The work delineated in this research thesis aims to achieve advances in understanding nuclear structure-function interrelationships. Two natural physiological processes---heat-shock and differentiation---have been used in order to provide a clearer understanding of the "big picture" that links structure and functions together. I have performed experiments using mild or severe heat-shock on HeLa S3 cells and I differentiated L6E9 myoblasts into skeletal muscle fibers. Microscopic and biochemical analysis were used in order to assess the effects of heat-shock and differentiation on nuclear morphology. A series of antibodies specific to the nuclear periphery such as: lam in-associated-polypeptides, emerin, lamins A/C and lamin B were used to analyze structural changes that occurs at the nuclear periphery. Similarly, nuclear matrix antigens such as the proliferating cell nuclear antigen or PCNA, the nucleocytoplasmic shuttling protein 2A7, 2H12 and fibrillarin, two nucleolar proteins, were used as tools to assess structural changes that occur in the nuclear morphology. (Abstract shortened by UMI.)

The tyrosine kinase Kit is expressed in oocytes and is involved in primordial germ cell (PGC) proliferation and oocyte growth. Our goal was to determine the involvement of the SHP-1 phosphatase in regulating PGC proliferation and oocyte growth by examining SHP-1 deficient motheaten mice. Ovaries from wild-type and motheaten mice were observed at 10--13 days of age and after transplantation under the kidney capsule of SCID mice. Ovaries were analyzed by histological and western analyses. SHP-1 was expressed in all ovarian cell types throughout follicle development. At 10--13 days, motheaten animals had smaller ovaries, increased numbers of PGCs and decreased granulosa cell proliferation compared to controls. After transplantation, both groups had formed large antral follicles in similar proportions. Interestingly, motheaten oocytes achieved larger sizes than controls. These results suggest that SHP-1 may interact with Kit to regulate PGC proliferation and oocyte growth, however, the loss of SHP-1 does not impair granulosa cell proliferation and follicle development.

Although the growth of native corneal epithelial cells over the anterior surface of an artificial corneal implant is desired, the growth of these cells on the interface located between the implant and the stromal layer of the host eye tissue (i.e. epithelial cell downgrowth) poses a significant problem to be overcome in developing a suitable implant. In this study the growth factor surface modification of a polymer substrate was examined as a means of inhibiting the proliferation of corneal epithelial cells while promoting corneal stromal cell growth. Two separate studies were conducted in which transforming growth factor-beta1 (TGF-beta1) and transforming growth factor-beta2 (TGF-beta2) respectively, were immobilized via a bifunctional poly (ethylene glycol) (PEG) spacer, MW 3400, to poly(dimethyl siloxane) surfaces (PDMS) that had been aminated by the plasma polymerization of allylamine. The modified surfaces were chemically and biologically characterized. The effect of the surface modification with TGF-beta1 and TGF-beta2 respectively, on interactions with corneal epithelial and corneal stromal cells was examined using in vitro cell culture. (Abstract shortened by UMI.)

The significance of the uncoupling protein-1 (UCP1) in brown adipose tissue (BAT) thermogenesis has been well defined. However, the absence of significant amounts of BAT in adult humans, and the desire to reveal the mechanisms modulating skeletal muscle thermogenesis, led to the search for other proteins homologous to UCP1 which could also mediate thermogenesis by uncoupling oxidative phosphorylation. This search led to the discovery of Ucp2 and Ucp3 genes, as well as Ucp4 and brain mitochondrial carrier protein-1 (BMCP1). Ucp3 is of particular interest as a potential mediator of thermogenesis because it is selectively expressed at moderately high levels in human skeletal muscle, a tissue that contributes significantly to resting energy expenditure. In this thesis we attempt to elucidate the true physiological role of the UCP1 homologues, and of UCP3 specifically, by examining mitochondrial proton leak and UCP3 protein expression in: (1) mice that do not express Ucp3 ( Ucp3-knockout mice), (2) transgenic mice that overexpress human Ucp3 in their skeletal muscle (UCP-3Tg mice), (3) and finally in a distinct population of women who differ in their rate of weight loss. Our studies with the Ucp3-knockout mice show that the mitochondrial proton conductance is reduced in skeletal muscle mitochondria when UCP3 is absent. This result supports the hypothesis that UCP3 is a significant contributor to proton leak in skeletal muscle, and that the remaining UCPs in muscle (e.g., UCP2) do not compensate for the loss of UCP3. Mice overexpressing human UCP3 in skeletal muscle have total Ucp3 mRNA expression that is 66-fold greater than normal, and despite an increase in food intake, they are more lean than controls. Our results from human studies have provided the first ever measurements of proton leak in human muscle mitochondria. We examined the role of UCP3 and mitochondrial proton leak in patients on an energy restricted diet (900 kcal/day) who were identified as either diet-responsive or diet-resistant. Our results indicate that the state 4 oxygen consumption is 51% higher in the diet-responsive subjects compared to the diet-resistant ones. In addition, Ucp3 mRNA expression was found to be 25% greater in skeletal muscle of diet-responsive compared to diet-resistant individuals, suggesting that the upregulation of this protein could be responsible for the differences seen in the rate of weight loss. (Abstract shortened by UMI.)

Although ovarian cancer is rare, it is the most deadly of gynaecological cancers. Unfortunately, still very little is known about the cells that give rise to 90% of ovarian cancers, the ovarian surface epithelial (OSE) cells, and much of the available data remains controversial. This project was designed to address the possible involvement of ovarian stromal/thecal cells in the regulation of OSE cell growth and Kit and KL expression. Such interactions are probably involved in normal OSE-stromal/thecal cell activities as well as in interactions occurring within inclusion cysts and leading to ovarian tumour formation. The regulation of rat OSE (ROSE) cell growth by theca-derived factors and gonadotropins was investigated by proliferation experiments and cell counts. The modulation of Kit and Kit ligand (KL) messenger ribonucleic acid (mRNA) expression in these cells by the same factors was investigated by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). (Abstract shortened by UMI.)

The effects of the active metabolite of vitamin D$\sb3$, 1,25 (OH)$\sb2$D$\sb3$, and of transforming growth factor $\beta$1, TGF$\beta$1, on the proliferation and differentiation of an epithelial cell line derived from the rat small intestine (IEC-6) were investigated. IEC-6 cells contain the vitamin D receptor, which is upregulated by serum. TGF$\beta$1 has no effect on the activities of two markers of differentiation, alkaline phosphatase and sucrase. However 1,25 (OH)$\sb2$D$\sb3$ elevates alkaline phosphatase activity 8.9-fold within 72 hours. Both 1,25 (OH)$\sb2$D$\sb3$ and TGF$\beta$1 inhibit DNA synthesis in a concentration-dependent manner. Within 24 hours, 0.8 ng/ml TGF$\beta$1 and 500 nM 1,25 (OH)$\sb2$D$\sb3$ reduce $\sp3$H-thymidine incorporation by 55% and 60% respectively. A transient time course of DNA synthesis results from TGF$\beta$1 treatment while 1,25 (OH)$\sb2$D$\sb3$ treatment produces a sustained effect. The effect of 1,25 (OH)$\sb2$D$\sb3$ on DNA synthesis is partially blocked with neutralizing antibodies to TGF$\beta$ which suggest that active TGF$\beta$ is produced in response to 1,25 (OH)$\sb2$D$\sb3$. TGF$\beta$1 induces an accumulation of cells in the G$\sb0$G$\sb1$ phase of the cell cycle while 1,25 (OH)$\sb2$D$\sb3$ does not. However both 1,25 (OH)$\sb2$D$\sb3$ and TGF$\beta$1 inhibit the phosphorylation of the product of the retinoblastoma tumor suppressor gene. In conclusion, we have shown that 1,25 (OH)$\sb2$D$\sb3$ is a potent inhibitor of intestinal epithelial cell proliferation and have obtained indirect evidence to support a role for TGF$\beta$ in this effect. We speculate that modulation of IEC-6 cell proliferation may involve TGF$\beta$, whereas effects of 1,25 (OH)$\sb2$D$\sb3$ on differentiated markers, such as alkaline phosphatase, are mediated independently of TGF$\beta$.

The objectives of this study were as follows: (1) to establish a technique to determine (Ca$\sp{2+}\rbrack\sb i$ in single enzymatically isolated vascular smooth muscle cells (VSMCs) from the rat tail artery by means of the fura-2 fluorescent dye, (2) to study the relationship between VSMC shortening and Ca$\sp{2+}$ mobilization by caffeine, (3) to determine the mechanism of caffeine-induced Ca$\sp{2+}$ mobilization by studying the effects of removal of extracellular Ca$\sp{2+}$ and agents that modulate Ca$\sp{2+}$ mobilization (ryanodine, TMB-8, nifedipine, thapsigargin), (4) to determine the possible physiological role of the caffeine-sensitive Ca$\sp{2+}$ store in VSMCs by its interaction with noradrenaline. We conclude that, the fura-2 technique may be successfully employed to measure (Ca$\sp{2+}\rbrack\sb i$ in single vascular smooth muscle cells from the rat tail artery. The (Ca$\sp{2+}\rbrack\sb i$ response was found to be reproducible irrespective of cell length. This is the first study to show this, although others have used single cells to study (Ca$\sp{2+}\rbrack\sb i$ changes, the question of whether cell morphology affects (Ca$\sp{2+}\rbrack\sb i$ has never been addressed. This allows comparative (Ca$\sp{2+}\rbrack\sb i$ studies to be performed on the same single vascular smooth muscle cell regardless of its contractile state. This is important as there can be great heterogeneity in the Ca$\sp{2+}$ signal response between different cells in the same population. On this basis, we have shown that ryanodine reduces the caffeine-induced Ca$\sp{2+}$ response; whereas other agents known to affect Ca$\sp{2+}$ regulation such as thapsigargin, TMB-8, and nifedipine had no effect on the caffeine-induced Ca$\sp{2+}$ response. Extracellular Ca$\sp{2+}$ was found to play an important role in the maintenance of resting and tonic (Ca$\sp{2+}\rbrack\sb i$ levels, as well as being essential to replenishment of the caffeine-sensitive Ca$\sp{2+}$ store. We have also shown that caffeine and noradrenaline are equally effective in releasing intracellular Ca$\sp{2+}$ and inducing a tonic increase in (Ca$\sp{2+}\rbrack\sb i.$ (Abstract shortened by UMI.)

Macrophages are the predominant cell type in the chronic inflammatory reaction associated with the development of mineral-dust-induced fibrosis. Macrophages are potent producers of cytokines, which have recently emerged as potentially important mediators in regulating inflammatory states. I investigated cytokine production by alveolar macrophages (AM), with a special emphasis on tumor necrosis factor-$\alpha$, in experimental lung fibrosis induced by mineral dusts. Exposure to fibrigenic dusts had a bimodal effect on TNF-$\alpha$ production by AM. First, suppressed TNF-$\alpha$ production after 1 and 3 weeks of exposure. Second, after 6 weeks of exposure TNF-$\alpha$ production was high. By contrast, interleukin-1 (IL-1) and interleukin-6 (IL-6) production was increased in animals with inflammation with and without fibrosis. Potentiations in IL-1 and IL-6 production were associated with the early stage of the inflammatory reaction and were inversely correlated with TNF-$\alpha$ changes. Interestingly, alterations in TNF-$\alpha$ production were associated with definite shifts in the distribution size of AM suggesting that the overall production of TNF-$\alpha$ may be regulated by the specific class of AM present at sites of inflammation. To our knowledge, our study brings the first evidence for a negative modulation of TNF-$\alpha$ during inflammatory reactions leading to lung fibrosis. Furthermore, experiments with neutralizing antibody to TGF-$\beta$ suggest a role for TGF-$\beta$ in down-regulating TNF-$\alpha$ production in our system.

A pure population of myogenic precursor cells was isolated by explaining single mature myofibres from adult mice together with their associated satellite cells and basal lamina into culture. The satellite cells contained within these myofibres subsequently proliferated and differentiated to form meshworks of mature myotubes given the appropriate growth conditions. This is the first report describing the isolation of a population of satellite cell progeny in a primary culture that was completely free of contaminating fibroblasts, macrophages or other non-myogenic cells. Furthermore, it is the report whereby satellite cells, and their progeny, derived from a single myofibre have been isolated. The purity of adult myogenic precursor cell populations obtained by isolating single myofibre gives this method the potential for being a ready source of pure myoblasts, given the appropriate medium conditions, which could be a viable vehicle for various gene therapy strategies. (Abstract shortened by UMI.)

The major objective of this work was the development of an in vitro model for liver homeostasis which would allow the future study of early events in cell proliferation and cell death. The model which was set up involves growing T51B rat liver epithelial cells with a single dose of 1nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers two days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level five days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers three to five days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, from the electrophoresis of T51B cell DNA and from the quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. Cell growth studies and EGF binding studies were carried out in order to try to determine which events, if any, are EGF specific. The results from these studies suggest that occupancy of the low affinity binding site of the EGF receptor is responsible for the hyperproliferation seen when the T51B cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by the down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems. Thus this work describes the development and characterization of an in vitro model of liver homeostasis which closely parallels in vivo systems where animals are given mitogenic stimuli, and it also provides a good system for studying the role of EGF in cell proliferation and apoptosis.