Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: implant; medical; atomic force microscopy; fungi; bacteria. Includes bibliographical references (p. 82-100).

Thesis (M. S.. in Biomedical Engineering)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 89-94).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 156-160).

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis--M. Med. Sc., University of Hong Kong, 1982.

Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.

The main theme in this dissertation is to develop Scanning Electrochemical Microscopy (SECM) based electroanalytical techniques to study quantitative biology in real time. The multidrug resistance (MDR) phenomenon in live cancer cells was studied using mimic drug molecules such as menadione with the aid of SECM. Real time quantitative detection of thiodione (menadione-conjugate) pumped out of the cells was determined to be 140 μM due to exposure of 500 μM menadione to the cells. Selective blocking of these MDR pumps in live intact cells was also achieved by small molecules such as MK571 as well as by the MDR specific antibody. An approximately 50% drop in thiodione flux was observed in both cases of MDR pumps inhibition. This SECM technique was also extended to measure the permeability of a highly charged hydrophilic molecule passing through the membrane of a single living cell. The permeability was measured to be 6.5 ± 2.0 × 10-6 m/s. Real time monitoring of morphological changes in a live HeLa cell due to addition of varying concentration of surfactant such as Triton X-100 was also demonstrated by SECM. This electroanalytical technique was also expanded to study quantitative microbiology. Real time quantitative detection of pyocyanin produced by Pseudomonas aeruginosa (PA14 strain) biofilm locally was determined to be 2.5 μM after 6 h. Pyocyanin (PYO) was also observed to be reduced by PA14 biofilm, thus maintaining a reduced atmosphere above the biofilm even in presence of oxygen. Spatial mapping of this reduced PYO showed that this reduced zone was only formed up to 500 μm above the biofilm. The cells are also able to modulate the height of the reduced PYO zone in accordance to the availability of Fe(III/II) in the solution to scavenge iron from the surrounding environment. Real time spatial mapping hydrogen peroxide across polymicrobial biofilm (Sg and Aa) was also achieved with the aid of SECM. The local peroxide concentration produced by Sg was measured to be 1 mM, which is significantly higher than the bulk peroxide concentration. This study also showed that the local concentration across the microbial film is more important than the bulk concentration since bacteria communicate locally in real world. / text

This study was conducted to investigate the biofilm formation by using advanced microscopic and high-throughput sequencing techniques. The major tasks were (1) to quantitatively evaluate the initial bacterial attachment processes by Atomic Force Microscopy (AFM); (2) to characterize the chemical variation during biofilm formation by Raman microscopy; (3) to analyze the microbial structure and functions in the wastewater and drinking water biofilms by metagenomic analysis. To determine the lateral detachment force for bacteria, a quantitative method using contact mode of AFM was developed. The established method had good repeatability and sensitivity to various bacteria and substrata, and was applied to evaluate the roles of bacterial surface polymers in Phase I and II attachment, i.e. lipopolysaccharides, type 1 fimbria and capsular colanic acid. The results indicated lipopolysaccharides largely enhanced Phases I and II attachment. Fimbriae increased Phase I attachment but not significantly influence the adhesion strength in Phase II. Moreover, colanic acid had negative effect on attachment in both of Phases I and II. Surface-enhanced Raman scattering was applied to evaluate the chemical components in the biofilm matrix at different growth phases, including initial attached bacteria, colonies and mature biofilm. Three model bacteria, including Escherichia coli, Pseudomonas putida, and Bacillus subtilis, were used to cultivate biofilms. The results showed that the content of carbohydrates, proteins, and nucleic acids in biofilm matrix increased significantly along with the biofilm growth of three bacteria judging from the intensities and appearance probabilities of related marker peaks in the spectra. The content of lipids, however, only increased in the Gram-negative biofilms. Moreover, metagenomic data, coupled with PCR-based 454 pyrosequencing reads, were generated for activated sludge and biofilm from a full-scale hybrid reactor to study the microbial taxonomic and functional differences/connections between activated sludge and biofilm. The results showed that the dominant bacteria co-existed in two samples. Global functions in activated sludge and biofilm metagenomes showed quite similar pattern, revealing the limited differences of overall functions existed in two samples. For nitrogen removal, the diversity and abundance of nitrifiers and denitrifiers in biofilm did not surpass that in activated sludge. Whilst, higher abundances of nitrification and denitrification genes were indeed found in biofilm, suggesting the increased nitrogen removal by applying biofilm might be attributed to removal efficiency rather than biomass accumulation of nitrogen removal bacteria. To investigate the bacterial structure and functions of drinking water biofilm, PCR-based 454 pyrosequencing of 16S rRNA gene and Illumina metagenomic data were generated and analyzed. Significant differences of bacterial diversity and taxonomic structure were found between biofilms formed on stainless steel and plastics. Moreover, ecological succession could be obviously observed during biofilm formation. The metabolic network analysis for drinking water biofilm constructed for the first time. Moreover, the occurrence and abundance of specific genes involving in the bacterial pathway of glutathione metabolism and production/degradation of extracellular polymeric substances were also evaluated. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

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