Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, 1922. / "Private edition, distributed by the University of Chicago libraries, Chicago, Illinois." "Reprinted from Biological bulletin, vol. XLV, no. I, July, 1923." Bibliography: p. 75-77. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, 1922. / Letterpress descriptive of opposite plate on verso of each plate. "Author's abstract of this paper issued by the [Wistar institute] Bibliographic service, March 14." Stamped on t.p.: Private edition, distributed by the University of Chicago libraries, Chicago, Illinois. "Reprinted from the Journal of experimental zoölogy, vol. 33, no. 1, May, 1921." "Literature cited": p. 40-41. Also available on the Internet.

The aim of this study was to investigate the role of signalling molecules during the development of the chick limb. First, it was demonstrated that a functional gradient of bioactive FGFs is present down the limb from distal to proximal. This functional FGF gradient decreased at stage 26, at the time of AER regression. Although morphogenetic gradients are of considerable theoretical importance in developmental biology, there are rather few practical demonstrations of their existence. The effects of prolonging the presence of active FGF on limb pattern formation were investigated. Application of ectopic FGF-4 to the distal tip of the limb at stage 26 had a number of effects on limb development. In particular, the cartilage structure conventionally labelled element "5" increased in size and in some instances acquired a digit-like morphology. The evolutionary considerations of this finding are briefly considered. Analysis, including 3D computer reconstruction, of the musculature and vasculature of limbs after FGF implants was carried out in the hope of establishing the identity of this digit like element. This proved not to be possible. However, both muscle mass and vascularisation had increased after the procedure. Known molecular pathways involving the proximo-distal patterning of the limb were then investigated. Whole mount in situ hybridisation studies were carried out with respect to FGF-4, sonic hedgehog, Hoxd-11 and FGF-8. These revealed that the shh/ FGF positive feedback loop was not involved in these changes. FGF-8 expression in late stage AERs was markedly increased. Hoxd-11 expression was not affected by ectopic FGF implants. Together, these findings suggest that the effect of FGF implants is mediated by a novel mechanism. The effect of FGF-4 implants on programmed cell death in the limb was examined. At stage 28 anterior necrotic zone was larger, and had shifted distally. However, the posterior necrotic zone was absent. The implications of these findings for limb development were discussed.

During cellular differentiation, gene expression is globally regulated through changes in the epigenome. How a single genome can give rise to a diversity of cell and tissue types remains a complex area of investigation, and here we sought to explore the molecular regulation of gene expression during the differentiation of skeletal muscle cells from committed myogenic progenitors. Using a systematic and integrated analysis of global transcriptional and epigenetic data, we characterized the regulation of gene expression in differentiating myoblasts and found that muscle-specific gene expression is regulated through differential activation of tissue-specific regulatory DNA elements by the myogenic transcription factor MyoD. In addition, the genome-wide localization of MyoD, and the mechanisms underlying its function in transcriptional regulation, varies between myogenic progenitors and differentiating myoblasts. Our study explores the recruitment and function of MyoD at regulatory elements of target genes and additionally describes a novel role for ligand-inducible signaling in the regulation of MyoD function and ultimately in myogenic differentiation.

During the haematogenous spread of tumours, the metastasizing cells must arrest within the blood vessels of the organs they colonize. There is still much debate upon the mechanism of such arrest, whether it is due to mechanical trapping or, more specifically, to adhesion of the tumour cells to the blood vessel wall. This work demonstrates that tumour cells are capable of adhering to blood vessel wall components. According to the hypothesis of specific adhesion, it is thought that metastasizing tumour cells would only come into contact with the vessel wall for a very short time and therefore their adhesion to the vessel wall must be extremely rapid. It has been shown in the past that tumour cells can adhere rapidly to components of the blood vessel wall such as exposed sub-endothelial matrix. Adhesion to endothelial cells was believed to occur at a much slower rate and therefore the involvement of the endothelium during the arrest phase of the metastatic process was thought to be marginal. The experiments carried out during this study show that, in vitro, tumour cells do adhere to the endothelium at a rate comparable to that for isolated components of the sub-endothelial matrix. Furthermore, this work provides some evidence that the molecular basis for such rapid adhesion to the endothelium may be different from the ones involved in the adhesion to known components of the sub-endothelial matrix.

The efficiency of a number of experimental techniques for the extraction of total RNA from various cells and tissues (including MDCK strain I cells) was assessed, and the optimal conditions for hybridisation of Na,K-ATPase isoform-specific DNA probes to this RNA were determined. The specificity of hybridisation of DNA probes for the Na,K-ATPase al, a2, a3, and beta1 isoforms was assessed using RNA isolated from rat tissues. The relative abundance of isoform mRNA's in rat kidney, brain, lung, and myocardial tissues was determined by Northern blotting. The abundance of Na,K-ATPase isoforms was also determined in the myocardial tissues of the Milan rat, a hypertensive animal model. Significant differences between the abundance of Na,K-ATPase isoform mRNA's in hypertensive rats and their age and sex matched controls were found. The relative abundance (per ng of total RNA) of al, a3, and beta1 mRNA's in left ventricle and, that of a1, and beta1 mRNA's in right ventricle were significantly decreased in hypertensive rats. The relative abundance (per mug of total RNA) of a2 and beta1 mRNA's in atria was significantly increased in hypertensive rats. These differences found in ventricles and atria were further accentuated by expression of the results per gram wet weight of tissue. The results from ventricular tissues were in contrast to those previously reported by Herrera et al. (1988) who found either increases or no change in the abundance of a1 and beta1 mRNA's in hypertensive rat aorta, skeletal muscle and left ventricle. The differences between these results may be related to the deoxy-corticosterone treatment and high salt diet of the hypertensive rat model used by Herrera et al. (1988). Na,K-ATPase isoform-specific DNA restriction endonuclease fragments were used to investigate the expression of the isoform mRNA's in MDCK strain I cells. Only a1 and beta1 mRNA's was detected on Northern blots, with no detectable a2 or a3 isoform mRNA signals being found in this cell line. [3H]-ouabain binding to cells was used, as an estimate of the cell surface expression of Na,K-ATPase. Possible factors affecting the expression of Na,K-ATPase during the normal cell growth of MDCK strain I cells were investigated. Factors such as cell seeding density, cell growth substrate and the volume of growth medium used, were all found to affect both the level and pattern of expression of Na,K-ATPase during the normal cell growth or culturing cycle. After 2 days of culture the large increases in the expression of Na,K-ATPase assayed in low density compared to high density seeded cells, were not correlated with concomitant changes in the relative abundance of Na,K-ATPase a subunit mRNA. These results indicate that the large changes in cell surface expression of Na,K- ATPase found during cell growth are probably controlled by post transcriptional processes. The effect of certain hormones or their agonists (aldosterone, deoxy-corticosterone, corticosterone, dexamethasone, and tri-iodo thyronine), on the expression of Na,K-ATPase in MDCK strain I cells was also briefly investigated. Under the conditions used, hormone treatment was not found to induce any measurable expression of a2 or a3 mRNA's. The mineralocorticoid aldosterone, and the glucocorticoid corticosterone, both produced small but significant increases in the level of Na,K-ATPase present on the cell membrane, however these increases were not correlated with similar increases in the abundance of both Na,K-ATPase a1 and beta1 mRNA's. The small size of increases in Na,K-ATPase enzyme abundance after hormone treatments and the inability of those treatments to induce consistent increases in Na,K-ATPase mRNA's further suggests that changes in the cell surface expression of Na,K-ATPase in MDCK cells is the result of regulation at a post transcriptional level.

This thesis is a study of the biology of Drosophila imaginal disc cells growing as continuous cell lines. Their morphological characteristics and cellular properties were analysed in order to characterise the cells in vitro. Morphological analysis of the cells revealed several cell types within the original cell lines perhaps representing a diversity in the cellular origin of these cells. A new cloning technique was devised which enabled a single imaginal disc cell to give rise to new cell line and indicated that a single cell could give rise to the different cell types seen in culture. This indicates that the diversity in morphology of the cells in culture was an indication of the conditions in culture rather than as a result of cellular diversity. The original cell lines and the newly derived cloned cell lines were subjected to the insect moulting hormone 20-HE in order to ascertain the degree of differentiation that was possible in culture. The cells showed a dramatic morphological response to hormone, they elongated, began to aggregate and threw out cell processes. This is combined with concomitant biochemical changes in the cell lines, including the induction of chitin synthesis and acetylcholinesterase. The cells in culture show a characteristic pattern of aggregation which was studied at the ultrastructural level using electron microscopy. These studies and also immunofluorescence of cell aggregates indicated the prevalence of cell processes and a role was postulated for their action in bringing about these aggregates. Aggregation was also correlated with the expression of PS integrins, which are well characterised Drosophila adhesion molecules. The adhesive properties of the cells were further characterised with reaggregation experiments in different media as a prelude to setting up cell sorting assays between wing and leg cell lines. This proved somewhat inconclusive but pointed to some sorting out occurring between wing and leg cells.

The Reproductive Homeobox X-linked, Rhox, genes encode transcription factors that are expressed exclusively in the testis, epididymis, placenta, and ovary. While there are 33 Rhox genes in mice, only Rhox5 and Rhox8 are expressed in Sertoli cells, suggesting that they alone regulate the expression of somatic-cell gene products crucial for testicular metabolism and germ-cell development. Targeted deletion of Rhox5, the founding member of the Rhox gene cluster, in mice, results in decreased expression of the insulin-2 gene (Ins2) and other metabolic genes, male subfertility via reduced sperm number, increased germ-cell apoptosis and a reduced proportion of sperm with normal motility. Davis et al. developed a Rhox8 siRNA knockdown transgenic model to study possible functional similarities between Rhox5 and Rhox8 and reveal compensatory actions via the breeding of Rhox5/Rhox8 double knockout mice. They observed that loss of Rhox8 results in downregulation of the sex-determining region Y gene (Sox9). Further analysis of the role of Rhox5 in testicular metabolism regulation was completed by development of mutant constructs encoding combinations of Rhox5 functional domains and subsequent analysis via qRT-PCR, luciferase assay and immunohistochemistry in cell lines transfected with expression plasmids containing these mutants. Our results indicated direct interaction of RHOX5 with the Ins2 promoter. The homeodomain and amino-terminal domain construct being sufficient for promoter activation albeit at a lower level than the full-length RHOX5 construct. MacLean et al. conducted qRT-PCR analysis of cells transfected with plasmids encoding the other Rhox genes revealed that Rhox8 and Rhox11 were also capable of upregulating Ins2 expression at a lower level than Rhox5. Our analysis of metabolic gene expression in the Rhox8 knockdown model also revealed decreased expression of Ins2 as well as insulin receptor-1 (InsR1). Continued analysis of the Rhox8-KD model and Rhox5/Rhox8 double knockout mice in young and aged (~12 months) male mice revealed a subfertility phenotype characterized by reduced litter frequency and size, reduced total spermatozoa and reduced sperm forward motility. This was reflected by a decrease in RHOX8 and SOX9 protein expression maintained in the aged mice that was more severe in the double knockout animals. qRT-PCR analysis of altered gene expression in the Rhox8 single knockdown male mice revealed significant expression downregulation of fellow Rhox genes Rhox5 and Rhox10 and expression upregulation of the growth differentiation factor-9 (Gdf9) gene normally expressed in the somatic cells of the ovary. Based on the regulation of sex specific genes Sox9 and Gdf9 in the Rhox8 knockdown model as well as data published by Daggag et al. revealing that Rhox8 is the only Rhox member expressed in the somatic cells of the embryonic testis, we sought to elucidate the possible role of Rhox8 in sex determination and testicular differentiation/development. Due to the limitations of our current knockdown model whereby siRNA production is initiated postnatally due to its androgen-dependent promoter, we elected to develop a new Rhox8 knockdown model to characterize the embryonic functions of Rhox8. Rhox8 siRNA were cloned into a Cre-recombinase activated expression vector in which a highly active U6 promoter drives expression of the shRNA. The constitutively active form of this vector exhibited knockdown of Rhox8 in stable Rhox8 overexpressing cell lines developed for this purpose. This developed vector is being used to develop a new transgenic line. While waiting for the development of the new Rhox8 knockdown transgenic mice, we chose to characterize the mRNA and protein expression of Rhox8 in the embryonic testis. Using qRT-PCR and immunohistochemistry, expression of Rhox8 was confirmed and exhibited continually increased expression in the Sertoli cells over the course of embryonic day ~13.5-18.5 (E13.5-E18.5). To examine possible functions of Rhox8 in the embryonic testis prior to receipt of the new knockdown model, we adapted a protocol to transfect the new constitutively active Rhox8 knockdown into embryonic gonads and cultured them up to 72 hours after electroporation. This protocol yielded successful expression of GFP from a GFP-expression plasmid and this was maintained for up to 72 hours in E13.5 gonads. qRT-PCR analysis of gonads transfected with the Rhox8 knockdown expression plasmid revealed significant knockdown of Rhox8 and Sox9 mRNA expression at the 48-hour and 72-hour time points.

No description available.