Browsing by Subject "histochemistry"
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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 Molecular and Biochemical Characterization of Hydrocarbon Production in the Green Microalga Botryococcus braunii(2012-10-19) Weiss, Taylor LeighBotryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 +/- 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 +/- 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 +/- 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600?1700 cm^-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm^-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm^-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells.