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In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue. In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix. In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure. To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis. The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion. Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis supervisor: Maureen D. Donovan. Includes bibliographic references (p. 239-247).

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliographical references.

Unlike most RNA and DNA containing viruses, which induce cytogenetic damage at random sites throughout the human genome, the highly oncogenic adenovirus type 12 is also capable of inducing damage at specific chromosomal sites. Infection of human embryonic retinal or kidney cells with Ad12 results in the induction of heterochromatic gaps at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosome. Previous work by Durnam et al. (1986) demonstrated that the viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. The objective of the present study was to 1) identify the Ad12 E1 gene product(s) required for the induction of aberrations in human diploid cells, and 2) to determine whether the same or different functions are involved in the induction of damage at specific and random sites. To this end, adenovirus type 12/adenovirus type 5 recombinants with hybrid E1 sequences as well as viruses with mutations in the Ad12 E1B genes were used to map the Ad12 E1 function(s) required for the induction of chromosomal aberrations. The results of this study indicate that the expression of E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55Kd protein but not the E1B 19Kd protein were found to affect the ability of the virus to induce both specific and random damage (Schramayr et al., 1990). / Thesis / Master of Science (MS)

Cathepsin Z is a cysteine proteinase found in lysosomes of human cells. It was detected in human cultured cell lines using the peptidyl diazomethane inhibitor Fmoc-Leu-Leu- [¹²⁵]Tyr-CHN₂. The labeling of cathepsin Z by the inhibitor was both time- and concentration-dependent, and the proteinase was found in all human cell lines examined. The characteristics of cathepsin Z were examined in U-937 cells, a human monocytic line. The labeling of cathepsin Z was blocked by pre-incubation of the cells either in non-iodinated inhibitor or in the epoxysuccinyl peptide inhibitor E-64d, a specific inhibitor of cysteine proteinases. Cathepsin Z was not immunoprecipitated by antisera specific for cathepsins B, L, or S. Cathepsin Z has been estimated to be at millimolar concentrations in lysosomes, suggesting that it is a major lysosomal proteinase. The molecular weight of cathepsin Z was calculated to be 22.4 kDa by SDS-PAGE and 45— 47 kDa by native PAGE and gel exclusion chromatography, indicating that it is dimeric. Cathepsin Z is susceptible to digestion by endoglycosidase H, and oligosaccharides comprise 3.1 kDa of the reduced molecular weight. The expression of cathepsin Z was not affected by differentiation of U-937 cells with phorbol ester, unlike the expression of cathepsins B and S. Undifferentiated U-937 cells express low levels of cathepsins B and S; after differentiation, expression of cathepsins B and S is greatly increased. Cathepsin Z was purified from U-937 cells by anion exchange chromatography (Mono Q), affinity chromatography (concanavalin A), and preparatory electrophoresis. N-terminal sequence analysis of both the purified protein and fragments of the protein from a V8 digest indicates that cathepsin Z is a member of the papain superfamily of cysteine proteinases. / Ph. D.

published_or_final_version / Anatomy / Master / Master of Philosophy

This project developed methodology for in vitro toxicity assessment of contaminated sites using the Promega?? MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay performed on human cells (HepG2 and Skin fibroblasts). The project included the development of a method for extracting contaminants from soil based on leaching and centrifugation. A number of solvents and surfactants were assessed for their suitability as extracting agents. The Zwitterionic surfactant CHAPS ({3[(3-Cholamidopropyl) dimethylammonio] propanesulphonic acid}), which is an irritant in vivo, was found suitable for in vitro toxicity assessment applications. CHAPS was found to be the least toxic surfactant in vitro when tested on skin fibroblasts (NOEC: 1800??577 ppm, IC50: 4000??577 ppm) and HepG2 cells (NOEC: 833??289 ppm, IC50: 5300??287 ppm). The chosen surfactant was used in three different methods for extraction of Toluene and Xylene spiked in 2 g and 10g soil. The combination comprising of 0.1% (s/w) CHAPS and cosolvent 1% (w/w) Isopropanol, at their respective NOEC (No Observed Effective Concentration) toxicity values, showed good recovery of the nonpolar organic compounds in comparison to the recovery by 0.1% CHAPS and 0.5% CHAPS. The study found additive interactions to be the most common form of toxicity for 16 concentration combinations of Formaldehyde (polar), Toluene and Xylene (nonpolar) when compared to predicted toxicity (R2=0.943, P&lt0.0001). When assessing the in vitro toxicity of unknown (blind) contaminated soil samples, the Hazard Index (HI) predicted from the chemical analyses results showed a relatively good correlation (R2&gt0.7062, n=26) when compared to the experimental toxicity results on HepG2 cells. Furthermore, the comparison of Australian Health Investigation Levels (HIL) with in vitro toxicity testing gave similar correlation (R2&gt0.6882, n=26) on HepG2 cells. The overall project suggests the potential application of the zwitterionic surfactant (CHAPS) in sampling contaminants from soils in an in vitro toxicity assessment. This study demonstrates the application of in vitro toxicity assessment using human cells for the prediction of toxic risk as a sentinel to human toxicity from a contaminated site.

Univ., Diss.--Göteborg, 2005. / Literaturverz. S. [289] - 313.

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