Beurteilung von nativen und aufgetauten Spermatozoen fertiler und subfertiler Hengste mit Hilfe der Phasenkontrast- und Transmissionselektronenmikroskopie. Institut für Veterinär-Pathologie der Veterinärmedizinischen Fakultät, Universität Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In dieser Arbeit wurde die Ultrastruktur von nativen und tiefgefrorenen Spermien mittels Phasenkontrast- und Transmissionselektronenmikroskopie (TEM) untersucht. Für die Beurteilung der Spermienmotilität und der Morphologie von in Formolzitrat fixierten Spermien standen jeweils drei Ejakulate von 50 Hannoveraner Hengsten des Niedersächsischen Landgestüts Celle zur Verfügung. Aus dieser Gruppe wurden drei fertile, drei subfertile Hengste und 6 Hengste mittlerer Fertilität ausgewählt, von denen sowohl die Nativ-Proben als auch eine Tiefgefrierprobe (TG-Probe) für die TEM im Institut für Veterinär-Pathologie der Universität Leipzig gemäß des Standardprotokolls des Institutes aufbereitet wurden. Die Spermien wurden gewaschen und das Seminalplasma der nativen Proben oder der Verdünner der TG-Proben abpipettiert und durch eine 5%-ige Glutaraldehydlösung in einem 0,1 M Kakodylatpuffer (pH 7,2) ersetzt. Die Fixierungslösung wurde anschließend entfernt und das Pellet gewaschen und danach mit Gelatine gemischt. Die spermienreichen Stellen wurden aus der Gelatine herausgeschnitten und in Glutaraldehyd aufbewahrt. Nach einer Nachfixierung in OsO4 und einer Entwässerung in Ethanollösungen erfolgte eine Einbettung in einer Eponmischung. Nach einer Polymerisation von 5 Tagen wurden die eingebetteten Eponblöckchen angetrimmt und die Semi- und Ultradünnschnitte angefertigt. Die Ultradünnschnitte wurden auf ein Kupfergrid gelegt, mit Uranylazetat und Bleizitrat kontrastiert und mit dem Transmissionselektronenmikroskop (Zeiss EM 900, Oberkochem) bei 80 kV analysiert. In den nativen Proben wurden insgesamt 360 Spermien pro Hengst beurteilt, in den TG-Proben 120 Spermien pro Hengst. Die Qualität der elektronenmikroskopischen Aufnahmen war sehr gut, doch die Plasmamembran zeigte fixierungsbedingte Artefakte. Nach dem Auftauen waren die Bilder heller und der Kontrast etwas geringer. Es gab eine Zunahme an Akrosomdefekten, akrosomreagierten Spermien und Beschädigungen der Plasmamembran, der Mitochondrien, sowie der Mantel- und Ringfasern. Durch die Membranbeschädigungen trat auch eine Verringerung der Anzahl proximaler und distaler Zytoplasmatropfen auf. Sowohl geschwollene Akrosome mit einer niedrigeren Dichte der akrosomalen Matrix als auch Mitochondrien mit einer zu hellen mitochondrialen Matrix waren typische Befunde in den TG-Proben. Die Studie der Ultrastruktur und die wahrgenommenen Defekte führten zur Erstellung eines Standardprotokolls für die transmissionselektronenmikroskopische Beurteilung von Hengstspermien. Die Beurteilung mittels TEM sollte aber nicht zu einer quantitativen, sondern zu einer qualitativen Aussage führen. Sie ermöglicht die Diagnose von Kern- (Kerndeformationen und Taschenbildung im Kern) und Akrosomabweichungen (deformierte Akrosome mit oder ohne Vakuolenbildung, abgehobene Akrosome und akrosomreagierte Spermien), Anomalien der Mitochondrien (Unterbrechung der Mitochondrienscheide, zu viele Mitochondrien, anormale Dichte der mitochondrialen Matrix), Defekten des Axonemas (Ordnung oder Anzahl der Mikrotubuli, Mantel- und Ringfasern) und der Anwesenheit immaturer Spermienvorstufen. Diese Methode eignet sich für die Diagnostik subfertiler Hengste mit normalen Spermienparametern bei der routinemäßige Spermienbeurteilung und kann sowohl in nativen als auch in TG-Proben angewendet werden. Im Vergleich zur Phasenkontrastmikroskopie waren die elektronenmikroskopischen Bilder wegen ihrer stärkeren Vergrößerung und der Darstellung innerer Spermienstrukturen viel aussagekräftiger. Für die Beurteilung von Halsansatzdefekten, abweichende Geißelformen und Mehrfachmißbildungen ist die Phasenkontrastmikroskopie die am besten geeignete Methode. / Evaluation of fresh and frozen-thawed semen samples of fertile and subfertile stallions by light microscopy and transmission electron microscopy. Institut of Pathology of the Faculty of Veterinary Medicine, University of Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In this study the ultrastructure of fresh and frozen-thawed semen samples of 50 stallions from the National Stud of Lower Saxony (Celle, Germany) were evaluated by light microscopy and transmission electron microscopy (TEM). Three ejaculates of each stallion were available for the motility analysis and the morphological analysis by lightmicroscopy after fixation in formol citrate. Based on the fertility data, the ejaculates of 12 stallions (3 fertile stallions, 3 subfertile stallions and 6 stallions of average fertility) were selected for the morphological analysis by TEM. The native samples and one frozen-thawed sample from these stallions were prepared for the TEM at the Institute of Pathology of the Faculty of Veterinary Medicine, Uni-versity of Leipzig. The sperm cells were washed and the seminal plasma from the native samples and the diluents of the frozen-thawed samples were replaced by a 5%-glutaraldehyde solution in a 0,1 M cacodylate buffer pH 7,2. The fixative was removed, the pellet was washed again and mixed with gelatin. The sperm rich fraction in the gelatin mass was excised and stored in glutaraldehyde. A second fixation in OsO4 was followed by a dehydratation in ethanol and a polymerization phase in epon. After 5 days of polymerization the starred samples were used for semi- and ultratight cuts. The latter were placed on a copper grid, contrasted with uranyl acetate and lead citrate and analyzed with the transmission electron micro-scope (EM 900) by 80 kV. In the fresh samples, 360 sperm cells were examined per stallion, whereas in the frozen-thawed samples only 120 sperm cells per stallion were evaluated. The microscopic pictures were of a high quality. However, the sperm plasma membrane showed some fixation artifacts. In the thawed samples a lower contrast was noticed than in the fresh samples. The sperm cells in the frozen-thawed samples showed an increase in acrosome defects, acrosome reactions, damage of the cell plasma membrane, mitochondria, fibrous sheet and outer dense fibers. The latter defect was associated with a decrease in proximal and distal cytoplasmatic droplets. Swollen acrosomes with a lower matrix density and a bright mitochondrial matrix were typically present in the cryopreserved samples. The ultrastructural defects in these samples, examined by TEM, have led to the development of a standard evaluation protocol with the most common sperm defects in stallion semen. TEM is an expensive and time consuming technique, which cannot be used to obtain quantitative results, but is considered as an accurate method for the qualitative examination of semen samples in cases of unexplained subfertility. TEM can especially be recommended for the diagnosis of nuclear (nuclear malformations and pouches) and acrosomal defects (acrosome deformations, acrosome vacuoles, detached acrosomes and acrosome reactions), mitochondrial (mitochondrial sheet defects, mitochondrial proliferation, decrease in mitochondrial matrix density) and axonema malformations (anormal position or quantity of microtubules and fibrous sheet or outer dense fibers defects) and the detection of immature sperm cells in ejaculates. The results of this study state that TEM can be useful for the evaluation of both fresh and frozen-thawed semen samples. Compared to the light microscopic evaluation of stallion sperm, the TEM images give more precise information because of their higher magnification rate and the ability to reveal internal sperm structures. However, light microscopy remains the best method to detect sperm neck defects, deformed tailes and sperm cells with multiple heads or tails.

The phase transformation of anodized Ti film has been studied. Although X-ray diffraction detected only the amorphous TiO2 phase, transmission electron microscopy analysis showed that TiO nanocrystallites less than 10 nm in size were also present, which was further supported by x-ray photoelectron spectroscopy analysis. Anatase was found to appear gradually by annealing the as-anodized specimen in air at 500¡V550 oC, which was accompanied by a simultaneous disappearance of TiO nanocrystallites. In contrast, only rutile is formed by annealing the Ti film at the same temperatures. The results indicate that TiO can induce the formation of anatase, which is explained by the close similarity between their structures. (Chapter 1) Anatase phase of TiO2 has been shown to have very good biocompability. It was frequently observed on Ti surfaces after anodizing and thermal annealing treatments. In this report the mechanisms of the Ti to TiO and the TiO to anatase phase transitions in anodizing and annealing treatments of Ti have been studied by transmission electron microscopy. Ti thin films of two strong textures were first grown on the (001)NaCl substrates. In addition to amorphous TiO2, the anodization treatment caused the formation of TiO with an orientation relationship of (11-20)Ti // (220)TiO with Ti. The subsequent thermal annealing treatment caused the TiO to anatase transition with an orientation relationship of {200}TiO //{200}A. Pure anatase film was prepared by this method. (Chapter 2)

The orientation relationship and interfaces of PtSn4 and PtSn with the Pt (001) and (111) surfaces have been studied with transmission electron microscopy. Pt was evaporated onto the NaCl (001) and (111) surfaces to form epitaxial Pt thin films and Sn was evaporated onto the Pt films at different temperature to form PtSn4 and PtSn. Pt was evaporated onto the NaCl (001) and (111) surfaces at 350 ¢J to form epitaxial Pt thin films of [001] and [111] zone axes, respectively. Some grains are in random orientation and other as ring pattern. The grain size was at about 10-20 nm. Sn was evaporated onto the Pt surface at 150 ¢J to form PtSn4 and at 200 ¢J to form PtSn. No good orientation relationships were formed on both the PtSn4/ Sn and the PtSn/ Sn interfaces. Heterogeneous nucleation theory, predicts that PtSn should form before PtSn4, but PtSn4 was observed to the first to form. The possible reasons were discussed. Keywords: PtSn4/ Sn interface, PtSn/ Sn interface, orientation relationships, thin films, evaporator, transmission electron microscopy

Amyloid fibrils results from a type of ordered polypeptide aggregation that is associated with ailments such as Alzheimer's disease (AD). Annually, millions of people in the United States alone develop and die from AD. Therefore, it is necessary to understand not only the process of amyloid formation, but also the causes of this specific type of aggregation. This study used ABeta(25-35) since it is a fragment of the Alzheimer?s peptide that behaves like the full length peptide found in patients with AD. To study the process of amyloid formation, several methods were used so that a more complete picture of the stepped aggregation process could be realized. Several oligomeric species were detected and described many of which could not have been observed without using the complete battery of methods utilized here. The oligomeric species detected included a novel 'rolled sheet' that appeared to be the immediate precursor of amyloid fibrils, and two supermolecular species that appear after amyloid fibrils were formed. In determining the causes of amyloid formation, two significant discoveries were made. First, by partial sequence randomization, truncation, and Ala scanning mutagenesis, the critical amyloidogenic region of ABeta(25-35) was found to be residues 30-35. This critical core region is important because it is thought to be the region that initiates amyloid formation, therefore knowing the residues involved in the region is a useful tool for developing methods of fibril formation prevention. Second, by inserting all naturally occurring amino acids into position 34 of ABeta(25-35), three distinct classes of variants were observed and the effect of several physiochemical properties on amyloidosis were examined. Hydrophobicity, solubility, and ?-strand propensity were found to affect aggregation to the greatest extent. Also within these two studies, our results suggest that early oligomers are the cytotoxic species as opposed to amyloid fibrils or other larger macromolecular assemblies.

Ants are capable of building close associations with plants, insects, fungi and bacteria. Symbionts can provide essential nutrients to their insect host, however, the development of new molecular tools has allowed the discovery of new microorganisms that manipulate insect reproduction, development and even provide defense against parasitoids and pathogens. In this study we investigated the presence of bacteria inside the Red Imported Fire Ant midgut using molecular tools and transmission electron microscopy. The midgut bacteria were also characterized by their morphology, biochemical activity, and antibiotic resistance profile. After isolation, culture, and characterization of these bacteria, the molecular analysis revealed ten unique profiles which were identified to at least the genus level, Enterococcus sp./durans, Klebsiella ornithinolytica, Kluyvera cryocrescens, Lactococ-cus garvieae, Pseudomonas aeruginosa, Achromobacter xylosoxidans, Bacillus pumilus, Listeria innucua, Serratia marcescens, and an uncultured bacterium from the Entero-bacteriaceae. New SEM and TEM techniques revealed a possible functional association of endosymbiotic bacteria with the insect host, and it also showed the absence of bacteriocytes in the epithelial cells of the midgut. The PCR results, from the bacteria abundance and distribution studies, showed that Enterococcus sp., Kluyvera cryocres-cens and Lactococcus garvieae are the most abundant species, but they are not consistently found in all sites throughout the southeastern United States. Kluyvera cryocrescens, Serratia marcescens, and an uncultured bacterium (isolate #38: Enterobacteriaceae) were genetically modified with the plasmid vector pZeoDsRed and successfully reintroduced into fire ant colonies. Strong fluorescence of DsRed was detected up to seven days after introduction. The transformed bacteria can still be rescued after pupal emergence; however most were passed out in the meconium. We further demonstrated that nurses contributed to the spread of the transformed bacteria within the colony by feeding the meconium to naive larvae. Although the role of midgut bacteria in the fire ant is still unknown, we have no indication that they cause any pathology. Studies emphasizing the role of these bacteria in fire ant physiology are still ongoing. These results are the foundation for a fire ant biological control program using endosymbiotic bacteria as vectors to introduce foreign genes that express proteins with insecticidal properties.

Iron is an essential element for life. It is involved in a number of biological processes, including iron sulfur (Fe/S) cluster assembly and heme biosynthesis. However it is also potentially toxic due to its ability to induce formation of reactive oxygen species (ROS) via Fenton chemistry. Therefore its uptake, trafficking and utilization must be regulated to avoid its toxicological effect. It has been recently discovered that Fe/S cluster biosynthesis machinery plays a key role in the cellular iron regulation and its disruption leads to impaired iron regulation and iron accumulation within mitochondria. The iron accumulation resulted from impaired Fe/S cluster assembly in the eukaryotic model organism Saccharomyces cerevisiae (baker’s yeast) was studied. Various biophysical (e.g. Mössbauer, EPR, UV-vis spectroscopy) and biochemical (e.g. Western blots, PCR, enzyme activity assay, etc.) techniques were used to characterize the iron content in yeast mitochondria isolated from several mutants strains. In these mutants one of the proteins involved in Fe/S cluster biosynthesis (Yah1p and Atm1p) is mutated and iron regulation and metabolism are disrupted. By integrating the results obtained from these different methods, it was determined that excess iron accumulates in the mutant mitochondria as inorganic phosphate Fe(III) nano-particles exhibiting superparamagnetic behaviors. Oxygen is required for iron accumulation and nanoparticle formation. The Fe(III) nano-particles can be chemically reduced to Fe(II) then largely exported from the mitochondria. These biophysical and biochemical methods were also used to examine the iron distribution in whole yeast cells of the Aft1-1up strain in which iron regulon genes are constitutively activated and compared to that of Yah1p-depleted and wild type yeast. Constitutive activation of iron regulon genes does not alter the cellular iron distribution significantly. However disruption of Fe/S cluster assembly by Yah1p depletion causes dramatic cellular iron redistribution: the vacuolar iron is largely evacuated and most of the cellular iron probably precipitates in mitochondria as Fe(III) nanoparticles. The results provide novel insights into iron trafficking and possible signal communications between organelles within cells.

Four denture base materials of poly(methyl methacrylate) (QC-20, Pladent-20, Hygenic, and Optilon-399) were prepared by convention water bath and microwave-energy cured methods. While the resin was in the dough stage, it was packed into two molds (65 mm ¡¦15 mm ¡¦10 mm) in the fiber reinforced plastic flask. The variation of temperature with time was recorded by two thermocouples during the microwave heating at 80, 160, and 240 watts, respectively. Microwave polymerization was carried out in the same equipment. The microwave flask containing the same size of resin blocks were processed at 80, 160, 240, and 560 watts for 15, 10, 7, and 2 min, separately. Then each flask was turned over, and cured an additional 2 min at 560 watts. In the case of water-bath method, the resin in the dough stage was packed in the Brass flask, and then cured at 70¢J for 9 hours. Ten specimens were prepared for each condition studied. The surface hardness, porosity, flexural properties and solubility of both process conditions were evaluated. The samples were sectioned by microtome and stained 2 % Osmiun tetroxide, then the morphology of Optilon-399 was observed by using TEM (Transmission electron microscopy) at 160 KV. The result indicate that the flexural strength for Optilon-399 specimens prepared by water-bath method was 20 MPa higher than that prepared in microwave oven, however, there were no obvious difference between the samples cured at different power. Phase separation in two different sizes was observed in all of the Optilon-399 specimens. The larger domain was with 0.18 mm~0.67 mm diameter has dispersed rubber phase surrounded by a rubber periphery. The smaller domain with 0.1 mm diameter is rich with rubber phase. The size and distribution of the larger domain were correlated with the microwave power and curing time. The sample cured by water-bath has the largest average domain diameter (0.395¡Ó0.068 mm). In the specimens prepared by microwave method, the domain size decreased with increasing power. In additions, the domain size varied across the specimen. The size difference between the largest and the smallest domain for specimens cured at 80W was 0.03 mm, and that for specimens cured at 560W was 0.05 mm. This indicated that the larger the power watt was, the higher the morphology difference was.

none

Advancements in the semiconductor industry require new materials with improved performance. With the introduction of copper as the interconnect material for integrated circuits, efficient diffusion barriers are required to prevent the diffusion of copper into silicon, which is primarily through grain boundaries. This dissertation reports the processing of high quality stoichiometric thin films of TiN, TaN and HfN, and studies their Cu diffusion barrier properties. Epitaxial metastable cubic TaN (B1-NaCl) thin films were grown on Si(001) using an ultra-thin TiN (B1-NaCl) seed layer which was as thin as 1 nm. The TiN/TaN stacks were deposited by Pulsed Laser Deposition (PLD), with the TiN thickness systematically reduced from 15 to 1 nm. Microstructural studies included X-ray diffraction (XRD), transmission electron microscopy (TEM) and high resolution TEM (HRTEM). Preliminary Cu diffusion experiments showed that the TiN seed layer thickness had little or no obvious effect on the overall microstructure and the diffusion barrier properties of the TaN/TiN stacks. Epitaxial and highly textured cubic HfN (B1-NaCl) thin films (~100 nm) were deposited on MgO(001) and Si(001) using PLD. Low resistivities (~40 mu omega-cm) were measured with a four point probe (FPP). Microstructural characterizations included XRD, TEM, and HRTEM. Preliminary Cu diffusion tests demonstrated good diffusion barrier properties, suggesting that HfN is a promising candidate for Cu diffusion barriers. Cubic HfN (B1-NaCl) thin films were grown epitaxially on Si(001) substrates by using a TiN (B1-NaCl) buffer layer as thin as ~10 nm. The HfN/TiN stacks were deposited by PLD with an overall thickness less than 60 nm. Detailed microstructural characterizations included XRD, TEM, and HRTEM. The electrical resistivity measured by FPP was as low as 70 mu omega-cm. Preliminary copper diffusion tests showed good diffusion barrier properties with a diffusion depth of 2~3 nm after vacuum annealing at 500 degrees C for 30 minutes. Additional samples with Cu deposited on top of the cubic HfN/TiN/Si(001) were vacuum annealed at 500 degrees C, 600 degrees C and 650 degrees C for 30 minutes. The diffusivity of copper in the epitaxial stack was investigated using HRTEM. The measured diffusion depths, 2 Dt , were 3, 4 and 5 nm at 500 degrees C, 600 degrees C and 650 degrees C respectively. Finally, the diffusivity of Cu into epitaxial HfN was determined to be D=D0 exp(-Q/kT)cm2s-1 with D0=2.3x10-14cm2s-1 and Q=0.52eV.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 107-109). Also available on the Internet.