Proefschrift - Utrecht.

Thesis (Ph. D.)--University of Hong Kong, 1982.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Inaug.-Diss.--Leipzig. / Vita.

The histogenesis of two squamous-carcinoma cell lines, originally derived from the same human cervical tumor but with different morphology in vitro and in vivo, was compared histochemically and electron microscopically. Inoculation into hamster cheek pouches was used to distinguish in vitro from in vivo responses in the two cell lines. Normal cervical epithelium was grown in vitro to determine which histogenetic characteristics were due to the malignant nature of the cell lines. Possible mechanisms underlying histogenetic variation were investigated by chemical and physical modifications of the in vitro environment. In vitro cells of line C-4c were more cohesive, less deformable and less adhesive to substrata than the cells of line C-4s and they formed more compact and highly stratified colonies. Line C-4s responded to crowding in vitro by cell separation (dispersal) while line C-4c increased stratification. Histochemically the cell lines were essentially similar but they differed in ultrastructure, particularly at the level of tissue organization. The C-4s cells were columnar, with a higher nucleo-cytoolasmic ratio, a polarity of the organelles, dispersed cytoplasmic filaments, little stratification and with terminal bars between superficial cells. In contrast, C-4c cells were oval, stratified and with superficial cells flattened, with a lower nucleo-cytoplas-mic ratio, without polarity of organelles, and with more cytoplasmic filaments condensed into bundles that were associated with desrnosomes. In both lines, but particularly in line C-4c, interdigitating microvilli provided the main intercellular contact and the cell surfaces were modified in association with both substrata and the growth medium. Most in vitro differences between the cell lines were retained in vivo, and, in addition, incomplete basement membranes were formed in both lines, though more extensively in line C-4.s. It appeared that the only major difference in vitro between benign squamous cells and the carcinoma cells lay in the ability of the latter to maintain an intercellular organization in the complete absence of any supporting tissue. Possible mechanisms underlying these observed differences in histogenesis were experimentally investigated. Ferritin uptake in vitro indicated that the difference in stratification was not due to more efficient intercellular circulation in the more highly stratified C-4c line. No difference in cell-surface charges or distribution of cell-surface acid mucosubstances could be demonstrated, suggesting no difference in contact inhibition to be associated with the stratification. It was found that C-4c cells were more cohesive and that this cohesion, in both lines, depended predominantly on the presence of divalent cations. In contrast, adhesion to the substrata required extracellular proteins, probably accompanied by the masking of acid groups on the cell surface. These and other described observations suggested a defect in deformability in these cell lines with the probability that the stronger cohesion in line C-4c was due to the more extensive microvillus population. The in vitro maintenance of cell shape, of tissue organization and of cell separation (dispersal) seemed to involve an interaction between these intercellular adhesive forces and the cytoplasmic filaments. On the other hand, the specific, modifications, such as terminal-bar formation and cell flattening, which appeared in relation to growth medium, to other cells and to substrata could be shown to be responses of the cells to the viscosity of the immediate environment and thus experimentally modifiable. The results of this investigation are discussed relative to the specific suggestion that much of the histogenetic variation could be explained by considering that the more cohesive cell line (C-4c) had retained properties of normal epithelium stratum spinosum cells, while the spreading cell line (C-4s) exhibited characteristics of basal cells, and relative to the interaction of differentiation capacity and malignancy in tumor development. / Science, Faculty of / Zoology, Department of / Graduate

Thesis (MApSc(BiomedicalScience)--University of South Australia, 2005.

A histological assessment allows aquatic scientists to assess fish health in polluted aquatic ecosystems at tissue and cellular level. However, a firm knowledge and understanding of normal histological structure is essential to ensure accurate and objective results. Hence, the histologist must be able to distinguish between toxicant induced lesions and the range of histological characteristics considered to be normal for that specific species. However, limited histological reference material and data describing normal conditions are available for southern African fish species. The aim of this baseline study was to establish reference material, both qualitative and quantitative, for two southern African freshwater fish species used as indicator species in toxicity studies at the University of Johannesburg, Clarias gariepinus and Oreochromis mossambicus. The reference material includes descriptive histology of selected target organs, and the associated values and intervals for related quantitative health aspects including somatic indices, condition factor, blood parameters (haematocrit and plasma proteins) and quantitative histological results. Essentially, this project originated from a need to better understand the normal histology of C. gariepinus and O. mossambicus, as a range of histological characteristics were identified in control groups in previous toxicity studies which could not be confirmed to be associated with normal conditions. To be able to conduct a baseline study, with the purpose of establishing reference material, it was essential that the history of the specimens be known and documented including age, sexual maturity, nutritional status and physical and chemical water quality data. The project therefore involved the breeding of fish specimens under controlled conditions. A subsequent necropsy and qualitative and quantitative histological assessments were executed on five target organs (usually included in toxicity studies within the department): liver, gills, gonads, heart and kidney. Fish of both species were bred in an environmental room in reconstituted, reverse osmosis water and reared until sexually mature. Twenty specimens with an approximately 50:50% sex ratio were collected for both species. A necropsy was performed on each specimen and tissue samples of the selected target organs were processed using standard techniques, and prepared for the qualitative and quantitative histological assessments using light microscopy. The results The results showed that no macroscopic abnormalities were identified during the necropsy regarding external features or internal organs of the specimens within the sample groups, and all fish appeared to be in good health. The target organs were then examined microscopically, followed by a description of the normal histological structure (qualitative assessment). In addition, the target organs were assessed by means of a specific quantitative histological assessment protocol, which provides a standardised methodology of criteria to objectively assess fish health (quantitative assessment). Histological alterations identified during this assessment were quantified and subsequent reference index values could be calculated for each organ. / Dr. G.M. Pieterse

Physicians are blind to the microscopic tissue structure that defines tissue type and pathologies during procedures. For diagnosis, tissue must be excised, fixed, and processed for histology, which can take anywhere from 20 minutes to days. This need for tissue excision and processing for microscopic visualization delays decision-making and necessitates repeat procedures. Limited sampling can also never fully eliminate the presence of disease. However, advances in optical sectioning techniques such as confocal and two-photon microscopy, which provide isotropic cellular-level resolution in bulk tissues, have obviated the need to physically section and process tissues for histology. Many optical imaging probes have been developed over the last three decades with the goal of demarcating tissue health in situ, either completely eliminating the need for tissue excision and processing for histopathology or guiding biopsy selection to reduce sampling bias. However, these techniques have faced major barriers to routine and widespread clinical use, including small 2D fields of view, limited contrast, slow imaging speeds and bulky laser sources. To address this critical need, SCAPE Microscopy, a light sheet-based microscopy technique recently developed in the Hillman lab, was developed for label-free, real-time, volumetric imaging at the point-of-care. SCAPE allows visualization of both cross-sectional and multilayer en face geometries in parallel and real-time, providing a more comprehensive view of tissue architecture than individual histology slides. Furthermore, tissues can be imaged label-free with structure shown through intrinsic fluorescence or in conjunction with intravenous or topical dyes. SCAPE’s video-rate speeds permit 3D stitching of large tissue areas and can withstand in vivo motion, which typically renders point-scanning techniques impractical. Most importantly, SCAPE is shown to allow 3D visualization of key histoarchitectural markers in human kidney biopsies through both endogenous and exogenous fluorophores. In this thesis work, a benchtop system is used for proof-of-concept imaging; however, miniaturized prototypes more suitable for clinical use are also presented. Further, high-throughput imaging of tissues is a critical but underserved need for bedside biopsy evaluation, as well as large-scale interrogation of structural organization and connectivity in the brain, retina and even whole model organisms. SCAPE provides near giga-voxel per second imaging rates that are well-suited for imaging large-scale ex vivo tissues at isotropic resolution at orders of magnitude faster speeds than point-scanning techniques. To this end, SCAPE was also developed as a versatile imaging platform for structural imaging of large-scale fresh, fixed, cleared and expanded samples for both bedside clinical evaluation and basic science research. It is demonstrated that planar samples of a few millimeters can be fully imaged at cellular resolution in just minutes by combining 3-axis stage-scanning and 3D stitching.

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