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No description available.

Circadian mitotic rhythms in epithelial cells of human epidermis (Schevirg, 1959), rat cornea (Scheving,1967) and intestinal epithelium of the mouse (Sidgestad,1972) have been reported in the literature. It is the attempt of this investigation to determine the presence of a circadian rhythm governing the mitotic activity of hair follicle cells in neonatal white mice. The animals used were reared under closely controlled conditions of temperature, humidity, lighting and food. A total of forty-eight, three to four day old mice were used, with eight animals being sampled every four hours over a twenty-four hour period. Skin sections were removed and fixed in Bouins fluid. The tissues were dehydrated in alcohols, cleared in xylol and infiltrated and embedded in paraffin. The sectioned tissues were placed on glass slides, rehydrated and stained with hematoxylin and eosin. To assess the mitotic activity over the twenty-four hour period, cell counts within the follicles were made from the slides. For each animal, 1000 hair follicle cells were counted. Cells undergoing mitosis were identified and specific stages were tallied. These stages were 1) prophase, 2) metaphase, 3) anaphase-telophase and 4) unidentifiable cells. Photomicrographs were made to record the histology and demonstrate the mitotic activity.When the data was statistically analyzed, an analysis of variance test failed to show a significant difference among the mean values of dividing cells from animals sampled during the different time periods. However, the F value for metaphase favorably approaches the table F value for the conditions of this study.By far, the strongest data to support the existence of a circadian rhythm within the hair follicle cells of mice is demonstrated when the mean values for each of the mitotic cell categories are plotted against time for the twenty-four hour period. Metaphase shows peaks in activity at 6:00 a.m. and 10:00 p.m. A similar pattern is also seen for the unidentifiable cells. The peak mitotic activities for prophase, and anaphase-telophase was seen to occur at 10:00 p.m.

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No description available.

No description available.

In the biopharmaceutical industry, essentially, there are three components that play the main role in producing biopharmaceutical products, the host cell, the expression vector and the bioreactor and/or production environment. To produce the highly valued and desired products, the choice of a suitable host is one of the most important aspects. The host required is not only required to produce the desired product, but also needs to demonstrate robustness in a bioreactor system. Constantly facing challenges in a bioreactor, cells often undergo apoptosis, a well-known limiting factor in biopharmaceutical production, which ultimately leads to low yield of valuable protein(s). We have genetically engineered a CHO-K1 cell line to constitutively express human insulin-like growth factor-1 (IGF-1) and murine polyoma large T-antigen (PyLT-Ag) to generate Super-CHO and CHO-T respectively, two cell lines that can potentially serve different niches in the biopharmaceutical industry. In the first part of the project, we hypothesised that suspension-adapted Super-CHO and CHO-T cells are both resilient cell lines relative to the suspension-adapted CHO-K1 (designated as CHO-XL-99) when facing nutrient depletion, one of the most common problems in a bioreactor. Furthermore, in the second part of this project, the suspension-adapted CHO cell lines were also tested against a cytotoxic heavy metal, cadmium. Without the protection of the metal-resistance element, metallothionein, both Super-CHO and CHO-T cells were also challenged with cadmium to demonstrate their robustness over the parental cell line, CHO-XL-99. In the subsequent study, this project also focussed on the transfection efficiency of each parental and engineered CHO cell lines. Different strategies have been employed in the past in an attempt to improve productivity in the biopharmaceutical industry, from alterations in vector construction, improved culture condition, down to enhanced product recovery. However, the transfer and expression of the gene-of-interest (GOI) has still proven to be the limiting factor for achieving increased specific productivity. In an effort to improve transfection efficiency, strategies including cell cycle synchronisation and various transfection methods to deliver the GOI into the cells have been employed. Thus, the third part of this project has used synchronising agents in conjunction with commercially available lipid- and polymer-based reagents as delivery vehicle for the model protein, EGFP. The combination of cell synchronisation and transfection vehicle on transfection efficiency is studied here, in addition to their individual or collective effect on cell growth, apoptosis and viability. In summary, this project demonstrates the incidence of apoptosis in the cell culture induced by nutrient depletion and heavy metal, and that the use of transfection reagents solely, or in combination with synchronising agents also correlates with the increase of apoptotic indices in the cell culture. The use of the robust cell lines for transfection is an important aspect, and the balance between cell viability and the effort for augmenting transfection efficiency has to be met in order to achieve the maximum biopharmaceutical yields.

Graduation date: 1997