The objective of the project is to use the RICS analysis technique in complement with confocal microscopy to determine the diffusion coefficient of the selectively labeled green fluorescent protein (GFP), GFP-EGFR and GFP-p53 in cervical cancer cells. This is a collaboration work with MD Anderson Cancer Center. The application of the study is to lay the foundation for further study in understanding the cell metabolism, subcellular morphologic and dynamic biochemical processes to aid in the diagnosis and to potentially screen cancers. Fluorescence microscopy techniques have been developed for the study of cellular processes and molecular signal pathway. However, the spatial resolution to distinguish and resolve the interactions of single molecular complexes or molecule in cells is limited by the wavelength. Hence, indirect image correlation methods complementary to the imaging techniques were developed to obtain the dynamic information within the cell. RICS is one such mathematical image processing method to determine the dynamics of the cell. HeLa cells are transfected with GFP to highlight the protein of interest. These samples were imaged with confocal microscope, Olympus FV1000 with a 60 x 1.2 NA water objective in the pseudo photon counting mode with an excitation of 488 nm argon ion laser. About 100 frames of scan area 256x256 pixels were collected from each sample at scan speed of 12.5 seconds per pixel. The stacks of images were processed with SimFCS software. The images were subjected to immobile subtraction algorithm to remove the immobile features. Consequently, each frame in the stack is subjected to 2D-correlation; then, the average 2D-spatial correlation is calculated. This 2D-spatial correlated data constitutes as RICS data which is then displayed and analyzed by fitting it to specific models. This generates a spatial temporal map of the molecular dynamics of fluorescently labeled probes within the cell. In summary, we apply RICS techniques based on correlation spectroscopy to the image data and quantify diffusion coefficient of protein of interest in cancerous cells with different treatments. This is expected to better understand cellular dynamics of cancerous cells and build better diagnostic biosensor devices for early screening.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

The reversible acetylation of histones is thought to play a role in chromatin processing, including transcription, replication and repair. Studies on the acetyltransferases, responsible for acetylating the nucleosomal core histones, have resulted in characterization of these enzymes. However, very little is known about the properties and distribution of histone deacetylase. The reversible inhibition of histone deacetylase by butyrate was employed to permit studies on the chromatin-bound histone deacetylase of HeLa cells using endogenous [3H]-acetyl labelled polynucleosomes containing the enzyme. These were prepared in the presence of 50mM butyrate and histone deacetylase was assayed upon removal of the inhibitor. It was found that active enzyme is present only in association with a high molecular weight complex. This deacetylase-containing complex is relatively resistant to digestion with micrococcal nuclease. No activity is found on mononucleosomes or oligonucleosomes. Up to 90% of labelled acetyl groups are removed from histone deacetylase complexes incubated in the absence of butyrate, indicating that denaturation of the histone deacetylase is kept to a minimum using the techniques developed in this study. Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex. Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from it during its reaction with acetylated core hisones. Under typical nuclease digestion conditions, the histone deacetylase complex contains DNA with a size distribution of 5-11 kilobase pairs and a variety of nonhistone proteins. Comparison of the protein composition of histone deacetylase complexes with that of nuclear matrix preparations shows some similarities. Taken together, the results on the chromatographic behaviour, the DNA fragment sizes, and the protein composition of the deacetylase complex suggest that protein-protein interactions may be important in maintaining its structure and also in the binding of the deacetylase itself to the complex Later research efforts were concerned with characterization of the histone deacetylase complex. The effect of J3-mercaptoethanol and neocuproine on histone deacetylase was examined in view of the fact that these reagents are known to disrupt chromosome scaffolds. HeLa cell histone deacetylase complex partially dissociates in 10 mM B-mercaptoethanol, resulting in a loss of non-histone proteins. The presence of 10 mM J3-mercaptoethanol during the partial micrococcal nuclease digestion of HeLa cell nuclei, results in a very low yield of histone deacetylase complex, with a correspondingly large increase in the production of small oligonucleosomes and mononucleosomes. Histone deacetylase activity on endogenous labelled histone is strongly inhibited by either 1 or 10 mM J3-mercaptoethanol or 3 mM neocuproine. The loss of histone deacetylase activities is not due to an inactivation of the enzyme, but appears to be a consequence of the disruption of the structure of the histone deacetylase complex. Histone H4 in histone deacetylase complex prepared from HeLa cell nuclei by micrococcal nuclease digestion was more highly acetylated than H4 in bulk nucleosomes. Restriction enzyme analysis of the DNA associated with the histone deacetylase complex revealed neither an enrichment nor depletion of major satellite sequences in this material. In view of these findings, histone deacetylase appears to be associated with a high molecular weight chromatin complex which may be a site of rapid acetyl group turnover. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Apurinic endonuclease activity in human fibroblasts had been previously resolved into aflow-through and a high-salt eluate species by phosphocellulose chromatography (Kuhnlein, U. et al., Nucl. Acid. Res. 5: 951-960, 1978). Enzyme activity in the flow-through species amounted to 20-30% that of the high-salt eluate species. The flow—through enzyme species was not found in-eel I lines of xeroderma pigmentosum complementation group D. In this thesis, apurinic endonuclease activity was analysed in Hela ceflls. Specific enzyme activity in crude extracts "of Hela cells was in the range of 400-800 units/mg protein, similar to that of , human fibroblasts which was between 380-680 units/mg protein. Three species of endonuclease activity for apurinic DNA were resolved by phosphocellulose chromatography. They were designated as Peak I, Peak If, and Peak III. Peak I did not adsorb to the phosphoceIIuIose column at' 10 mM KP04 (pH 7.4) (flow-through activity), Peak II eluted from the column at about 210 mM KP0₄ (pH 7.4) and Peak I I I at 260 mM KPO₄ (pH 7.4). Based on their affinity to phosphoceIIuIose, we presumed Peak I and Peak III corresponded to the flow-through and high-salt eluate species in human fibroblasts respectively. Under our experimental conditions, the flow-through enzyme activity in both Hela cells and normaS human fibroblasts was only 2-4% of the activity of high-salt eluate species. We suspect that tissue culture conditions may affect the cellular level of the flow-through species of apurinic endonuclease. Peaks l-lII were optimally active at pH 7.5-8.0 and 5-10 mM MgCI, They were 'inhibited by increasing concentrations of KCI and NaCI except Peak III which was slightly stimulated by 20-40 mM KCI. The three species were distinguished by their thermosensitivities in a 50 mM KPO. buffer. Peak I was stable at 45°C. Peak III was heat-labile, having a half-life of 2-3 min at 45°C. Peak II seemed to contain two components, one with a ha If-life of 2-3 min at 45°C, and the other with a half-life of 25 min. In human fibroblasts, both the flow-through and high-salt eluate species of apu-rinic endonuclease were reported to be stimulated to 2.5-fold by 10 mM KCI. They had a ha If-life of 6 min at 45°C in a 230 mM KP0₄ (pH 7.4) buffer. Thus, Peaks I-lI I and enzyme species from human fibroblasts had a similar pH optimum, and Mg²⁺ requirement, but they differed in their thermosensitivities and inhibition by higher salt concentration. We do not know as yet whether these differences reflect the neoplastic nature of Hela cells or the different tissue origins of Hela cells and human fibroblasts. When either Peak I or Peak I I I was rechromatographed on the phosphoceIIulose column, activity was recovered in both the flow-through and high-salt eluate fractions. The result suggested an interconversion phenomenon between the flow-through and high-salt eluate species of apurinic endonuclease, This was further supported by molecular weight determinations of the apurinic endonucI eases In Peaks l-lII. Apurinic endonuclease activity in Peak III and Peak II had a molecular weight of 35,000-40,000 and 22,000-25,000 respectively. Peak I had two components with molecular weights similar to those of Peak II and Peak III. An understanding of the conversion between the different apurinic endonuclease species may help in elucidating the molecular defects of xeroderma pigmentosum complementation group D. Apurinic endonuclease activity in Peaks l-lII was found to be associated with a high molecular weight complex. The complex could be dissociated by high salt treatment. The possible biological significance of the high molecular weight complex Is discussed. We also found that apurinic endonuclease could adsorb to the Sephadex gel. The adsorption would lead to an aberrant estimation of molecular weight of the protein. The problem was solved with an elution buffer of high ionic strength. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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To the reader <p> Since I worked on two separate projects towards my doctorate thesis, the arrangement of my thesis is rather unusual. The reader will find that my thesis is divided into four parts. Part 1 is dedicated to a very general introduction about the basic knowledge needed to guide you, the reader, through the rest of the thesis. Within this part, different sections focus on different fundamental aspects of Biophysics related to my work. In Part 2, I discuss my studies of the distribution and dynamics of the nuclear protein Ran in live interphase HeLa cells. This part contains a background section specific to this project, the materials and methods used for this study, experimental results, a discussion of our findings, and it ends with conclusions. Part 3 is dedicated to the study of the dynamical mechanisms responsible for the establishment of the Bed protein concentration gradient along the anterior-posterior axis in live Drosophila melanogaster embryos. Again, a specific background section is included in this part, followed by the materials and methods used to perform this research, results, discussions and finally I will summarize my results to conclude this work. The last part, part 4, is rather short and contains the summary of the overall results of my work on both nuclear proteins with some emphasis on the similarities and differences in their dynamical behavior.</p> / Thesis / Doctor of Philosophy (PhD)

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by Lui Po Yee Pauline. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 211-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.