Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

M.S. / Applied Physics / A noble gas ion laser with strong transitions in the 196-225 nm wavelength region has been developed for use as an illuminator in a photoelectron microscope. The laser is pulsed, and it can be operated at repetition rates up to 200 Hz to produce average output powers up to 5.0 mW at 219 nm. This is comparable to the output of the brightest available incoherent source, a Hg-Xe-Cd arc lamp that produces 2.6 mW of usable light in the 221-226 nm range. The laser has the advantage that it can be focused to produce much higher intensities than the arc lamp, and less total power is necessary. But the pulsed laser has a low duty cycle (~ 10[superscript minus 5]), and the corresponding peak powers (~ 300 watts) result in a space-charge-limited resolution of approximately 500 Å when the laser illuminates a phthalocyanine target. The magnitude of this aberration is proportional to beam current. Consequently, the resolution o can be improved to about 50 Å by decreasing the input power, or increasing the duty cycle, by a factor of 100-1000. Techniques for achieving such an improvement are suggested.

The effect of electron beam irradiation upon microbiological quality and safety of fresh-cut tomatoes was studied. Preliminary studies were conducted to ensure reliability of the rifampicin-resistant strain versus the parent strain of Salmonella serovar Montevideo for use in this study. Growth curve, heat tolerance and lactic acid resistance studies were performed, all of which showed no differences in behavior between the organisms. Fresh tomatoes were obtained from a local supplier and then cut into cubes with stem scars being separated. Both cubes and stem scars were inoculated with a rifampicin- resistant strain of either Salmonella Montevideo or Salmonella Agona, separated into treatment groups and treated by electron beam irradiation at 0.0 kGy (control), 0.7 kGy or 0.95 kGy. The effect of electron beam irradiation was determined for Salmonella, yeast, mold, and lactic acid bacteria (LAB) populations as well as pH on tomato cubes and stem scars over a 15-day storage period at 4??C. Results indicated that while irradiation treatment significantly reduced most microbial populations on tomato samples, there were no differences in the microbial populations between treatments of 0.7 kGy or 0.95 kGy. Irradiation at either dose resulted in a significant reduction of Salmonella Montevideo when compared to the control, with an initial reduction of 1.8 and 2.2 log10 CFU/g on tomatoes for 0.7 kGy and 0.95 kGy, respectively. LAB, yeasts and molds were more resistant to the treatment than Salmonella. Populations present on stem scars and tomato cubes did experience some differences in log reductions, possibly due to the protective effect of the stem scar on microorganisms. However, no differences were detected between the two Salmonella serotypes in response to irradiation treatment. This study indicates that doses of irradiation greater than 1 kGy should be used in fresh-cut tomatoes to eliminate significant populations of pathogens, as well as to ensure the microbial quality of the product. Additional studies also need to be conducted to examine the effects of higher irradiation doses on the sensory qualities of fresh-cut tomatoes.

Three-dimensional (3D) structural determination of biological macromolecules is not only critical to understanding their mechanisms, but also has practical applications. Combining the high resolution imaging of transmission electron microscopy (TEM) and efficient computer processing, protein structures in solution or in two-dimensional (2D) crystals can be determined. The lipid monolayer technique uses the high affinity binding of 6His-tagged proteins to a Ni-nitrilotriacetic (NTA) lipid to create high local protein concentrations, which facilitates 2D crystal formation. In this study, several proteins have been crystallized using this technique, including the SARS virus Nsp15 endonuclease and the human Toll-like receptor (TLR) 3 extracellular domain (ECD). Single particle analysis can determine protein structures in solution without the need for crystals. 3D structures of several protein complexes had been solved by the single particle method, including IniA from Mycobacterium tuberculosis, Nsp15 and TLR3 ECD. Determining the structures of these proteins is an important step toward understanding pathogenic microbes and our immune system.

Electron paramagnetic resonance (EPR) and Mössbauer spectroscopy were used to examine the distribution of iron in mitochondria from Saccharomyces cerevisiae. These organelles were packed into EPR and Mössbauer cuvettes, affording spectra with unprecedented signal/noise ratios. EPR spectra of as-isolated intact mitochondria exhibited fourteen distinct signals, some of which were assigned according to previously reported g-values obtained using isolated proteins. Signals from adventitious manganese (II) and iron (III) were largely removed when mitochondria were isolated in buffers supplemented with the metal chelators EDTA or EGTA. Signals were simulated and intensities were quantified to afford spin concentrations and estimates of the concentration of EPR-active species in mitochondria. The effects of treating samples with chemical modifiers were examined. Packed samples were analyzed for protein and metal content, affording averaged values of 50 mg/mL [protein], 590 µM [Fe], 340 µM [Cu], and 17 µM [Mn]. 57Fe-enriched intact mitochondria isolated in the presence of metal chelators exhibited Mössbauer spectra dominated by three components. Approximately 60% of the 57Fe in the sample gave rise to a quadrupole doublet, most of which was diamagnetic. The parameters of this doublet are typical of S = 0 [4Fe-4S]2+ clusters and S = 0 ferrous heme groups. Spectra of samples reduced with dithionite, pH 8.5, suggested that at least half of this doublet arose from [4Fe-4S]2+ clusters. The second major component exhibited in the Mössbauer spectra arose from high-spin ferrous ions (10%-30%). The third major component (15%) came from iron exhibiting magnetic hyperfine interactions and is likely reflected in the Fe-containing species observed by EPR. The results presented here suggest that mitochondria contain ~ 600 µM of Fe overall, ~ 200 – 400 µM organized as [4Fe-4S]2+ clusters, with about 25 µM due to the [4Fe-4S]2+ cluster of aconitase. Approximately 60 µM – 200 µM of the Fe in mitochondria is high-spin ferrous ions, ~ 40 µM as the Rieske S = 1/2 [2Fe-2S]+ cluster of cytochrome bc1, and ~20 µM as the S = 1/2 [2Fe-2S]+ cluster of succinate dehydrogenase. The high-spin ferric hemes of the a3:CuB site of cytochrome oxidase and cytochrome c peroxidase each account for ~ 4 µM of Fe.

Double 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.

Mesoporous silica crystals have a large variety of structures mainly due to the versatility of their structure template. The configuration and the chemical state of the templating micellar surfactants, together with the kinetic process of silica will determine the final outcome of the synthesis. Increasing the understanding of the complex formation processes involved will enable a possibilityto fine tune the material for specific uses, today focused into the fields of photoniccrystals, drug delivery, catalysis and separation technology. In this thesis emphasis is put on (1) increasing the understanding the formation mechanism yielding the different species of mesoporous silica crystals through an in depth study of quasicrystallinity (2) Characterization and description of the structural complexity through various characterization techniquesand also by studying the kinetic structural transformation phenomenon related to the minimal G- and D-surfaces. (3) The structural studies of the versatile surfactant liquid crystals for establishing a thermodynamically stable basis to evaluate the kinetic mesoporous silica growth processes. Furthermorethe thesis both enlightens the possibilities of and contributes to the developmentof electron microscopy characterization techniques. In these studies, electron microscopy is largely employed in the characterization to give a thorough picture of the mesoporous structures. This is combined with the sample preparation techniques cross-section polishing and ionslicing. Low voltage scanning electron microscopy is utilized for studying the surfaces and cross-sections of various materials at the limit of the resolution. Here, a deep understanding of the electron beam-material interaction is used for a better interpretation of the detected signals. Transmission electron microscopyis combined with electron crystallographic reconstruction to yield a three dimensional structural model. For determination of the quasicrystallinity level for a structure of dodecagonal tiling, revealed in the scope of this study,a phason strain analysis was made. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 7: Manuscript.</p>

The goal of fully decoding how the brain works requires a detailed wiring diagram of the brain network that reveals the complete connectivity matrix. Recent advances in high-throughput 3D electron microscopy (EM) image acquisition techniques have made it possible to obtain high-resolution 3D imaging data that allows researchers to follow axons and dendrites and to identify pre-synaptic and post-synaptic sites, enabling the reconstruction of detailed neural circuits of the nervous system at the level of synapses. However, these massive data sets pose unique challenges to structural reconstruction because the inevitable staining noise, incomplete boundaries, and inhomogeneous staining intensities increase difficulty of 3D reconstruction and visualization. In this dissertation, a new set of algorithms are provided for reconstruction of neuronal morphology from stacks of serial EM images. These algorithms include (1) segmentation algorithms for obtaining the full geometry of neural circuits, (2) interactive segmentation tools for manual correction of erroneous segmentations, and (3) a validation method for obtaining a topologically correct segmentation when a set of segmentation alternatives are available. Experimental results obtained by using EM images containing densely packed cells demonstrate that (1) the proposed segmentation methods can successfully reconstruct full anatomical structures from EM images, (2) the editing tools provide a way for the user to easily and quickly refine incorrect segmentations, (3) and the validation method is effective in combining multiple segmentation results. The algorithms presented in this dissertation are expected to contribute to the reconstruction of the connectome and to open new directions in the development of reconstruction methods.

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