Alterações na expressão do Hormônio Concentrador de Melanina (MCH) na área hipotalâmica lateral do rato ao longo do desenvolvimento pós-natal e envelhecimento

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The noradrenergic projection from the locus coeruleus to the cochlear root neurons in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

10 Å cryo-EM structure of Drosophila melanogaster hexamerin and Limulus polyphemus hemocyanin

In the present study, the quaternary structures of Drosophila melanogaster hexamerin LSP-2 and Limulus polyphemus hemocyanin, both proteins from the hemocyanin superfamily, were elucidated to a 10 Å resolution with the technique of cryo-EM 3D-reconstruction. Furthermore, molecular modelling and rigid-body fitting allowed a detailed insight into the cryo-EM structures at atomic level. The results are summarised as follows: Hexamerin 1. The cryo-EM structure of Drosophila melanogaster hexamerin LSP-2 is the first quaternary structure of a protein from the group of the insect storage proteins. 2. The hexamerin LSP-2 is a hexamer of six bean-shaped subunits that occupy the corners of a trigonal antiprism, yielding a D3 (32) point-group symmetry. 3. Molecular modelling and rigid-body fitting of the hexamerin LSP-2 sequence showed a significant correlation between amino acid inserts in the primary structure and additional masses of the cryo-EM structure that are not present in the published quaternary structures of chelicerate and crustacean hemocyanins. 4. The cryo-EM structure of Drosophila melanogaster hexamerin LSP-2 confirms that the arthropod hexameric structure is applicable to insect storage proteins. Hemocyanin 1. The cryo-EM structure of the 8×6mer Limulus polyphemus hemocyanin is the highest resolved quaternary structure of an oligo-hexameric arthropod hemocyanin so far. 2. The hemocyanin is build of 48 bean-shaped subunits which are arranged in eight hexamers, yielding an 8×6mer with a D2 (222) point-group symmetry. The 'basic building blocks' are four 2×6mers that form two 4×6mers in an anti-parallel manner, latter aggregate 'face-to-face' to the 8×6mer. 3. The morphology of the 8×6mer was gauged and described very precisely on the basis of the cryo-EM structure. 4. Based on earlier topology studies of the eight different subunit types of Limulus polyphemus hemocyanin, eleven types of interhexamer interfaces have been identified that in the native 8×6mer sum up to 46 inter-hexamer bridges - 24 within the four 2×6mers, 10 to establish the two 4×6mers, and 12 to assemble the two 4×6mers into an 8×6mer. 5. Molecular modelling and rigid-body fitting of Limulus polyphemus and orthologous Erypelma californicum sequences allowed to assign very few amino acids to each of these interfaces. These amino acids now serve as candidates for the chemical bonds between the eight hexamers. 6. Most of the inter-hexamer contacts are conspicuously histidine-rich and evince constellations of amino acids that could constitute the basis for the allosteric interactions between the hexamers. 7. The cryo-EM structure of Limulus polyphemus hemocyanin opens the door to a fundamental understanding of the function of this highly cooperative protein.

Leopoli-Cencelle beyond virtual reality Documentazione, interpretazione e comprensione di una città medievale

La città medievale di Leopoli-Cencelle (fondata da Papa Leone IV nell‘854 d.C. non lontano da Civitavecchia) è stata oggetto di studio e di periodiche campagne di scavo a partire dal 1994. Le stratigrafie investigate con metodi tradizionali, hanno portato alla luce le numerose trasformazioni che la città ha subìto nel corso della sua esistenza in vita. Case, torri, botteghe e strati di vissuto, sono stati interpretati sin dall’inizio dello scavo basandosi sulla documentazione tradizionale e bi-dimensionale, legata al dato cartaceo e al disegno. Il presente lavoro intende re-interpretare i dati di scavo con l’ausilio delle tecnologie digitali. Per il progetto sono stati utilizzati un laser scanner, tecniche di Computer Vision e modellazione 3D. I tre metodi sono stati combinati in modo da poter visualizzare tridimensionalmente gli edifici abitativi scavati, con la possibilità di sovrapporre semplici modelli 3D che permettano di formulare ipotesi differenti sulla forma e sull’uso degli spazi. Modellare spazio e tempo offrendo varie possibilità di scelta, permette di combinare i dati reali tridimensionali, acquisiti con un laser scanner, con semplici modelli filologici in 3D e offre l’opportunità di valutare diverse possibili interpretazioni delle caratteristiche dell’edificio in base agli spazi, ai materiali, alle tecniche costruttive. Lo scopo del progetto è andare oltre la Realtà Virtuale, con la possibilità di analizzare i resti e di re-interpretare la funzione di un edificio, sia in fase di scavo che a scavo concluso. Dal punto di vista della ricerca, la possibilità di visualizzare le ipotesi sul campo favorisce una comprensione più profonda del contesto archeologico. Un secondo obiettivo è la comunicazione a un pubblico di “non-archeologi”. Si vuole offrire a normali visitatori la possibilità di comprendere e sperimentare il processo interpretativo, fornendo loro qualcosa in più rispetto a una sola ipotesi definitiva.

Molekularbiologische Charakterisierung der beiden Untereinheiten des Mega-Hämocyanins der Schnecke Melanoides tuberculata

Bei dem 2010 von unserer Arbeitsgruppe entdeckten Mega-Hämocyanin handelt es sich um einen stark abgewandelten Typ des respiratorischen Proteins Hämocyanin, bestehend aus zwei flankierenden regulären Dekameren und einem zentralen Mega-Dekamer. Diese sind aus zwei immunologisch verschiedenen Untereinheiten mit ~400 bzw. ~550 kDa aufgebaut, die in unserer Arbeitsgruppe bereits proteinbiochemisch charakterisiert wurden. Im Zuge dieser Untersuchungen konnte zudem eine 3D-Rekonstruktion des Oligomers (13,5 MDa) mit einer Auflösung von 13Å erstellt werden. Das Ziel der vorliegenden Arbeit war die Aufklärung der Primärstruktur beider Polypeptide bei der Schnecke Melanoides tuberculata (MtH). Es gelang, die cDNAs der beiden Untereinheiten vollständig zu sequenzieren. Die zu typischen Dekameren assemblierende MtH400-Untereinheit umfasst 3445 Aminosäuren und besitzt eine theoretische Molekularmasse von 390 kDa. Nach dem Signalpeptid von 23 Aminosäuren Länge folgen die für Gastropoden-Hämocyanine typischen funktionellen Einheiten FU-a bis FU-h. Insgesamt verfügt die MtH400-Untereinheit über sechs potentielle N-Glykosylierungsstellen. Die MtH550-Untereinheit, welche mit 10 Kopien das Mega-Dekamer bildet, umfasst 4999 Aminosäuren und besitzt eine theoretische Molekularmasse von 567 kDa. Damit handelt es sich bei dieser Untereinheit um die zweitgrößte jemals bei einem Protein detektierte Polypeptidkette. Die MtH550-Untereinheit besteht aus einem Signalpeptid von 20 Aminosäuren Länge und den typischen Wand-FUs (FU-a bis FU-f). Daran anschließend folgen sechs weitere Varianten der FU-f (FU-f1 bis FU-f6). Die MtH550-Untereinheit verfügt über insgesamt zwölf potentielle N-Glykosylierungsstellen. Anhand der ermittelten Primärstrukturdaten wird klar, dass der auffällig vergrößerte Kragenbereich des Mega-Dekamers aus je 10 Kopien der FU-f1 bis FU-f6 besteht. Die ermittelten Sequenzdaten der beiden MtH-Untereinheiten weisen im Vergleich zu anderen Hämocyanin Sequenzen einige sehr charakteristische Indels sowie unübliche N-Glykosylierungsstellen auf. Es war zudem möglich, anhand einer molekularen Uhr den Entstehungszeitpunkt des Mega-Hämocyanins zu datieren (145 ± 35 MYA). Sowohl die Topologie als auch die berechneten Trennungszeitpunkte des an allen Verzweigungen gut unterstützten Stammbaums stimmen mit den bisher publizierten und auf Hämocyanindaten basierenden molekularen Uhren überein.

The scintillating fiber focal plane detector for the use of Kaos as a double arm spectrometer

The upgrade of the Mainz Mikrotron (MAMI) electron accelerator facility in 2007 which raised the beam energy up to 1.5,GeV, gives the opportunity to study strangeness production channels through electromagnetic process. The Kaon Spectrometer (KAOS) managed by the A1 Collaboration, enables the efficient detection of the kaons associated with strangeness electroproduction. Used as a single arm spectrometer, it can be combined with the existing high-resolution spectrometers for exclusive measurements in the kinematic domain accessible to them.rnrnFor studying hypernuclear production in the ^A Z(e,e'K^+) _Lambda ^A(Z-1) reaction, the detection of electrons at very forward angles is needed. Therefore, the use of KAOS as a double-arm spectrometer for detection of kaons and the electrons at the same time is mandatory. Thus, the electron arm should be provided with a new detector package, with high counting rate capability and high granularity for a good spatial resolution. To this end, a new state-of-the-art scintillating fiber hodoscope has been developed as an electron detector.rnrnThe hodoscope is made of two planes with a total of 18432 scintillating double-clad fibers of 0.83 mm diameter. Each plane is formed by 72 modules. Each module is formed from a 60deg slanted multi-layer bundle, where 4 fibers of a tilted column are connected to a common read out. The read-out is made with 32 channels of linear array multianode photomultipliers. Signal processing makes use of newly developed double-threshold discriminators. The discriminated signal is sent in parallel to dead-time free time-to-digital modules and to logic modules for triggering purposes.rnrnTwo fiber modules were tested with a carbon beam at GSI, showing a time resolution of 220 ps (FWHM) and a position residual of 270 microm m (FWHM) with a detection efficiency epsilon>99%.rnrnThe characterization of the spectrometer arm has been achieved through simulations calculating the transfer matrix of track parameters from the fiber detector focal plane to the primary vertex. This transfer matrix has been calculated to first order using beam transport optics and has been checked by quasielastic scattering off a carbon target, where the full kinematics is determined by measuring the recoil proton momentum. The reconstruction accuracy for the emission parameters at the quasielastic vertex was found to be on the order of 0.3 % in first test realized.rnrnThe design, construction process, commissioning, testing and characterization of the fiber hodoscope are presented in this work which has been developed at the Institut für Kernphysik of the Johannes Gutenberg - Universität Mainz.

Geomorphological and statistical analysis on Ravenna dune fields changes, based on Terrestrial Laser Technology

Coastal sand dunes represent a richness first of all in terms of defense from the sea storms waves and the saltwater ingression; moreover these morphological elements constitute an unique ecosystem of transition between the sea and the land environment. The research about dune system is a strong part of the coastal sciences, since the last century. Nowadays this branch have assumed even more importance for two reasons: on one side the born of brand new technologies, especially related to the Remote Sensing, have increased the researcher possibilities; on the other side the intense urbanization of these days have strongly limited the dune possibilities of development and fragmented what was remaining from the last century. This is particularly true in the Ravenna area, where the industrialization united to the touristic economy and an intense subsidence, have left only few dune ridges residual still active. In this work three different foredune ridges, along the Ravenna coast, have been studied with Laser Scanner technology. This research didn’t limit to analyze volume or spatial difference, but try also to find new ways and new features to monitor this environment. Moreover the author planned a series of test to validate data from Terrestrial Laser Scanner (TLS), with the additional aim of finalize a methodology to test 3D survey accuracy. Data acquired by TLS were then applied on one hand to test some brand new applications, such as Digital Shore Line Analysis System (DSAS) and Computational Fluid Dynamics (CFD), to prove their efficacy in this field; on the other hand the author used TLS data to find any correlation with meteorological indexes (Forcing Factors), linked to sea and wind (Fryberger's method) applying statistical tools, such as the Principal Component Analysis (PCA).

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF(164) levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Do women have impaired regional systolic function in hypertensive heart disease? A 3-dimensional echocardiography study

AIMS: In pressure overload left ventricular (LV) hypertrophy, gender-related differences in global LV systolic function have been previously reported. The goal of this study was to determine regional systolic function of the left ventricle in male and female patients with hypertensive heart disease. METHODS AND RESULTS: Regional LV function was analyzed from multiplane transesophageal echocardiography with three-dimensional (3D) reconstruction of the left ventricle. In 24 patients (13 males and 11 females), four parallel (2 basal and 2 apical) equidistant short axis cross-sections from base to apex were obtained from the reconstructed LV. In each short axis 24 wall-thickness measurements were carried out at 15 degrees intervals at end-diastole and end-systole. Thus, a total of 192 measurements were obtained in each patient. Wall thickening was calculated as difference of end-diastolic and end-systolic wall thickness, and fractional thickening as thickening divided by end-diastolic thickness. Fractional thickening and wall stress were inversely related to end-diastolic wall thickness in both, males and females. Females showed less LV systolic function when compared to males (p<0.001). However, when corrected for wall stress, which was higher in females, there was no gender difference in systolic function. CONCLUSION: There are regional differences in LV systolic function in females and males which are directly related to differences in wall stress. Thus, gender-related differences in LV regional function are load-dependent and not due to structural differences.

Digital forensic osteology--possibilities in cooperation with the Virtopsy project

The present study was carried out to check whether classic osteometric parameters can be determined from the 3D reconstructions of MSCT (multislice computed tomography) scans acquired in the context of the Virtopsy project. To this end, four isolated and macerated skulls were examined by six examiners. First the skulls were conventionally (manually) measured using 32 internationally accepted linear measurements. Then the skulls were scanned by the use of MSCT with slice thicknesses of 1.25 mm and 0.63 mm, and the 33 measurements were virtually determined on the digital 3D reconstructions of the skulls. The results of the traditional and the digital measurements were compared for each examiner to figure out variations. Furthermore, several parameters were measured on the cranium and postcranium during an autopsy and compared to the values that had been measured on a 3D reconstruction from a previously acquired postmortem MSCT scan. The results indicate that equivalent osteometric values can be obtained from digital 3D reconstructions from MSCT scans using a slice thickness of 1.25 mm, and from conventional manual examinations. The measurements taken from a corpse during an autopsy could also be validated with the methods used for the digital 3D reconstructions in the context of the Virtopsy project. Future aims are the assessment and biostatistical evaluation in respect to sex, age and stature of all data sets stored in the Virtopsy project so far, as well as of future data sets. Furthermore, a definition of new parameters, only measurable with the aid of MSCT data would be conceivable.

Development of the premature infant nose throat-model (PrINT-Model): an upper airway replica of a premature neonate for the study of aerosol delivery

Clinical efficacy of aerosol therapy in premature newborns depends on the efficiency of delivery of aerosolized drug to the bronchial tree. To study the influence of various anatomical, physical, and physiological factors on aerosol delivery in preterm newborns, it is crucial to have appropriate in vitro models, which are currently not available. We therefore constructed the premature infant nose throat-model (PrINT-Model), an upper airway model corresponding to a premature infant of 32-wk gestational age by three-dimensional (3D) reconstruction of a three-planar magnetic resonance imaging scan and subsequent 3D-printing. Validation was realized by visual comparison and comparison of total airway volume. To study the feasibility of measuring aerosol deposition, budesonide was aerosolized through the cast and lung dose was expressed as percentage of nominal dose. The airway volumes of the initial magnetic resonance imaging and validation computed tomography scan showed a relative deviation of 0.94%. Lung dose at low flow (1 L/min) was 61.84% and 9.00% at high flow (10 L/min), p < 0.0001. 3D-reconstruction provided an anatomically accurate surrogate of the upper airways of a 32-wk-old premature infant, making the model suitable for future in vitro testing.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

Registration of Sub-Sequence and Multi-Camera Reconstructions for Camera Motion Estimation

This paper presents different application scenarios for which the registration of sub-sequence reconstructions or multi-camera reconstructions is essential for successful camera motion estimation and 3D reconstruction from video. The registration is achieved by merging unconnected feature point tracks between the reconstructions. One application is drift removal for sequential camera motion estimation of long sequences. The state-of-the-art in drift removal is to apply a RANSAC approach to find unconnected feature point tracks. In this paper an alternative spectral algorithm for pairwise matching of unconnected feature point tracks is used. It is then shown that the algorithms can be combined and applied to novel scenarios where independent camera motion estimations must be registered into a common global coordinate system. In the first scenario multiple moving cameras, which capture the same scene simultaneously, are registered. A second new scenario occurs in situations where the tracking of feature points during sequential camera motion estimation fails completely, e.g., due to large occluding objects in the foreground, and the unconnected tracks of the independent reconstructions must be merged. In the third scenario image sequences of the same scene, which are captured under different illuminations, are registered. Several experiments with challenging real video sequences demonstrate that the presented techniques work in practice.

A complementary radiographic projection of the equine temporomandibular joint

The complexity of the equine skull makes the temporomandibular joint a difficult area to evaluate radiographically. The goal of this study was to determine the optimal angle for a complementary radiographic projection of the equine temporomandibular joint based on a computed tomography (CT) cadaver study. CT was performed on six equine cadaver heads of horses that were euthanized for other reasons than temporomandibular joint disease. After the CT examination, 3D reconstruction of the equine skull was performed to subjectively determine the angle for a complementary radiographic projection of the temporomandibular joint. The angle was measured on the left and right temporomandibular joint of each head. Based on the measurements obtained from the CT images, a radiographic projection of the temporomandibular joint in a rostra-145 degrees ventral-caudodorsal oblique (R45 degrees V-CdDO) direction was developed by placing the X-ray unit 30 degrees laterally, maintaining at the same time the R45 degrees V-CdDO angle (R45 degrees V30 degrees L-CdDLO). This radiographic projection was applied to all cadaver heads and on six live horses. In three of the live horses abnormal findings associated with the temporomandibular joint were detected. We conclude that this new radiographic projection of the temporomandibular joint provides superior visualization of the temporomandibular joint space and the articular surface of the mandibular condyle.

Patient-specific spinal stiffness in AIS: a preoperative and noninvasive method.

INTRODUCTION The clinical tests currently used to assess spinal biomechanics preoperatively are unable to assess true mechanical spinal stiffness. They rely on spinal displacement without considering the force required to deform a patient's spine. We propose a preoperative method for noninvasively quantifying the three-dimensional patient-specific stiffness of the spines of adolescent idiopathic scoliosis patients. METHODS The technique combines a novel clinical test with numerical optimization of a finite element model of the patient's spine. RESULTS A pilot study conducted on five patients showed that the model was able to provide accurate 3D reconstruction of the spine's midline and predict the spine's stiffness for each patient in flexion, bending, and rotation. Statistically significant variation of spinal stiffness was observed between the patients. CONCLUSION This result confirms that spinal biomechanics is patient-specific, which should be taken into consideration to individualize surgical treatment.