Charakterisierung und Expression zweier multimerer Hämolympheproteine der Schnecke Biomphalaria glabrata

Die tropische Süsswasserschnecke Biomphalaria glabrata gehört zu der Familie der Planorbidae, welche als einziges Taxon der Gastropoden Hämoglobin als Sauerstofftransportprotein verwenden. Als Zwischenwirt des Bilharzioseerregers Schistosoma mansoni ist B. glabrata von tropenmedizinischer Interesse. Das extrazelluläre BgHb zeigt sich mit einem Anteil von 95% als Hauptprotein in der Hämolymphe. Dieses setzt sich aus Polypeptidketten mit je 240kDa zusammen. Diese wiederrum lassen sich in 13-Häm-Domänen und eine deutlich kleinere N-terminalen nicht Häm-Domäne untergliedern. Die Sequenzierung von zwei der drei Untereinheiten des BgHb (BgHb1, BgHb2) ermöglichte die rekombinante Expression ganzer Untereinheiten in Insektenzellen, und die Expression einiger BgHb2-Konstrukte in E. coli Zellen. Im Rahmen meiner Arbeit gelang es, BgHb1 in biologisch aktiver Form in Insektenzellen zu exprimieren. Das aus dem Überstand der Insektenzellen aufgereinigte rekombinante BgHb1 zeigte eine immunologische Identität mit nativen BgHb. Strukturelle Analysen belegten zudem die Assemblierung des rekombinanten BgHb1 zu einer dem nativen Protein gleichenden Quartärstruktur. Demnach konnte in meiner Arbeit der Nachweis erbracht werden, dass eine einzelne Isoform in der Lage ist, zur Quartärstruktur zu assemblieren. Zusätzlich ergaben Sauerstoffbindungsanalysen, dass das rekombinante BgHb1 reversibel Sauerstoff binden kann.rnIn den restlichen 5% der B. glabrata Hämolymphe zeigt sich ein rudimentäres Hämocyanin, welches für den Sauerstofftransport keine Rolle zu spielen scheint, und ein rosettenförmiges Protein, das es aufzuklären galt. Durch massenspektrometrische Analysen erhaltene Peptidfragmente zeigten eine hohe Sequenzähnlichkeit zu den löslichen Acetylcholin -Bindeproteinen anderer Mollusken. Diese AChBP zeigen eine hohe Sequenzähnlichkeit zur Ligandenbindedomäne von Rezeptoren der Cys-Loop-Proteinfamilie.rnDatenbankrecherchen deckten die Existenz zweier Isoformen auf

Investigation of the Hygroscopic and Morphological Properties of Atmospheric Aerosols

The first objective of this thesis was to examine the hygroscopic and morphological nature of various substances through the use of an Environmental Scanning Electron Microscope (ESEM). The hygroscopic growth and changes in morphology for pure-component aerosols were studied for particles greater than 2 µm in size. Hygroscopic growth was observed through changes in relative humidity (RH) and hygroscopic growth curves were created. The second objective of this thesis, the hygroscopic growth of multi-component aerosol mixtures, was studied using Hygroscopic Tandem Differential Mobility Analysis(HTDMA). The size distribution for an aerosol stream was determined before and after the stream was subjected to an increase in relative humidity.

Cryoultramicrotomy

Cryoultramicrotomy allows the sectioning of vitrified biological samples. These biological samples are preserved at the atomic level and represent the real structure at the moment of freezing. Cryoultramicrotomy produces ultra-thin cryosections that are investigated in a cryoelectron microscope. The necessity of working during the whole preparation at temperatures less than −140°C results in some difficulties, including the cryosection transfer from the knife-edge to the electron microscropy grid; the grid handling in the cryochamber and the grid transfer into the cryoholder of the electron microscope. Furthermore, ice crystal contamination (from air humidity) can obscure the structures of interest in the sections. It is mainly know-how and experience that will prevent the contamination of ice crystals and the recrystallization of the sections during the manipulations. Here, we describe the tips, tricks, the tools, and methods that help to overcome these burdens and pave the path for successful cryoultramicrotomy.

Evaluation of cavitations in proximal caries lesions at various magnification levels in vitro

OBJECTIVES: The aim of this study was to evaluate the cavitation rate of proximal caries using different magnification aids in vitro. METHODS: Radiographs of 285 extracted teeth were taken and the proximal surfaces were graded to the criteria R0 (no radiolucency), R1 (radiolucency confined to the outer half of enamel), R2 (inner half of enamel) and R3 (outer half of dentin). Subsequently, the proximal surfaces were checked for the presence of cavitations with the naked eye (NE), and by using 4.3 x magnification eyeglasses (ME), a stereo microscope (SM, 10x), or a scanning electron microscope (SEM, up to 2000 x magnification). RESULTS: In surfaces with R3 caries, cavitations were visible in 56 of 59 cases with the naked eye. When using SEM, all surfaces revealed cavitations (100%). Regarding the surfaces with R2 lesion, 36 of 46 cases showed cavitations (NE); the corresponding values were 39/46 (ME), 41/46 (SM), and 46/46 (SEM); in the latter, in most cases deep defects could be observed. With regard to R1 lesions, 36/60 (NE), 43/60 (ME), 45/60 (SM), and 58/60 (SEM) cases revealed cavitations. A breakdown of radiographically sound surfaces (R0) was present in some 10% of the examined surfaces (24/261, NE; 33/261, SEM). CONCLUSIONS: Cavitations (defined as breakdown of the surface) are present in significantly more cases than previously reported. This might be an explanation why even small radiolucencies tend to progress, albeit slowly. Thus, close follow-ups should strongly be recommended when considering a preventive treatment regimen with small radiolucencies.

Immunodetection of bone-related proteins in microcalcifications of human dental pulp

Objective: Root canal obliterations may pose esthetic and clinical problems or may even be a risk factor for tooth survival. Microcalcifications in the pulp can be so extensive that the entire root canal system becomes obliterated. Since bone sialoprotein (BSP) and osteopontin (OPN) are involved in both physiological and pathological mineralization processes, our hypothesis was that these two bone-related noncollagenous proteins are present in microcalcifications of the pulp. The purpose of this study was, therefore, to characterize the nature of microcalcifications in the pulp of aged human teeth. Methods: From a large collection of human teeth, 10 were found to exhibit pulpal microcalcifications. The teeth were extracted for periodontal reasons from 39-60 year old patients. After fixation in aldehydes and decalcification, teeth were processed for embedding in LR White resin for analysis in the light and transmission electron microscope. For the detection of BSP and OPN, post-embedding high resolution immunocytochemistry was applied. Results: The microcalcifications were round or elongated, occasionally coalescing, and intensely stained with toluidine blue. Collagen fibrils were found in most but not all microcalcifications. All microcalcifications were immunoreactive for both antibodies and showed an identical labeling pattern. Gold particle labeling was extensively found throughout the interfibrillar ground substance of the microcalcifications, whereas the dentin matrix lacked immunolabeling. Conclusion: BSP and OPN appear to be major matrix constituents of pulp microcalcifications and may thus, like in other mineralized tissues, be involved in their mineralization process.

Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.

Integration of computational models and experimental characterization to study internal frost damage in cementitious materials

The objective of this doctoral research is to investigate the internal frost damage due to crystallization pore pressure in porous cement-based materials by developing computational and experimental characterization tools. As an essential component of the U.S. infrastructure system, the durability of concrete has significant impact on maintenance costs. In cold climates, freeze-thaw damage is a major issue affecting the durability of concrete. The deleterious effects of the freeze-thaw cycle depend on the microscale characteristics of concrete such as the pore sizes and the pore distribution, as well as the environmental conditions. Recent theories attribute internal frost damage of concrete is caused by crystallization pore pressure in the cold environment. The pore structures have significant impact on freeze-thaw durability of cement/concrete samples. The scanning electron microscope (SEM) and transmission X-ray microscopy (TXM) techniques were applied to characterize freeze-thaw damage within pore structure. In the microscale pore system, the crystallization pressures at sub-cooling temperatures were calculated using interface energy balance with thermodynamic analysis. The multi-phase Extended Finite Element Modeling (XFEM) and bilinear Cohesive Zone Modeling (CZM) were developed to simulate the internal frost damage of heterogeneous cement-based material samples. The fracture simulation with these two techniques were validated by comparing the predicted fracture behavior with the captured damage from compact tension (CT) and single-edge notched beam (SEB) bending tests. The study applied the developed computational tools to simulate the internal frost damage caused by ice crystallization with the two dimensional (2-D) SEM and three dimensional (3-D) reconstructed SEM and TXM digital samples. The pore pressure calculated from thermodynamic analysis was input for model simulation. The 2-D and 3-D bilinear CZM predicted the crack initiation and propagation within cement paste microstructure. The favorably predicted crack paths in concrete/cement samples indicate the developed bilinear CZM techniques have the ability to capture crack nucleation and propagation in cement-based material samples with multiphase and associated interface. By comparing the computational prediction with the actual damaged samples, it also indicates that the ice crystallization pressure is the main mechanism for the internal frost damage in cementitious materials.

Preparation of highly-ordered TiO₂ nanotube arrays and their applications in Dye-sensitized solar cells

Titanium oxide is an important semiconductor, which is widely applied for solar cells. In this research, titanium oxide nanotube arrays were synthesized by anodization of Ti foil in the electrolyte composed of ethylene glycol containing 2 vol % H2O and 0.3 wt % NH4F. The voltages of 40V-50V were employed for the anodizing process. Pore diameters and lengths of the TiO2 nanotubes were evaluated by field emission scanning electron microscope (FESEM). The obtained highly-ordered titanium nanotube arrays were exploited to fabricate photoelectrode for the Dye-sensitized solar cells (DSSCS). The TiO2 nanotubes based DSSCS exhibited an excellent performance with a high short circuit current and open circuit voltage as well as a good power conversion efficiency. Those can be attributed to the high surface area and one dimensional structure of TiO2 nanotubes, which could hold a large amount of dyes to absorb light and help electron percolation process to hinder the recombination during the electrons diffusion in the electrolyte.

In vitro precision of fit of computer-aided design and computer-aided manufacturing titanium and zirconium dioxide bars

OBJECTIVES Optical scanners combined with computer-aided design and computer-aided manufacturing (CAD/CAM) technology provide high accuracy in the fabrication of titanium (TIT) and zirconium dioxide (ZrO) bars. The aim of this study was to compare the precision of fit of CAD/CAM TIT bars produced with a photogrammetric and a laser scanner. METHODS Twenty rigid CAD/CAM bars were fabricated on one single edentulous master cast with 6 implants in the positions of the second premolars, canines and central incisors. A photogrammetric scanner (P) provided digitized data for TIT-P (n=5) while a laser scanner (L) was used for TIT-L (n=5). The control groups consisted of soldered gold bars (gold, n=5) and ZrO-P with similar bar design. Median vertical distance between implant and bar platforms from non-tightened implants (one-screw test) was calculated from mesial, buccal and distal scanning electron microscope measurements. RESULTS Vertical microgaps were not significantly different between TIT-P (median 16μm; 95% CI 10-27μm) and TIT-L (25μm; 13-32μm). Gold (49μm; 12-69μm) had higher values than TIT-P (p=0.001) and TIT-L (p=0.008), while ZrO-P (35μm; 17-55μm) exhibited higher values than TIT-P (p=0.023). Misfit values increased in all groups from implant position 23 (3 units) to 15 (10 units), while in gold and TIT-P values decreased from implant 11 toward the most distal implant 15. SIGNIFICANCE CAD/CAM titanium bars showed high precision of fit using photogrammetric and laser scanners. In comparison, the misfit of ZrO bars (CAM/CAM, photogrammetric scanner) and soldered gold bars was statistically higher but values were clinically acceptable.

In vitro precision of fit of computer-aided designed and computer-aided manufactured titanium screw-retained fixed dental prostheses before and after ceramic veneering

OBJECTIVE To compare the precision of fit of full-arch implant-supported screw-retained computer-aided designed and computer-aided manufactured (CAD/CAM) titanium-fixed dental prostheses (FDP) before and after veneering. The null-hypothesis was that there is no difference in vertical microgap values between pure titanium frameworks and FDPs after porcelain firing. MATERIALS AND METHODS Five CAD/CAM titanium grade IV frameworks for a screw-retained 10-unit implant-supported reconstruction on six implants (FDI tooth positions 15, 13, 11, 21, 23, 25) were fabricated after digitizing the implant platforms and the cuspid-supporting framework resin pattern with a laser scanner (CARES(®) Scan CS2; Institut Straumann AG, Basel, Switzerland). A bonder, an opaquer, three layers of porcelain, and one layer of glaze were applied (Vita Titankeramik) and fired according to the manufacturer's preheating and fire cycle instructions at 400-800°C. The one-screw test (implant 25 screw-retained) was applied before and after veneering of the FDPs to assess the vertical microgap between implant and framework platform with a scanning electron microscope. The mean microgap was calculated from interproximal and buccal values. Statistical comparison was performed with non-parametric tests. RESULTS All vertical microgaps were clinically acceptable with values <90 μm. No statistically significant pairwise difference (P = 0.98) was observed between the relative effects of vertical microgap of unveneered (median 19 μm; 95% CI 13-35 μm) and veneered FDPs (20 μm; 13-31 μm), providing support for the null-hypothesis. Analysis within the groups showed significantly different values between the five implants of the FDPs before (P = 0.044) and after veneering (P = 0.020), while a monotonous trend of increasing values from implant 23 (closest position to screw-retained implant 25) to 15 (most distant implant) could not be observed (P = 0.169, P = 0.270). CONCLUSIONS Full-arch CAD/CAM titanium screw-retained frameworks have a high accuracy. Porcelain firing procedure had no impact on the precision of fit of the final FDPs. All implant microgap measurements of each FDP showed clinically acceptable vertical misfit values before and after veneering. Thus, the results do not only show accurate performance of the milling and firing but show also a reproducible scanning and designing process.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

In situ micropillar compression reveals superior strength and ductility but an absence of damage in lamellar bone

Ageing societies suffer from an increasing incidence of bone fractures. Bone strength depends on the amount of mineral measured by clinical densitometry, but also on the micromechanical properties of the hierarchical organization of bone. Here, we investigate the mechanical response under monotonic and cyclic compression of both single osteonal lamellae and macroscopic samples containing numerous osteons. Micropillar compression tests in a scanning electron microscope, microindentation and macroscopic compression tests were performed on dry ovine bone to identify the elastic modulus, yield stress, plastic deformation, damage accumulation and failure mechanisms. We found that isolated lamellae exhibit a plastic behaviour, with higher yield stress and ductility but no damage. In agreement with a proposed rheological model, these experiments illustrate a transition from a ductile mechanical behaviour of bone at the microscale to a quasi-brittle response driven by the growth of cracks along interfaces or in the vicinity of pores at the macroscale.

Resin composites: Modulus of elasticity and marginal quality

OBJECTIVE To investigate how the modulus of elasticity of resin composites influences marginal quality in restorations submitted to thermocyclic and mechanical loading. METHODS Charisma, Filtek Supreme XTE and Grandio were selected as they were found to possess different moduli of elasticity but quite similar polymerization contraction. MOD cavities (n=30) were prepared in extracted premolars, restored and then subjected to thermocyclic and mechanical loading. Marginal quality of the restorations before and after loading was analyzed on epoxy replicas under a scanning electron microscope. The percentage of gap-free margins and occurrence of paramarginal fractures were registered. Modulus of elasticity and polymerization contraction were analyzed with parametric and margins with nonparametric ANOVA and post hoc Tukey HSD or Wilcoxon rank-sum tests, respectively. The number of paramarginal fractures was analyzed with exact Fisher tests (α=0.05). RESULTS Grandio demonstrated significantly more gap-free enamel margins than Charisma and Filtek Supreme XTE, before and after loading (p<0.01), whereas there was no difference between Charisma and Filtek Supreme XTE (p>0.05). No significant effect of resin composite (p=0.81) on the quality of dentine margins was observed, before or after loading. Deterioration of all margins was evident after loading (p<0.0001). More paramarginal enamel fractures were observed after loading in teeth restored with Grandio when compared to Charisma (p=0.008). CONCLUSIONS The resin composite with the highest modulus of elasticity resulted in the highest number of gap-free enamel margins but with an increased incidence of paramarginal enamel fractures. CLINICAL SIGNIFICANCE The results from this study suggest that the marginal quality of restorations can be improved by the selection of a resin composite with modulus of elasticity close to that of dentine, although an increase in paramarginal enamel fractures can result as a consequence.

Experimental, theoretical and numerical investigation of the nonlinear micromechanical properties of bone

Aging societies suffer from an increasing incidence of bone fractures. Bone strength depends on the amount of mineral measured by clinical densitometry, but also on the micromechanical properties of the bone hierarchical organization. A good understanding has been reached for elastic properties on several length scales, but up to now there is a lack of reliable postyield data on the lower length scales. In order to be able to describe the behavior of bone at the microscale, an anisotropic elastic-viscoplastic damage model was developed using an eccentric generalized Hill criterion and nonlinear isotropic hardening. The model was implemented as a user subroutine in Abaqus and verified using single element tests. A FE simulation of microindentation in lamellar bone was finally performed show-ing that the new constitutive model can capture the main characteristics of the indentation response of bone. As the generalized Hill criterion is limited to elliptical and cylindrical yield surfaces and the correct shape for bone is not known, a new yield surface was developed that takes any convex quadratic shape. The main advantage is that in the case of material identification the shape of the yield surface does not have to be anticipated but a minimization results in the optimal shape among all convex quadrics. The generality of the formulation was demonstrated by showing its degeneration to classical yield surfaces. Also, existing yield criteria for bone at multiple length scales were converted to the quadric formulation. Then, a computational study to determine the influence of yield surface shape and damage on the in-dentation response of bone using spherical and conical tips was performed. The constitutive model was adapted to the quadric criterion and yield surface shape and critical damage were varied. They were shown to have a major impact on the indentation curves. Their influence on indentation modulus, hardness, their ratio as well as the elastic to total work ratio were found to be very well described by multilinear regressions for both tip shapes. For conical tips, indentation depth was not a significant fac-tor, while for spherical tips damage was insignificant. All inverse methods based on microindentation suffer from a lack of uniqueness of the found material properties in the case of nonlinear material behavior. Therefore, monotonic and cyclic micropillar com-pression tests in a scanning electron microscope allowing a straightforward interpretation comple-mented by microindentation and macroscopic uniaxial compression tests were performed on dry ovine bone to identify modulus, yield stress, plastic deformation, damage accumulation and failure mecha-nisms. While the elastic properties were highly consistent, the postyield deformation and failure mech-anisms differed between the two length scales. A majority of the micropillars showed a ductile behavior with strain hardening until failure by localization in a slip plane, while the macroscopic samples failed in a quasi-brittle fashion with microcracks coalescing into macroscopic failure surfaces. In agreement with a proposed rheological model, these experiments illustrate a transition from a ductile mechanical behavior of bone at the microscale to a quasi-brittle response driven by the growth of preexisting cracks along interfaces or in the vicinity of pores at the macroscale. Subsequently, a study was undertaken to quantify the topological variability of indentations in bone and examine its relationship with mechanical properties. Indentations were performed in dry human and ovine bone in axial and transverse directions and their topography measured by AFM. Statistical shape modeling of the residual imprint allowed to define a mean shape and describe the variability with 21 principal components related to imprint depth, surface curvature and roughness. The indentation profile of bone was highly consistent and free of any pile up. A few of the topological parameters, in particular depth, showed significant correlations to variations in mechanical properties, but the cor-relations were not very strong or consistent. We could thus verify that bone is rather homogeneous in its micromechanical properties and that indentation results are not strongly influenced by small de-viations from the ideal case. As the uniaxial properties measured by micropillar compression are in conflict with the current literature on bone indentation, another dissipative mechanism has to be present. The elastic-viscoplastic damage model was therefore extended to viscoelasticity. The viscoelastic properties were identified from macroscopic experiments, while the quasistatic postelastic properties were extracted from micropillar data. It was found that viscoelasticity governed by macroscale properties has very little influence on the indentation curve and results in a clear underestimation of the creep deformation. Adding viscoplasticity leads to increased creep, but hardness is still highly overestimated. It was possible to obtain a reasonable fit with experimental indentation curves for both Berkovich and spherical indenta-tion when abandoning the assumption of shear strength being governed by an isotropy condition. These results remain to be verified by independent tests probing the micromechanical strength prop-erties in tension and shear. In conclusion, in this thesis several tools were developed to describe the complex behavior of bone on the microscale and experiments were performed to identify its material properties. Micropillar com-pression highlighted a size effect in bone due to the presence of preexisting cracks and pores or inter-faces like cement lines. It was possible to get a reasonable fit between experimental indentation curves using different tips and simulations using the constitutive model and uniaxial properties measured by micropillar compression. Additional experimental work is necessary to identify the exact nature of the size effect and the mechanical role of interfaces in bone. Deciphering the micromechanical behavior of lamellar bone and its evolution with age, disease and treatment and its failure mechanisms on several length scales will help preventing fractures in the elderly in the future.

Microstructural and mechanical characterization of contemporary lingual orthodontic brackets.

OBJECTIVES To investigate the composition and the microstructural and mechanical characterization of three different types of lingual brackets. MATERIALS AND METHODS Incognito™ (3M Unitek), In-Ovation L (DENTSPLY GAC) and STb™ (Light Lingual System, ORMCO) lingual brackets were studied under the scanning electron microscope employing backscattered electron imaging and their elemental composition was analysed by energy-dispersive X-ray microanalysis. Additionally, Vickers hardness was assessed using a universal hardness-testing machine, and the indentation modulus was measured according to instrumented indentation test. Two-way analysis of variance was conducted employing bracket type and location (base and wing) as discriminating variable. Significant differences among groups were allocated by post hoc Student-Newman-Keuls multiple comparison analysis at 95% level of significance. RESULTS Three different phases were identified for Incognito and In-Ovation L bracket based on mean atomic number contrast. On the contrary, STb did not show mean atomic contrast areas and thus it is recognized as a single phase. Incognito is a one-piece bracket with the same structure in wing and base regions. Incognito consists mainly of noble metals while In-Ovation L and STb show similar formulations of ferrous alloys in wing and base regions. No significant differences were found between ferrous brackets in hardness and modulus values, but there were significant differences between wing and base regions. Incognito illustrated intermediate values with significant differences from base and wing values of ferrous brackets. CONCLUSIONS/IMPLICATIONS Significant differences exist in microstructure, elemental composition, and mechanical properties among the brackets tested; these might have a series of clinical implications during mechanotherapy.