Browsing by Subject "electron microscopy"
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Item A Study of the Process and Causes of Abeta(25-35) Amyloid Formation(2011-02-22) Ridinger, Katherine V.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.Item Immunohistochemical fiber typing, ultrastructure, and morphometry of harbor seal skeletal muscle(Texas A&M University, 2004-09-30) Watson, Rebecca ReikoThere is strong evidence that the skeletal muscles of pinnipeds are adapted for an aerobic, lipid-based metabolism under the hypoxic conditions associated with breath-hold diving. However, regional variations in mitochondrial density are unknown, and the few fiber typing studies performed on pinniped skeletal muscles are not consistent with an aerobic physiological profile. Thus, the objectives of this study were to (1) reexamine the fiber type distribution throughout the primary locomotory muscles of the harbor seal, and (2) to better understand the density and distribution of mitochondria in the locomotory muscles. Multiple samples from transverse sections of the epaxial muscles and a single sample of the pectoralis muscle of wild harbor seals were analyzed using immunohistochemical fiber typing and electron microscopy. Fiber typing results indicated that harbor seal epaxial muscles are composed of 47.4% type I (slow twitch, oxidative) fibers and 52.8%, IIa (fast twitch, oxidative) fibers. No fast twitch, glycolytic (type IIb) fibers were detected in the epaxial muscles or the pectoralis muscle. Mean volume density of mitochondria [Vv(mt,f)] was 5.6%, which is elevated over what would be predicted for a terrestrial mammal of similar mass. The elevated Vv(mt,f) had a high proportion of intermyofibrillar mitochondria, a trait not normally found in the muscles of terrestrial mammals with elevated Vv(mt,f). These results provide further evidence that the elevated mitochondrial volume density in pinniped muscle decreases the oxygen diffusion distance between myoglobin and mitochondria to facilitate aerobic respiration in working muscles. In addition, analyses of heterogeneity revealed that the regions of the epaxial muscles that were located deep within the muscle showed a significantly higher Vv(mt,f) relative to those regions that were superficially-located. In contrast, there was no significant heterogeneity of fiber type detected in either plane of the epaxial muscles. Thus, there was a fine-scale pattern of spatial heterogeneity of Vv(mt,f) within the epaxial muscles that does not manifest in fiber type distribution, indicating that the fibers have similar oxidative capacities.Item Structural studies of the bacteriophage lambda holin and M. tuberculosis secA translocase(2009-05-15) Savva, George ChristosDouble stranded DNA bacteriophages achieve release of phage progeny by disrupting the cell envelope of the host cell. This is accomplished by two phage-encoded proteins, the holin and the endolysin. In bacteriophage lambda, the S holin is a small three TMD membrane protein that creates a lesion in the inner membrane of the host at a specific time, programmed in its primary structure. Lesion formation permits the cytoplasmic endolysin R access to the murein cell wall for degradation and cell lysis. Although it has been shown that S oligomerizes in the membrane, the structural nature of this complex has not been elucidated. In this study the S holin was purified using a mild non-ionic detergent and the structure of a ring complex formed by the holin was determined by electron microscopy and single particle analysis at a resolution of 2.6 nm. Biochemical characterization of the rings suggests that such a complex might represent the assembly formed by S in the membrane. Protein translocation in all organisms allows the export of proteins destined for localization outside the cytoplasm. In eubacteria, newly synthesized proteins are directed to the heterotrimeric membrane complex SecYEG by signals embedded in their sequence. The driving force through this complex is provided by the cytoplasmic ATPase SecA which combines ATP hydrolysis to mechanically insert proteins through the protein conducting channel. Using electron microscopy and single particle analysis we have obtained the structure of SecA from M. tuberculosis. The structure indicates that four SecA monomers assemble to form an elongated molecule with D2 symmetry. Docking of the EM map to the crystal structure of tb SecA confirms this arrangement of the subunits. This finding, that M. tuberculosis SecA forms a tetramer raises intriguing possibilities about SecA function.Item Study of Midgut Bacteria in the Red Imported Fire Ant, Solenopsis invicta B?ren (Hymenoptera: Formicidae)(2011-08-08) Medina, FrederAnts 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.