Enzyme Microheterogeneous Hydration and Stabilization in Supercritical Carbon Dioxide

Supercritical carbon dioxide is a promising green-chemistry solvent for many enzyme-catalyzed chemical reactions, yet the striking stability of some enzymes in such unconventional environments is not well understood. Here, we investigate the stabilization of the Candida antarctica Lipase B (CALB) in supercritical carbon dioxide-water biphasic systems using molecular dynamics simulations. The preservation of the enzyme structure and optimal activity depend on the presence of small amounts of water in the supercritical dispersing medium. When the protein is at least partially hydrated, water molecules bind to specific sites on the enzyme surface and prevent carbon dioxide from penetrating its catalytic core. Strikingly, water and supercritical carbon dioxide cover the protein surface quite heterogeneously. In the first solvation layer, the hydrophilic residues at the surface of the protein are able to pin down patches of water, whereas carbon dioxide solvates preferentially hydrophobic surface residues. In the outer solvation shells, water molecules tend to cluster predominantly on top of the larger water patches of the first solvation layer instead of spreading evenly around the remainder of the protein surface. For CALB, this exposes the substrate-binding region of the enzyme to carbon dioxide, possibly facilitating diffusion of nonpolar substrates into the catalytic funnel. Therefore, by means of microheterogeneous solvation, enhanced accessibility of hydrophobic substrates to the active site can be achieved, while preserving the functional structure of the enzyme. Our results provide a molecular picture on the nature of the stability of proteins in nonaqueous media.

Oral health profile in patients infected with HTLV-1: Clinical findings, proviral load, and molecular analysis from HTLV-1 in saliva

Human T-lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has also been implicated in several disorders, including periodontal disease. The proviral load is an important biological marker for understanding HTLV-1 pathogenesis and elucidating whether or not the virus is related to the clinical manifestation of the disease. This study describes the oral health profile of HTLV-1 carriers and HAM/TSP patients in order to investigate the association between the proviral load in saliva and the severity of the periodontal disease and to examine virus intra-host variations from peripheral blood mononuclear cells and saliva cells. It is a cross-sectional analytical study of 90 individuals carried out from November 2006 to May 2008. Of the patients, 60 were HTLV-1 positive and 30 were negative. Individuals from the HTLV-1 positive and negative groups had similar mean age and social-economic status. Data were analyzed using two available statistical software packages, STATA 8.0 and SPSS 11.0 to conduct frequency analysis. Differences of P?<?0.05 were considered statistically significant. HTLV-1 patients had poorer oral health status when compared to seronegative individuals. A weak positive correlation between blood and saliva proviral loads was observed. The mean values of proviral load in blood and saliva in patients with HAM/TSP was greater than those in HTLV-1 carriers. The HTLV-1 molecular analysis from PBMC and saliva specimens suggests that HTLV-1 in saliva is due to lymphocyte infiltration from peripheral blood. A direct relationship between the proviral load in saliva and oral manifestations was observed. J. Med. Virol. 84:1428-1436, 2012. (c) 2012 Wiley Periodicals, Inc.

EFFECTS OF MUSCULAR STRETCHING AND SEGMENTAL STABILIZATION ON FUNCTIONAL DISABILITY AND PAIN IN PATIENTS WITH CHRONIC LOW BACK PAIN: A RANDOMIZED, CONTROLLED TRIAL

Objective: The purpose of this study was to compare the effects of 2 exercise programs, segmental stabilization exercises (SSEs) and stretching of trunk and hamstrings muscles, on functional disability, pain, and activation of the transversus abdominis muscle (TrA), in individuals with chronic low back pain. Methods: A total of 30 participants were enrolled in this study and randomly assigned to 1 of 2 groups as a function of intervention. In the segmental stabilization group (SS), exercises focused on the TrA and lumbar multifidus muscles, whereas in the stretching group (ST), exercises focused on stretching the erector spinae, hamstrings, and triceps surae. Severity of pain (visual analog scale and McGill pain questionnaire) and functional disability (Oswestry disability questionnaire) and TrA muscle activation capacity (Pressure Biofeedback Unit, or PBU) were compared as a function of intervention. Interventions lasted 6 weeks, and sessions happened twice a week (30 minutes each). Analysis of variance was used for intergroup and intragroup comparisons. Results: As compared with baseline, both treatments were effective in relieving pain and improving disability (P < .001). Those in the SS group had significantly higher gains for all variables. The stretching group did not effectively activate the TrA (P = .94). Conclusion: Both techniques improved pain and reduced disability. In this study, SS was superior to muscular stretching for the measured variables associated with chronic low back pain. (J Manipulative Physiol Ther 2012;35:279-285)

CD3e expression in HTLV-1-infected individuals is associated with proviral load and Tax expression

Financial support: FUNDHERP, CTC, INCTC, FAPESP, CNPq and CAPES.

Influence of INF-gamma gene polymorphism in HTLV-1 proviral load

Financial Support FAPESP, CNPq, CTC/FUNDHERP and INCTC.

Microbial load in instruments used in surgeries classified as clean

Introduction / objectives The number of orthopedic surgery, especially surgery of total hip and knee, have been more frequent due to technological advances. This study aims to determine the microbial load in the instruments used in clean surgeries, quantifying and identifying the genus and species of microbial growth.Methods Orthopedic surgical instruments were immersed, after use, in sterile water, sonicated in ultrasonic washer and consecutively shaken. Then, the lavage was filtered through a 0.45micron membrane, the result was incubated in aerobic medium, anaerobic medium and medium for fungi and yeasts. Results In clean surgeries, results showed that 47% of used instruments had microbiological growth in the range of 1 to 100 CFU/instrument. The most prevalent organism was Staphylococcus coagulase negative (28%), followed by Bacillus subtilis (11%).This study refuted the hypothesis that clean surgeries happen in micro-organismsfree surgery field. Conclusion The microbiological findings reinforce the importance of antibiotic prophylaxis, practice already well established for this category of surgical procedure.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Nutritional composition of rice bran submitted to different stabilization procedures

In order to inactivate enzymatic deterioration, whole rice bran samples were subjected to two stabilization methods. Changes in nutritional value in terms of, concerning chemical composition, minerals and fatty acid content, were evaluated to supplement existing data and promote the utilization of rice bran in the human diet. The following homemade heat treatments were applied: roasting on a conventional stove or heating in a microwave oven. Based on the results, the different heating methods affected sample composition, since the levels of some nutrients of treated samples showed significant changes (p<0.05) compared to corresponding raw samples. The rice bran treated on a conventional stove produced products with lower moisture (5.14±0.10 g/100 g) and nutrients such as sodium 11.8%; palmitic acid 9.9% and stearic acid 8.1%. The microwave oven procedure resulted in better nutrient preservation, with slightly higher moisture content (6.28±0.10 g/100 g), and appears to be a practical and rapid tool for home heat stabilization of rice bran.

Towards the stabilization of the low density elements in topology optimization with large deformation

This work addresses the treatment of lower density regions of structures undergoing large deformations during the design process by the topology optimization method (TOM) based on the finite element method. During the design process the nonlinear elastic behavior of the structure is based on exact kinematics. The material model applied in the TOM is based on the solid isotropic microstructure with penalization approach. No void elements are deleted and all internal forces of the nodes surrounding the void elements are considered during the nonlinear equilibrium solution. The distribution of design variables is solved through the method of moving asymptotes, in which the sensitivity of the objective function is obtained directly. In addition, a continuation function and a nonlinear projection function are invoked to obtain a checkerboard free and mesh independent design. 2D examples with both plane strain and plane stress conditions hypothesis are presented and compared. The problem of instability is overcome by adopting a polyconvex constitutive model in conjunction with a suggested relaxation function to stabilize the excessive distorted elements. The exact tangent stiffness matrix is used. The optimal topology results are compared to the results obtained by using the classical Saint Venant–Kirchhoff constitutive law, and strong differences are found.

Primary stability in cementless total hip replacement: measurement techniques and aided-surgery

Primary stability of stems in cementless total hip replacements is recognized to play a critical role for long-term survival and thus for the success of the overall surgical procedure. In Literature, several studies addressed this important issue. Different approaches have been explored aiming to evaluate the extent of stability achieved during surgery. Some of these are in-vitro protocols while other tools are coinceived for the post-operative assessment of prosthesis migration relative to the host bone. In vitro protocols reported in the literature are not exportable to the operating room. Anyway most of them show a good overall accuracy. The RSA, EBRA and the radiographic analysis are currently used to check the healing process of the implanted femur at different follow-ups, evaluating implant migration, occurance of bone resorption or osteolysis at the interface. These methods are important for follow up and clinical study but do not assist the surgeon during implantation. At the time I started my Ph.D Study in Bioengineering, only one study had been undertaken to measure stability intra-operatively. No follow-up was presented to describe further results obtained with that device. In this scenario, it was believed that an instrument that could measure intra-operatively the stability achieved by an implanted stem would consistently improve the rate of success. This instrument should be accurate and should give to the surgeon during implantation a quick answer concerning the stability of the implanted stem. With this aim, an intra-operative device was designed, developed and validated. The device is meant to help the surgeon to decide how much to press-fit the implant. It is essentially made of a torsional load cell, able to measure the extent of torque applied by the surgeon to test primary stability, an angular sensor that measure the relative angular displacement between stem and femur, a rigid connector that enable connecting the device to the stem, and all the electronics for signals conditioning. The device was successfully validated in-vitro, showing a good overall accuracy in discriminating stable from unstable implants. Repeatability tests showed that the device was reliable. A calibration procedure was then performed in order to convert the angular readout into a linear displacement measurement, which is an information clinically relevant and simple to read in real-time by the surgeon. The second study reported in my thesis, concerns the evaluation of the possibility to have predictive information regarding the primary stability of a cementless stem, by measuring the micromotion of the last rasp used by the surgeon to prepare the femoral canal. This information would be really useful to the surgeon, who could check prior to the implantation process if the planned stem size can achieve a sufficient degree of primary stability, under optimal press fitting conditions. An intra-operative tool was developed to this aim. It was derived from a previously validated device, which was adapted for the specific purpose. The device is able to measure the relative micromotion between the femur and the rasp, when a torsional load is applied. An in-vitro protocol was developed and validated on both composite and cadaveric specimens. High correlation was observed between one of the parameters extracted form the acquisitions made on the rasp and the stability of the corresponding stem, when optimally press-fitted by the surgeon. After tuning in-vitro the protocol as in a closed loop, verification was made on two hip patients, confirming the results obtained in-vitro and highlighting the independence of the rasp indicator from the bone quality, anatomy and preserving conditions of the tested specimens, and from the sharpening of the rasp blades. The third study is related to an approach that have been recently explored in the orthopaedic community, but that was already in use in other scientific fields. It is based on the vibration analysis technique. This method has been successfully used to investigate the mechanical properties of the bone and its application to evaluate the extent of fixation of dental implants has been explored, even if its validity in this field is still under discussion. Several studies have been published recently on the stability assessment of hip implants by vibration analysis. The aim of the reported study was to develop and validate a prototype device based on the vibration analysis technique to measure intra-operatively the extent of implant stability. The expected advantages of a vibration-based device are easier clinical use, smaller dimensions and minor overall cost with respect to other devices based on direct micromotion measurement. The prototype developed consists of a piezoelectric exciter connected to the stem and an accelerometer attached to the femur. Preliminary tests were performed on four composite femurs implanted with a conventional stem. The results showed that the input signal was repeatable and the output could be recorded accurately. The fourth study concerns the application of the device based on the vibration analysis technique to several cases, considering both composite and cadaveric specimens. Different degrees of bone quality were tested, as well as different femur anatomies and several levels of press-fitting were considered. The aim of the study was to verify if it is possible to discriminate between stable and quasi-stable implants, because this is the most challenging detection for the surgeon in the operation room. Moreover, it was possible to validate the measurement protocol by comparing the results of the acquisitions made with the vibration-based tool to two reference measurements made by means of a validated technique, and a validated device. The results highlighted that the most sensitive parameter to stability is the shift in resonance frequency of the stem-bone system, showing high correlation with residual micromotion on all the tested specimens. Thus, it seems possible to discriminate between many levels of stability, from the grossly loosened implant, through the quasi-stable implants, to the definitely stable one. Finally, an additional study was performed on a different type of hip prosthesis, which has recently gained great interest thus becoming fairly popular in some countries in the last few years: the hip resurfacing prosthesis. The study was motivated by the following rationale: although bone-prosthesis micromotion is known to influence the stability of total hip replacement, its effect on the outcome of resurfacing implants has not been investigated in-vitro yet, but only clinically. Thus the work was aimed at verifying if it was possible to apply to the resurfacing prosthesis one of the intraoperative devices just validated for the measurement of the micromotion in the resurfacing implants. To do that, a preliminary study was performed in order to evaluate the extent of migration and the typical elastic movement for an epiphyseal prosthesis. An in-vitro procedure was developed to measure micromotions of resurfacing implants. This included a set of in-vitro loading scenarios that covers the range of directions covered by hip resultant forces in the most typical motor-tasks. The applicability of the protocol was assessed on two different commercial designs and on different head sizes. The repeatability and reproducibility were excellent (comparable to the best previously published protocols for standard cemented hip stems). Results showed that the procedure is accurate enough to detect micromotions of the order of few microns. The protocol proposed was thus completely validated. The results of the study demonstrated that the application of an intra-operative device to the resurfacing implants is not necessary, as the typical micromovement associated to this type of prosthesis could be considered negligible and thus not critical for the stabilization process. Concluding, four intra-operative tools have been developed and fully validated during these three years of research activity. The use in the clinical setting was tested for one of the devices, which could be used right now by the surgeon to evaluate the degree of stability achieved through the press-fitting procedure. The tool adapted to be used on the rasp was a good predictor of the stability of the stem. Thus it could be useful for the surgeon while checking if the pre-operative planning was correct. The device based on the vibration technique showed great accuracy, small dimensions, and thus has a great potential to become an instrument appreciated by the surgeon. It still need a clinical evaluation, and must be industrialized as well. The in-vitro tool worked very well, and can be applied for assessing resurfacing implants pre-clinically.

Shear behavior of reinforced concrete slabs under concentrated load: an investigation through Sequentially Linear Analysis

ABSTRACT (italiano) Con crescente attenzione riguardo al problema della sicurezza di ponti e viadotti esistenti nei Paesi Bassi, lo scopo della presente tesi è quello di studiare, mediante la modellazione con Elementi Finiti ed il continuo confronto con risultati sperimentali, la risposta in esercizio di elementi che compongono infrastrutture del genere, ovvero lastre in calcestruzzo armato sollecitate da carichi concentrati. Tali elementi sono caratterizzati da un comportamento ed una crisi per taglio, la cui modellazione è, da un punto di vista computazionale, una sfida piuttosto ardua, a causa del loro comportamento fragile combinato a vari effetti tridimensionali. La tesi è incentrata sull'utilizzo della Sequentially Linear Analysis (SLA), un metodo di soluzione agli Elementi Finiti alternativo rispetto ai classici approcci incrementali e iterativi. Il vantaggio della SLA è quello di evitare i ben noti problemi di convergenza tipici delle analisi non lineari, specificando direttamente l'incremento di danno sull'elemento finito, attraverso la riduzione di rigidezze e resistenze nel particolare elemento finito, invece dell'incremento di carico o di spostamento. Il confronto tra i risultati di due prove di laboratorio su lastre in calcestruzzo armato e quelli della SLA ha dimostrato in entrambi i casi la robustezza del metodo, in termini di accuratezza dei diagrammi carico-spostamento, di distribuzione di tensioni e deformazioni e di rappresentazione del quadro fessurativo e dei meccanismi di crisi per taglio. Diverse variazioni dei più importanti parametri del modello sono state eseguite, evidenziando la forte incidenza sulle soluzioni dell'energia di frattura e del modello scelto per la riduzione del modulo elastico trasversale. Infine è stato effettuato un paragone tra la SLA ed il metodo non lineare di Newton-Raphson, il quale mostra la maggiore affidabilità della SLA nella valutazione di carichi e spostamenti ultimi insieme ad una significativa riduzione dei tempi computazionali. ABSTRACT (english) With increasing attention to the assessment of safety in existing dutch bridges and viaducts, the aim of the present thesis is to study, through the Finite Element modeling method and the continuous comparison with experimental results, the real response of elements that compose these infrastructures, i.e. reinforced concrete slabs subjected to concentrated loads. These elements are characterized by shear behavior and crisis, whose modeling is, from a computational point of view, a hard challenge, due to their brittle behavior combined with various 3D effects. The thesis is focused on the use of Sequentially Linear Analysis (SLA), an alternative solution technique to classical non linear Finite Element analyses that are based on incremental and iterative approaches. The advantage of SLA is to avoid the well-known convergence problems of non linear analyses by directly specifying a damage increment, in terms of a reduction of stiffness and strength in the particular finite element, instead of a load or displacement increment. The comparison between the results of two laboratory tests on reinforced concrete slabs and those obtained by SLA has shown in both the cases the robustness of the method, in terms of accuracy of load-displacements diagrams, of the distribution of stress and strain and of the representation of the cracking pattern and of the shear failure mechanisms. Different variations of the most important parameters have been performed, pointing out the strong incidence on the solutions of the fracture energy and of the chosen shear retention model. At last a confrontation between SLA and the non linear Newton-Raphson method has been executed, showing the better reliability of the SLA in the evaluation of the ultimate loads and displacements, together with a significant reduction of computational times.

Psychobiological correlates of allostatic load

Introduction: The term Clinimetric was introduced by Feinstein in 1982, who first noticed that despite all the improvements in the assessment methods, a number of clinical phenomena were still unconsidered during the evaluation process. Yet today clinical phenomena, such as stress, relevant in diseases progression and course, are not completely evaluated. Only recently, according to the clinimetric approach, Fava and colleagues have introduced specific criteria for evaluating the allostatic overload in clinical setting. Methods: Participants were 240 blood donors recruited from May 2007 to December 2009 in 4 different blood Centers (AVIS) in Italy. Blood samples from each participant were collected for laboratory test the same day the self-rating instruments were administered (Psychosocial Index, Symptom Questionnaire, Psychological well-being scales, Temperament and Character inventory, Self-Report Altruism scale). The study explore different aspects describing sample characteristics and correlates of stress in the total sample (part I), new selection criteria applied to existing instruments to identify individuals reporting allostatic load (part II), and differences on biological correlates between subjects with vs without AL. Results: Significant differences according to gender and past illnesses have been found in different dimensions of well-being and distress. Further, distress was explained for more than 60% by 4 main factors such as anxiety, somatic symptoms, environmental mastery and persistence. According to the new criteria, 98 donors reported AL. Allostatic load individuals reported to engage in less altruistic behaviours. Also they differ in personality traits and characters from controls. In the last part, results showed significant differences among donors according to allostatic load on diverse biological parameters (RBC, MCV, immune essay). Conclusion: This study presents obvious limitations due to its preliminary nature. Further research are need to confirm that these new criteria may lead to identify high risk individuals reporting not only stressful situations but also vulnerabilities.

Dynamic stabilization of Rayleigh-Taylor instability of ablation fronts in inertial confinement fusion

One of the most important problems in inertial confinement fusion is how to find a way to mitigate the onset of the Rayleigh-Taylor instability which arises in the ablation front during the compression. In this thesis it is studied in detail the possibility of using for such a purpose the well-known mechanism of dynamic stabilization, already applied to other dynamical systems such as the inverted pendulum. In this context, a periodic acceleration superposed to the background gravity generates a vertical vibration of the ablation front itself. The effects of different driving modulations (Dirac deltas and square waves) are analyzed from a theoretical point of view, with a focus on stabilization of ion beam driven ablation fronts, and a comparison is made, in order to look for optimization.