Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), SmA, is a vaccine candidate against, S. mansoni and F hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine. (C) 2008 Elsevier B.V. All rights reserved.

Vertical Root Fracture in Upper Premolars with Endodontic Posts: Finite Element Analysis

Upper premolars restored with endodontic posts present a high incidence of vertical root fracture (VRF). Two hypotheses were tested: (1) the smaller mesiodistal diameter favors stress concentration in the root and (2) the lack of an effective bonding between root and post increases the risk of VRF. Using finite element analysis, maximum principal stress was analyzed in 3-dimensional intact upper second premolar models. From the intact models, new models were built including endodontic posts of different elastic modulus (E = 37 or E = 200 GPa) with circular or oval cross-section, either bonded or nonbonded to circular or oval cross-section root canals. The first hypothesis was partially confirmed because the conditions involving nonbonded, low-modulus posts showed lower tensile stress for oval canals compared to circular canals. Tensile stress peaks for the nonbonded models were approximately three times higher than for the bonded or intact models, therefore confirming the second hypothesis. (J Endod 2009;35:117-120)

Surgical planning for resection of an ameloblastoma and reconstruction of the mandible using a selective laser sintering 3D biomodel

Ameloblastoma is a benign locally aggressive infiltrative odontogenic lesion. It is characterized by slow growth and painless swelling. The treatment for ameloblastoma varies from curettage to en bloc resection, and the reported recurrence rates after treatment are high; the safety margin of resection is important to avoid recurrence. Advances in technology brought about great benefits in dentistry; a new generation of computed tomography scanners and 3-dimensional images enhance the surgical planning and management of maxillofacial tumors. The development of new prototyping systems provides accurate 3D biomodels on which surgery can be simulated, especially in cases of ameloblastoma, in which the safety margin is important for treatment success. A case of mandibular follicular ameloblastoma is reported where a 3D biomodel was used before and during surgery.

Cervical range of motion discriminates between asymptomatic persons and those with whiplash

Study Design. A comparative study of cervical range of motion in asymptomatic persons and those with whiplash. Objectives. To compare the primary and conjunct ranges of motion of the cervical spine in asymptomatic persons and those with persistent whiplash-associated disorders, and to investigate the ability of these measures of range of motion to discriminate between the groups. Summary of Background. Evidence that range of motion is an effective indicator of physical impairment in the cervical spine is not conclusive. Few studies have evaluated the ability to discriminate between asymptomatic persons and those with whiplash on the basis of range of motion or compared three-dimensional in vivo measures of range of motion in asymptomatic persons and those with whiplash-associated disorders. Methods. The study participants were 89 asymptomatic volunteers (41 men, 48 women; mean age 39.2 years) and 114 patients with persistent whiplash-associated disorders (22 men, 93 women; mean age 37.2 years) referred to a whiplash research unit for assessment of their cervical region. Range of cervical motion was measured in three dimensions with a computerized, electromagnetic, motion-tracking device. The movements assessed were flexion, extension, left and right lateral flexion, and left and right rotation. Results. Range of motion was reduced in all primary movements in patients with persistent whiplash-associated disorder. Sagittal plane movements were proportionally the most affected. On the basis of primary and conjunct range of motion, age, and gender, 90.3% of study participants could be correctly categorized as asymptomatic or as having whiplash (sensitivity 86.2%, specificity 95.3%). Conclusions. Range of motion was capable of discriminating between asymptomatic persons and those with persistent whiplash-associated disorders.

Effects of temporal association on recognition memory

The influence of temporal association on the representation and recognition of objects was investigated. Observers were shown sequences of novel faces in which the identity of the face changed as the head rotated. As a result, observers showed a tendency to treat the views as if they were of the same person. Additional experiments revealed that this was only true if the training sequences depicted head rotations rather than jumbled views: in other words, the sequence had to be spatially as well as temporally smooth. Results suggest that we are continuously associating views of objects to support later recognition, and that we do so not only on the basis of the physical similarity, but also the correlated appearance in time of the objects.

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current, study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways. Cell Motil. Cytoskeleton 49:93-103, 2001. (C) 2001 Wiley-Liss, Inc.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

Usefulness of quantitative echocardiographic techniques to predict recovery of regional and global left ventricular function after acute myocardial infarction

The left ventricular response to dobutamine may be quantified using tissue Doppler measurement of myocardial velocity or displacement or 3-dimensional echocardiography to measure ventricular volume and ejection fraction. This study sought to explore the accuracy of these methods for predicting segmental and global responses to therapy. Standard dobutamine and 3-dimensional echocardiography were performed in 92 consecutive patients with abnormal left ventricular function at rest. Recovery of function was defined by comparison with follow-up echocardiography at rest 5 months later. Segments that showed improved regional function at follow-up showed a higher increment in peak tissue Doppler velocity with dobutamine therapy than in nonviable segments (1.2 +/- 0.4 vs 0.3 +/- 0.2 cm/s, p = 0.001). Similarly, patients who showed a > 5% improvement of ejection fraction at follow-up showed a greater displacement response to dobutamine (6.9 +/- 3.2 vs 2.1 +/- 2.3 mm, p = 0.001), as well as a higher rate of ejection fraction, response to dobutamine (9 +/- 3% vs 2 +/- 2%, p = 0.001). The optimal cutoff values for predicting subsequent recovery of function at rest were an increment of peak velocity > 1 cm/s, >5 mm of displacement, and a >5% improvement of ejection fraction with low-dose dobutamine. (C) 2003 by Excerpta Medica, Inc.

Structural and functional studies of PpcA: a key protein in the electron transfer pathways of Geobacter sulfurreducens

Dissertação para obtenção do Grau de Doutor em Bioquímica, ramo de Biotecnologia

Structural investigation of the Bacillus subtilis morphogenic factor RodZ

A thesis to obtain a Master degree in Structural and Functional Biochemistry

Follow the red road of triheme cytochromes in Geobacter sulfurreducens

The bacterium Geobacter sulfurreducens (Gs) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membrane in a process designated as extracellular electron transfer. The Gs genome was fully sequenced and a family composed by five periplasmic triheme cytochromes c7 (designated PpcA-E) was identified. These cytochromes play an important role in the reduction of extracellular acceptors. They contain approximately 70 amino acids, three heme groups with bis-histidinyl axial coordination, and share between 57 and 77% sequence identity. The triheme cytochrome PpcA is highly abundant in Gs and is most likely the reservoir of electrons destined for outer surface. In addition to its role in electron transfer pathways this protein can perform e-/H+ energy transduction, a process that is disrupted when the strictly conserved aromatic residue phenylalanine 15 is replaced by a leucine (PpcAF15L). This Thesis focuses on the expression, purification and characterization of these proteins using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The orientations of the heme axial histidine ring planes and the orientation of the heme magnetic axis were determined for each Gs triheme cytochrome. The comparison of the orientations obtained in solution with the crystal structures available showed significant differences. The results obtained provide the paramagnetic constraints to include in the future refinement of the solution structure in the oxidized state. In this work was also determined the solution structure and the pH-dependent conformational changes of the PpcAF15L allowing infer the structural origin for e-/H+ energy transduction mechanism as shown by PpcA. Finally, the backbone and side chain NH signals of PpcA were used to map interactions between this protein and the putative redox partner 9,10-anthraquinone-2,6-disulfonate (AQDS). In this work a molecular interaction was identified for the first time between PpcA and AQDS, constituting the first step toward the rationalization of the Gs respiratory chain.

EFFECT – Efficient finite element code

The theme of this dissertation is the finite element method applied to mechanical structures. A new finite element program is developed that, besides executing different types of structural analysis, also allows the calculation of the derivatives of structural performances using the continuum method of design sensitivities analysis, with the purpose of allowing, in combination with the mathematical programming algorithms found in the commercial software MATLAB, to solve structural optimization problems. The program is called EFFECT – Efficient Finite Element Code. The object-oriented programming paradigm and specifically the C ++ programming language are used for program development. The main objective of this dissertation is to design EFFECT so that it can constitute, in this stage of development, the foundation for a program with analysis capacities similar to other open source finite element programs. In this first stage, 6 elements are implemented for linear analysis: 2-dimensional truss (Truss2D), 3-dimensional truss (Truss3D), 2-dimensional beam (Beam2D), 3-dimensional beam (Beam3D), triangular shell element (Shell3Node) and quadrilateral shell element (Shell4Node). The shell elements combine two distinct elements, one for simulating the membrane behavior and the other to simulate the plate bending behavior. The non-linear analysis capability is also developed, combining the corotational formulation with the Newton-Raphson iterative method, but at this stage is only avaiable to solve problems modeled with Beam2D elements subject to large displacements and rotations, called nonlinear geometric problems. The design sensitivity analysis capability is implemented in two elements, Truss2D and Beam2D, where are included the procedures and the analytic expressions for calculating derivatives of displacements, stress and volume performances with respect to 5 different design variables types. Finally, a set of test examples were created to validate the accuracy and consistency of the result obtained from EFFECT, by comparing them with results published in the literature or obtained with the ANSYS commercial finite element code.

Does abdominoplasty with liposuction of the love handles yield a shorter scar? An analysis with abdominal 3D laser scanning.

The aim of this study was to evaluate the combination of abdominoplasty with liposuction of both flanks with regards to length of scar, complications, and patient's satisfaction. A retrospective analysis of 35 patients who underwent esthetic abdominoplasty at our institution between 2002 and 2004 was performed. Thirteen patients underwent abdominoplasty with liposuction of both flanks, 22 patients underwent conventional abdominoplasty. Liposuction of the flanks did not increase the rate of complications of the abdominoplasty procedures. We found a tendency toward shorter scars in patients who underwent abdominoplasty combined with liposuction of the flanks. Implementation of 3-dimensional laser surface scanning to objectify the postoperative outcomes, documented a comparable degree of flatness of the achieved body contouring in both procedures. 3-dimensional laser surface scanning can be a valuable tool to objectify assessment of postoperative results.

Dimensionality of drinking consequences - cross-cultural comparability and stability over time

Despite the long tradition for asking about the negative social and health consequences of alcohol consumption in surveys, little is known about the dimensionality of these consequences. Analysing cross-sectional and longitudinal data from the Nordic Taxation Study collected for Sweden, Finland, and Denmark in two waves in 2003 and 2004 by means of an explorative principal component analysis for categorical data (CATPCA), it is tested whether consequences have a single underlying dimension across cultures. It further tests the reliability, replicability, concurrent and predictive validity of the consequence scales. A one-dimensional solution was commonly preferable. Whereas the two-dimensional solution was unable to distinguish clearly between different concepts of consequences, the one-dimensional solution resulted in interpretable, generally very stable scales within countries across different samples and time.

Targetting of the ventro-intermediate nucleus using ultra-high field (7T) MRI for gamma knife surgery purposes: A pilot in vivo study on healthy subjects.

INTRODUCTION: Gamma Knife surgery (GKS) is a non-invasive neurosurgical stereotactic procedure, increasingly used as an alternative to open functional procedures. This includes targeting of the ventro-intermediate nucleus of the thalamus (e.g. Vim) for tremor. We currently perform an indirect targeting, as the Vim is not visible on current 3Tesla MRI acquisitions. Our objective was to enhance anatomic imaging (aiming at refining the precision of anatomic target selection by direct visualisation) in patients treated for tremor with Vim GKS, by using high field 7T MRI. MATERIALS AND METHODSH: Five young healthy subjects were scanned on 3 (T1-w and diffusion tensor imaging) and 7T (high-resolution susceptibility weighted images (SWI)) MRI in Lausanne. All images were further integrated for the first time into the Gamma Plan Software(®) (Elekta Instruments, AB, Sweden) and co-registered (with T1 was a reference). A simulation of targeting of the Vim was done using various methods on the 3T images. Furthermore, a correlation with the position of the found target with the 7T SWI was performed. The atlas of Morel et al. (Zurich, CH) was used to confirm the findings on a detailed analysis inside/outside the Gamma Plan. RESULTS: The use of SWI provided us with a superior resolution and an improved image contrast within the basal ganglia. This allowed visualization and direct delineation of some subgroups of thalamic nuclei in vivo, including the Vim. The position of the target, as assessed on 3T, perfectly matched with the supposed one of the Vim on the SWI. Furthermore, a 3-dimensional model of the Vim-target area was created on the basis of the obtained images. CONCLUSION: This is the first report of the integration of SWI high field MRI into the LGP, aiming at the improvement of targeting validation of the Vim in tremor. The anatomical correlation between the direct visualization on 7T and the current targeting methods on 3T (e.g. quadrilatere of Guyot, histological atlases) seems to show a very good anatomical matching. Further studies are needed to validate this technique, both by improving the accuracy of the targeting of the Vim (potentially also other thalamic nuclei) and to perform clinical assessment.