorbance measured by UV-Visible detection, E is a constant, l means thickness of absorption layer which generally is 1 cm, and C means concentration. The data were detected by UV-Visible detection and linear regression were performed in the concentration  by peak area (A) that equation was: A = 0.2244 + 0.098C  were obtained from the Chinese Academy of Sciences Cell Bank . Cells were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under 37 °C and 5% CO® Aqueous One Solution Cell Assay Kit . 2 × 103 cells were plated into each well of 96-well plates and incubated in regular culture medium for 6 h. Then, cells were treated with indicated concentrations  of drugs for 48 h. 20 μL MTT reagents were added into each well and incubated at 37 °C for 2 h. The absorbance was measured at 490 nm by enzyme-linked immunosorbent meter. Experiments were performed in triplicate.The cellular cytotoxic effect was evaluated using Cell Titer96p < 0.05 was considered statistically significant using SPSS 22.0.The data from individual experiments were presented as the mean ± SD. Statistical comparisons between groups were done using one-way ANOVA followed by Dunnett post hoc testing.


This paper aims to demonstrate the self-protection and self-sensing functionalities of self-compacted concrete (SCC) containing carbon nanotubes (CNT) and carbon microfibers (CMF) in a hybrid system. The ability for self-sensing at room temperature and that of self-protection after thermal fatigue cycles is evaluated. A binder containing a high volume of supplementary mineral additions (30%BFSand20%FA) and different type of aggregates  are used. The self-diagnosis is assessed measuring electrical resistivity (ER) and piezoresistivity (PZR) in compression mode within the elastic region of the concrete. Thermal fatigue is evaluated with mechanical and crack measurements after heat cycles (290–550 °C). SCC withstands high temperature cycles. The protective effect of the hybrid additive (CNT+CMF) notably diminishes damage by keepinghigher residual strength and lessmicrocracking of the concrete. Significant reductions in ER are detected. The self-diagnosis ability of functionalized SCC isconfirmed with PZR. A content of the hybrid functional additive (CNT+CMF) in the percolation region is recommended to maximize the self-sensing sensitivity. Other parameters as sample geometry, sensor location, power supply, and load level have less influence. The optimizationof the harvesting and distribution of energy from renewable sources needs specific attention for sustainablegrowth. However, to guarantee the implementation of a determined renewable energy system, which is sufficiently durable for its service life, the parallel advance ofthe construction of the most suitable infrastructure is fundamental. The operating conditions of most renewable energy systems occur in extreme conditions . OffshorFor solar energy, in the case of solar thermal electricity (STE), the concrete has been considered for the heat storage system, and the severe operating conditions are the high temperatures it has to withstand . The useConcrete exposed to high temperatures follows a series of physico-chemical changes , such asThe major concerns in relation to the use of concrete for heat storage in aCSP is failure due to the risk of explosion, and its stability during the repetitive heatcharge and discharge cycles.The type of binder and aggregate chosen have been shown to affect the cycling thermal resistance of concrete ,6 but crRegarding the design of thermal concrete for STE use, several alternatives have been suggested to improve the resistance of concrete to thermal fatigue: The use of suitable components ,6, such Fiber reinforced concretes (FRC) are increasingly used. The incorporation of fibers to reinforce the concrete matrix is also beneficial for concrete exposed to high temperatures . PolyproResearch is needed to demonstrate if advanced concrete technologies, such as self-compacted concrete and the integration of hybrid functional additives such as CNT and CMF, can contribute to the self-protection of the reinforced concrete matrix and improve the performance at the high and cyclic operative temperatures of the STE, to extend the service life of heat storage infrastructures.As indicated above, several renewable energy systems withstand extreme mechanical operation conditions that result in early damage due to mechanical fatigue. The modification of the electrical resistivity of concrete with the incorporation of conductive additives has added a self-diagnostic property of the mechanical damage of concrete ,16. InteThe self-sensing property in cementitious materials is usually assessed by measuring the electrical resistivity changes in the matrix caused by the incorporation of the conductive additive  and withElectrical conduction in a cementitious material takes place, first, through the liquid water in the pores, that is, the mobility of ions in the pore solution. This type of conductivity is identified as the ionic conductivity  of a cemMost studies of PZR related to self-sensing assessment have been conducted in cement paste ,21,22,23Based on the literature from previous analysis, the main objective of this paper is the assessment of functional additives in SCC with carbon nanotubes and carbon microfibers as a hybrid reinforcement of the cement paste matrix from the nano-scale to the micro-scale. The demonstration of the self-sensingcapability and the self-protection performance of the functionalized SCC is made with use ofperformance indicators tailored to each functionality. For self-sensing of concrete electrical resistivity and PZR are usedand forthe enhancement of self-protectionto thermal fatigue, concrete mechanical strength, and cracking are considered.SCC have been designed to deal with these objectives. The type of cement used was a CEM II/B-S 52.5, containing mineral additions such as 30% ultra-fine blast furnace slag. This cement was also blended with 20% fly ash (FA). The chemical compositions are included in The mixing was carried out in a planetary mixer with a capacity of 120L. The mixing procedurewas: First, all dry components werepremixed for 30 s. PPF and CMF were added into the dry materials. 70% of the mixing water was added until a homogeneous mixture was achieved (approximately 40s). Immediately after, the chemical additives and CNT were added one by one to the evenly distributed remaining mixing water (30%). For the CNT, the pre-dispersion with the water was carried out with magnetic stirring for 15 min. All the components were mixedfor 5 minafter the last component had been added. Cylindrical samples of 75by150mm were prepared for the evaluationof enhancement self-protection to thermal fatigue, while prismatic geometry, samples of 40by40by160 and 70by70by 280mm, were more suitable for sensor location for the self-sensing characterization of the SCC with CNT+CMF.The sample size ratios (lower surface length/maximum aggregate size) were, 6.25, 3.33,and 5.83, respectively which allowedfor adequate component distribution in the SCC matrix.All concrete specimens were cured and kept, until testing, in a moisture chamber, at 20+2°C and 98% R.H.The thermal fatigue conditions were simulated by designing a heating protocol in three stages, as shown in The mechanical response of the heated concrete was evaluated in aresidual state, after cooling thesamples to room temperature. The compressive strength and ultrasonic pulse velocity (UPV) were measured for mechanical performance analysis after 0, 1, 5, 25, 50, and 75 heating/coolingcycles for samples of 75by150mm. For crack analysis, slices of 75by25mm were cutand exposed to heat cycles. The crack level of the concrete surface was determined with an optical microscope, Nikon SMZ-2T, coupled to a camera for taking the photos. Two magnifications were used: a) one covering an area of 14.43 mm by 11.25 mm for the definition of the pattern and crack area, and b) another covering an area of 4.81 mm by 3.75 mm for a better measure of the crack width. Using these photos, the evolution of crack width and crack area were analyzed using the photography software GIMP by counting pixels forming cracks.The self-sensing tests were carried out in the functionalized SCC with CNT+CMF. Two methods for showingthe self-sensing sensitivityof the concrete were followed: 1) the measurement of the intrinsic electrical resistivity (ER) was carried out after the concrete had matured for 28 days in the chamber at 98%RH and 20°C, in accordance with the UNE 83988-1standard: Direct method. The basic data included in the PZR calculation and measured during the test are: Current (A), voltage (V), and load (kN).2) as the effective area of the sensor.The resistivity,ƿ, of the functionalized concrete with CNT+CMF to assess the suitability of PZR is obtained through the Equation (1).0 as the electrical resistivity of the concrete before loading and ρas the electrical resistivity under a specific load.The basic parameter that reflects the PZR sensitivity of a self-sensing functionalized cementitious material is the fractional change of electrical resistivity (FCR), obtained through the Equation (2)Compression load changes were used for PZR. A universal compression-testing machine—MEH series—Ibertest MEH—3000—HM 3000kN. For the electrical source, a power supplier HP E3611A DC current, with a voltage range capacity ofup to 35V, connected to a data logger Agilent 349-70A, were used. The configuration of the test set-up is shown in the 0, as it follows a linear change over time. This phenomenon is also associated with the polarization of the cementitious material. Preliminary tests suggested 30min, at least, for the application of the electrical field before launching the load to achieve a more or less stable equilibrium ofthe current, as shown in When a cementitious material is exposed to an electrical field, the current flows mainly through the pore water solution. The high electrical resistance of a concrete induces a current drop. This phenomenon is identified by some authors as a consequence of the polarization of the ions of the water pore solution under the electrical field ,18,23. Tcs: 46.04GPa (young dynamic modulus). The maximum resistance reference for testing within the elastic limit (yield point) was set at 0.85fck (43.35 Mpa), from the stress-strain curve. The compression loads used were below the maximum strength in orderto notaffect irreversibly the material and to test the repeatability of the cycles in the same specimen. The loads for the 40by40by160mm were: 10kN , 15 kN , and25kN  and for the 70by70by280 75kN ,per unit area. The compression load mode was selected for the PZR test. The load ratewas0.1kN/s. The studies of PZR with cementitious materials ,20,24 sucs), 3) functional additive content, and4) sensor distance and sample size. At least two repetitive PZR cycles were performed to assess each variable in two identical samples; the repeatability of the cycle response for each influencing parameterhas been analyzed.The PZR performance of the functionalized concretes with CNT+CMF was shown through the analysis of a series of influencing variables, described in The variation in the mechanical properties of the SCCsregarding thermal fatigue performance is shown in The shrinkage suffered by the cement paste during dehydration, and the changes in the volume of the aggregates during the repetitive charge–discharge heat cycles favour the generation of cracking in the concrete matrix. The evolution of crack growth, identified by the maximum crack width and the percentage of the area of the concreteaffected by the crackafter 1.25, and 75 heat/cool cycles, identified with the digital image system, is presented in The electrical resistance of the functionalized concretes has been considered as a feasible parameter for the self-sensing capability of the SCC. The changes in the electrical conductivity of the functionalized concretes with different hybrid functional additive (CNT+CMF) contents are presented in The heating of the functionalized SCC with CNT+CMF was also affectedby the moisture content in the pores . An incrAs indicated in cs), and sensor location 40/80/40 mm are shown in The power between sensors was varied, supplying different voltages . The power was applied by decreasing the level from 30volts.The results obtained for the functionalized concrete with 0.2CNT+1.4CMF, load range 15kN  for the current flow was: 40 and 70, respectively.PZR was detected with both sample sizes. The location of the sensors embedded in the concrete guarantees the adhesion of the mesh to the concreteand eliminateany of the effects of de-bonding during testing, and any adverse decay of the PZR response. Thesensor configurationthat has the highest aspect ratiogavelowerFCR changes with a constant load cycle.When cementitious materials are exposed to temperatures above 100 °C, there is a progressive dehydration of cement paste. This process is accompanied by decreases in mechanical strength, which occurs above 300 °C in most concrete typologies. This phenomenon is well known in fire performance assessment for concrete . The levThe aggregate type and its content and size, have a significant influence on the performance of concrete at high temperatures. Cement paste shrinks due to dehydration while aggregates expandwith heat . These pAnother relevant aspect which must be highlighted is the more efficient control of cracking with the use of different types and length of fibers to reinforce the SCC matrix, which has shown a positive contribution to the performance of the concrete at high temperatures and in repetitive heat cycles. The bond of the fiber to the cement matrix governs the failure of fiber-reinforced cement composites ,14. The This beneficial effect of the hybrid functional additive CNT+CMF for self-protection at high temperatures and thermal cycles can be seen in The good performance of CMF at high temperatures showsthe suitability of this concrete forenergy infrastructure that operates at extremely high temperatures, asin the case of CSP, if the design of the concrete is appropriately carried out with adequate thermal components as in this study.The self-diagnosis property is a function of the test conditions and the optimal dosage of concrete with the self-sensing additives and the electrical resistivity of the concrete matrix, shown in In this work, hybrid self-sensing concrete has been used with the aim of covering the deformations in the concrete induced by compressive external loads from the nanoscale to the microscale. Aspreviously mentioned, there is a lack of information on self-sensing capabilities in concrete ,28. ConcThe electrical conduction of conventional concrete is controlled by the water pore content and composition. This type of electrical conduction alone is not able to show the self-sensing performance of concrete in a PZR test, which also occurredin the non-functionalized SCC, the results of which are not included in this work. However, the ionic conduction of an electrical field is probably critical if non-continuous contact conduction can be guaranteed under external loads.Both an optimal design for the concrete and a suitable PZR test arrangement are neededto demonstrate the self-sensing sensitivity of the concrete and the expectations for the level of the external loads in the operating conditions of a specific structure. The level of voltage between sensors is relevant both in the sensitivity level and in the repeatability of the FCR response. As seen in Certain scatter in the response of FCR during the load cycle is appreciated, although quite acceptable reversibility between cycles can be observed. The repeatability between the first and second cycle of the FCR signal can also be affected for the rest ofthe variables analyzed in The highest sensitivity detected for the hybrid additive content in the region of percolationis better than after the percolation level although the lower electrical resistivity of the concrete was measured with the highest CMF content (1.4%). Two reasons could explain this: (i) a lack in the dispersion of the sensing additives and (ii) the higher probability for contact conduction could negatively affect the detection of FCR changes at increasing loads, resulting in lower sensitivity.The need for a certain contact conduction in combination with tunnelling or field conduction, that show lower electrical conductivity in unload conditions, but higher self-sensing under loads. Some authors have also postulated better self-sensing capabilities in the percolation region in cement pastes ,31.The most efficient parameter in the self-sensing response of the SCC has been shown to be the content of the self-sensing additive, in The fitted functionused for adjustment of the results FCR versus load from senscoeff), as described in [The sensitivity of the functionalized concrete to the PZR can be clearly seen in ribed in  determin0-ρ)/ρ the fractional change in resistivity, named FCR.With: L as load applied and , power level and sensor location also affect the PZR response of the self-sensing functionalized SCC, but at a lower scale. The ER significantly increases with the temperature and the PZR response could be affected in the same way, an aspect that needs further study. Moreover, the improvement for better understancing of repeatability of FCR response.