Evaluation of the Integrity of Horse Hoof Dermal and Epidermal Tissues Collected by Dorsal Transmural Access

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of Excitation Signal on Impedance-based Structural Health Monitoring

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural, Spectroscopic (NMR, IR, and Raman), and DFT Investigation of the Self-Assembled Nanostructure of Pravastatin-LDH (Layered Double Hydroxides) Systems

Layered double hydroxide (LDH) nanocontainers, suitable as carriers for anionic drugs, were intercalated with Pravastatin drug using magnesium-aluminum and zinc-aluminum in a M-II/Al molar ratio equal 2 and different Al3+/Pravastatin molar ratios. Postsynthesis treatments were used in order to increase the materials crystallinity. Hybrid materials were characterized by a set of physical chemical techniques: chemical elemental analysis, X-ray diffraction (XRD), mass coupled thermal analyses, vibrational infrared and Raman spectroscopies, and solid-state C-13 nuclear magnetic resonance (NMR). Results were interpreted in light of computational density functional theory (DFT) calculations performed for Sodium Pravastatin in order to assign the data obtained for the LDH intercalated materials. XRD peaks of LDH-Pravastatin material and the one-dimensional (1D) electron density map pointed out to a bilayer arrangement of Pravastatin in the interlayer region, where its associated carboxylate and vicinal hydroxyl groups are close to the positive LDH. The structural organization observed for the stacked assembly containing the unsymmetrical and bulky monoanion Pravastatin and LDH seems to be promoted by a self-assembling process, in which local interactions are maximized and chloride ion cointercalation is required. It is observed a high similarity among vibrational and C-13 NMR spectra of Na-Pravastatin and LDH-Pravastatin materials. Those features indicate that the intercalation preserves the drug structural integrity. Spectroscopic techniques corroborate the nature of the guest species and their arrangement between the inorganic layers. Changes related to carboxylate, alcohol, and olefinic moieties are observed in both vibrational Raman and C-13 NMR spectra after the drug intercalation. Thus, Pravastatin ions are forced to be arranged as head to tail through intermolecular hydrogen bonding between adjacent organic species. The thermal decomposition profile of the hybrid samples is distinct of that one observed for Na-Pravastatin salt, however, with no visible increase in the thermal behavior when the organic anion is sequestrated within LDH gap.

Rapid assessment of internodal myelin integrity in central nervous system tissue

Monitoring pathology/regeneration in experimental models of de-/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X-ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited "blind" samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced +/-10% variation in periodicity and +/-40% variation in relative amount of myelin. For samples having the most native-like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well-fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de- and remyelination.

3CAPS – a structural AP-site analogue as a tool to investigate DNA base excision repair

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3′-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^

Structural health monitoring network system with wireless communications inside closed aerospace structures

Structural Health Monitoring (SHM) requires integrated "all in one" electronic devices capable of performing analysis of structural integrity and on-board damage detection in aircraft?s structures. PAMELA III (Phased Array Monitoring for Enhanced Life Assessment, version III) SHM embedded system is an example of this device type. This equipment is capable of generating excitation signals to be applied to an array of integrated piezoelectric Phased Array (PhA) transducers stuck to aircraft structure, acquiring the response signals, and carrying out the advanced signal processing to obtain SHM maps. PAMELA III is connected with a host computer in order to receive the configuration parameters and sending the obtained SHM maps, alarms and so on. This host can communicate with PAMELA III through an Ethernet interface. To avoid the use of wires where necessary, it is possible to add Wi-Fi capabilities to PAMELA III, connecting a Wi-Fi node working as a bridge, and to establish a wireless communication between PAMELA III and the host. However, in a real aircraft scenario, several PAMELA III devices must work together inside closed structures. In this situation, it is not possible for all PAMELA III devices to establish a wireless communication directly with the host, due to the signal attenuation caused by the different obstacles of the aircraft structure. To provide communication among all PAMELA III devices and the host, a wireless mesh network (WMN) system has been implemented inside a closed aluminum wingbox. In a WMN, as long as a node is connected to at least one other node, it will have full connectivity to the entire network because each mesh node forwards packets to other nodes in the network as required. Mesh protocols automatically determine the best route through the network and can dynamically reconfigure the network if a link drops out. The advantages and disadvantages on the use of a wireless mesh network system inside closed aerospace structures are discussed.

Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure–function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al3+ sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure–function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al3+ sensing.

Smart aeronautical structures: development and experimental validation of a structural health monitoring system for damage detection

En muchas áreas de la ingeniería, la integridad y confiabilidad de las estructuras son aspectos de extrema importancia. Estos son controlados mediante el adecuado conocimiento de danos existentes. Típicamente, alcanzar el nivel de conocimiento necesario que permita caracterizar la integridad estructural implica el uso de técnicas de ensayos no destructivos. Estas técnicas son a menudo costosas y consumen mucho tiempo. En la actualidad, muchas industrias buscan incrementar la confiabilidad de las estructuras que emplean. Mediante el uso de técnicas de última tecnología es posible monitorizar las estructuras y en algunos casos, es factible detectar daños incipientes que pueden desencadenar en fallos catastróficos. Desafortunadamente, a medida que la complejidad de las estructuras, los componentes y sistemas incrementa, el riesgo de la aparición de daños y fallas también incrementa. Al mismo tiempo, la detección de dichas fallas y defectos se torna más compleja. En años recientes, la industria aeroespacial ha realizado grandes esfuerzos para integrar los sensores dentro de las estructuras, además de desarrollar algoritmos que permitan determinar la integridad estructural en tiempo real. Esta filosofía ha sido llamada “Structural Health Monitoring” (o “Monitorización de Salud Estructural” en español) y este tipo de estructuras han recibido el nombre de “Smart Structures” (o “Estructuras Inteligentes” en español). Este nuevo tipo de estructuras integran materiales, sensores, actuadores y algoritmos para detectar, cuantificar y localizar daños dentro de ellas mismas. Una novedosa metodología para detección de daños en estructuras se propone en este trabajo. La metodología está basada en mediciones de deformación y consiste en desarrollar técnicas de reconocimiento de patrones en el campo de deformaciones. Estas últimas, basadas en PCA (Análisis de Componentes Principales) y otras técnicas de reducción dimensional. Se propone el uso de Redes de difracción de Bragg y medidas distribuidas como sensores de deformación. La metodología se validó mediante pruebas a escala de laboratorio y pruebas a escala real con estructuras complejas. Los efectos de las condiciones de carga variables fueron estudiados y diversos experimentos fueron realizados para condiciones de carga estáticas y dinámicas, demostrando que la metodología es robusta ante condiciones de carga desconocidas. ABSTRACT In many engineering fields, the integrity and reliability of the structures are extremely important aspects. They are controlled by the adequate knowledge of existing damages. Typically, achieving the level of knowledge necessary to characterize the structural integrity involves the usage of nondestructive testing techniques. These are often expensive and time consuming. Nowadays, many industries look to increase the reliability of the structures used. By using leading edge techniques it is possible to monitoring these structures and in some cases, detect incipient damage that could trigger catastrophic failures. Unfortunately, as the complexity of the structures, components and systems increases, the risk of damages and failures also increases. At the same time, the detection of such failures and defects becomes more difficult. In recent years, the aerospace industry has done great efforts to integrate the sensors within the structures and, to develop algorithms for determining the structural integrity in real time. The ‘philosophy’ has being called “Structural Health Monitoring” and these structures have been called “smart structures”. These new types of structures integrate materials, sensors, actuators and algorithms to detect, quantify and locate damage within itself. A novel methodology for damage detection in structures is proposed. The methodology is based on strain measurements and consists in the development of strain field pattern recognition techniques. The aforementioned are based on PCA (Principal Component Analysis) and other dimensional reduction techniques. The use of fiber Bragg gratings and distributed sensing as strain sensors is proposed. The methodology have been validated by using laboratory scale tests and real scale tests with complex structures. The effects of the variable load conditions were studied and several experiments were performed for static and dynamic load conditions, demonstrating that the methodology is robust under unknown load conditions.

Structural recovery in lesioned adult mammalian spinal cord by x-irradiation of the lesion site.

Mechanical injury to the adult mammalian spinal cord results in permanent morphological disintegration including severance/laceration of brain-cord axons at the lesion site. We report here that some of the structural consequences of injury can be averted by altering the cellular components of the lesion site with x-irradiation. We observed that localized irradiation of the unilaterally transected adult rat spinal cord when delivered during a defined time-window (third week) postinjury prevented cavitation, enabled establishment of structural integrity, and resulted in regrowth of severed corticospinal axons through the lesion site and into the distal stump. In addition, we examined the natural course of degeneration and cavitation at the site of lesion with time after injury, noting that through the third week postinjury recovery processes are in progress and only at the fourth week do the destructive processes take over. Our data suggest that the adult mammalian spinal cord has innate mechanisms required for recovery from injury and that timed intervention in certain cellular events by x-irradiation prevents the onset of degeneration and thus enables structural regenerative processes to proceed unhindered. We postulate that a radiation-sensitive subgroup of cells triggers the delayed degenerative processes. The identity of these intrusive cells and the mechanisms for triggering tissue degeneration are still unknown.

Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

Final report of a 1991 Plymouth Acclaim rear impact CNG fuel tank integrity.

National Highway Traffic Safety Administration, Washington, D.C.

Light truck side structure evaluation. Volume II: documentation of occupant seating position and structural components. Final report.

Mode of access: Internet.

Sensor networks optimization and software development for Structural Health Monitoring based on ultrasonic guided waves

The Structural Health Monitoring (SHM) research area is increasingly investigated due to its high potential in reducing the maintenance costs and in ensuring the systems safety in several industrial application fields. A growing demand of new SHM systems, permanently embedded into the structures, for savings in weight and cabling, comes from the aeronautical and aerospace application fields. As consequence, the embedded electronic devices are to be wirelessly connected and battery powered. As result, a low power consumption is requested. At the same time, high performance in defects or impacts detection and localization are to be ensured to assess the structural integrity. To achieve these goals, the design paradigms can be changed together with the associate signal processing. The present thesis proposes design strategies and unconventional solutions, suitable both for real-time monitoring and periodic inspections, relying on piezo-transducers and Ultrasonic Guided Waves. In the first context, arrays of closely located sensors were designed, according to appropriate optimality criteria, by exploiting sensors re-shaping and optimal positioning, to achieve improved damages/impacts localisation performance in noisy environments. An additional sensor re-shaping procedure was developed to tackle another well-known issue which arises in realistic scenario, namely the reverberation. A novel sensor, able to filter undesired mechanical boundaries reflections, was validated via simulations based on the Green's functions formalism and FEM. In the active SHM context, a novel design methodology was used to develop a single transducer, called Spectrum-Scanning Acoustic Transducer, to actively inspect a structure. It can estimate the number of defects and their distances with an accuracy of 2[cm]. It can also estimate the damage angular coordinate with an equivalent mainlobe aperture of 8[deg], when a 24[cm] radial gap between two defects is ensured. A suitable signal processing was developed in order to limit the computational cost, allowing its use with embedded electronic devices.

Hydrothermal Stability of Modified Silica Membranes for Gas Separation

A new class of hybrid molecular sieve silica (MSS) membranes is developed and tested against standard and organic templated membranes. The hybrid membrane is synthesized by the standard sol-gel process, integrating a template (methyltriethoxysilane - MTES) and a C6 surfactant (triethylhexylammonium bromide) into the silica film matrix. After hydro treatment under a relative humidity of 96% for 50h, the hybrid membrane shows no changes in its gas separation capabilities or energy of mobility. The structural characteristics and integrity of the hybrid membrane are retained due to a high concentration of organophilic functional groups and alkoxides observed using 29 Si NMR. In contrast, the structural integrity of the membranes prepared with non-templated films deteriorated during the hydro treatment due to a large percentage of silanol groups (Si-OH) which react with water. The hybrid membranes underwent a decrease in the H2/CO2 selectivity of only 1% whereas for the non-templated membrane a 21% decrease was observed. The transport mechanism of the hybrid membranes is activated as permeation increased with temperature. The activation energy for the permeation of H2 is positive while negative for CO2. The H2 permeation obtained was 3x 10 -8 mol.m -2 .s -1 .Pa -1 and permselectivities for H2/CO2 and H2/N2 varied between 1-7 and 31-34, respectively.