I have pursued a breadth of research that explored the functional genomic study of eukaryotic transcriptional regulation. I have utilized two model organisms, many experimental methodologies, and have developed a suite of computational resources to study the interaction of transcription factors with regulated targets. In Saccharomyces cerevisiae I worked with my collaborator Dr. Zhanzhi (Mike) Hu to characterize the whole-genome transcriptional response of 263 individual transcription factor deletions. We utilized a sophisticated error model and directed-weighted graphs to model a network of high-confidence targets for each transcription factor profiled. We then used regulatory epistasis to elucidate the true set of primary KO-regulated targets and construct a functional transcriptional regulatory network. This network was analyzed for ontological and sequence motif enrichment in order to gain insight into the biological functions represented by transcription factors studied. Functional validation was performed to evaluate the probability of novel functional characterizations. Significant insight was gained from this study with regard to the nature of regulatory cascades and the inability for DNA binding events to predict regulation. This set of analysis was performed with a novel bioinformatic server called ArrayPlex. ArrayPlex is a software package that centrally provides a large number of flexible toolsets useful for functional genomics including microarray data storage, quality assessments, data visualization, gene annotation retrieval, statistical tests, genomic sequence retrieval and motif analysis. It uses a client-server architecture based on open source components, provides graphical, command-line, as well as programmatic access to all needed resources, and is extensible by virtue of a documented API. Using many of the techniques and computational resources developed, I pursued the study of microRNA transcriptional abundance and targeting in H. sapiens cell cultures. Utilizing custom-fabricated microarrays, I measured the whole-genome response of both mRNAs and microRNAs under serum stimulation, c-Myc overexpression, and c-Myc siRNA-mediated knockdown. I then characterized the regulatory interactions between the sets of regulated microRNAs and coordinately regulated transcription factors. Using analytical methods sensitive to regulatory directionality of both populations I was able to determine high-confidence relationships between transcription factors and regulated microRNAs as well as microRNAs and regulated gene targets.

The enzyme adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyses the first reaction of the histidine biosynthetic pathway. ATP-PRT also represents a metabolic control point, directing the flux of metabolites through this energetically expensive pathway. Two distinctly different forms of ATP-PRT exist, the long form and the short form, which differ in the presence of a C-terminal regulatory domain. In the short form, where this domain is absent, it is functionally replaced by a regulatory protein, called HisZ. ATP-PRT activity is modulated by two layers of regulation: active site inhibition by adenosine monophosphate, which reflects cellular energy levels, and pathway end product feedback inhibition by histidine. In the long form ATP-PRT histidine binds to the allosteric site at the regulatory domain, but the exact nature of the inhibitory mechanism is still debated. This thesis characterises a new member of the ATP-PRT long form from Campylobacter jejuni (CjeATP-PRT) and investigates the molecular mechanisms involved in the feed back inhibition by histidine. Chapter 2 describes the characterisation of the CjeATP-PRT including a detailed description of its crystal structure. The C. jejuni enzyme is similar to the previously described enzymes of the ATP-PRT long form, but exists only as hexameric species under experimental conditions, which contradicts previous assumptions that the hexamer is exclusively inactive. Chapter 3 investigates the catalytic apparatus of CjeATP-PRT by separating the catalytic and regulatory domains of the enzyme for individual study. The isolated catalytic portion of the enzyme, the CjeATP-PRT Core mutant, forms a dimeric species with very limited catalytic capabilities but high substrate and product affnities. The CjeATP-PRT Core characteristics suggest that it exists in a permanently inhibited conformation, highlighting the requirement of the regulatory domain not only for feedback regulation but also for enzyme function. Additionally this supports the evolutionary need for the recruitment of a regulatory apparatus. In chapter 4 a potential intramolecular communication pathway from the allosteric to the active site is probed by the generation of several single site mutations. One of these, CjeATP-PRT R216A, is completely insensitive to histidine inhibition, although this ligand is still able to bind at the allosteric site, which is consistent with the involvement of R216 in the allosteric signal communication. The catalytic abilities of CjeATP-PRT R216A are largely impaired, leading to the assumption that this mutation causes a permanent inhibitory response. In summary this thesis supports the existence of a simple physical regulatorymechanism for the feedback inhibition of the ATP-PRT long form, the change between two different hexamer conformations depending on the presence of the allosteric effector.

Osteoclasts are multinucleated, terminally differentiated cells found in bone tissue, which have the unique ability to resorb calcified substrates. The study of human osteoclasts has been restricted in the past due to difficulties in obtaining these cells. Recently, however, cell culture techniques have been devised that allow human osteoclasts to be grown in culture from their mononuclear precursors found in peripheral blood, therefore providing a constant supply of these cells. These cultures are based on the discovery of RANKL (receptor activator for NFJ(B ligand), which has recently been shown to playa central role in osteoclast formation and activation. This thesis has initially characterised cells grown in such cultures to confirm that they are authentic human osteoclasts, possessing osteoclast markers and with the ability to resorb calcified substrates. Once these cultures were established, and the authenticity of human osteoclasts grown in these cultures was confirmed, these cells were used to study the effects of extracellular nucleotides on human osteoclast acti vity. Adenosine-5' -triphosphate (ATP) is well known as an intracellular energy source. This and other nucleotides, however, also exist extracellularly, where they are agonists at a group of receptors termed P2 receptors. This receptor family is subdivided into P2X ligand gated ion channels, and P2Y G-protein coupled receptors. Bone-forming osteoblast cells, and boneresorbing osteoclast cells both express multiple SUbtypes of these receptors. Studies examining the effects of extracellular nucleotides on osteoclasts have been largely limited to non-human cells in the past due to the difficulty in obtaining human osteoclasts. This thesis, therefore, has examined the effects of extracellular nucleotides on human osteoclast activity. Human osteoclasts and their precursors were shown to express mRNA for nearly all of the P2Y and P2X receptor subtypes. ATP was found to both activate human osteoclast formation, by acting directly on P2X receptors expressed by osteoclast precursors, and to stimulate osteoclast resorption indirectly by acting at osteoblast-expressed P2Y) receptors to induce elevated RANKL expression by these cells. Activation of P2Y receptors expressed by osteoclasts was shown to result in downstream activation of MAPKinase pathways. Parathyroid hormone (PTH) is considered to be one of, if not the most important systemic factor in the regulation of bone. Co-stimulation of UMR-I 06 osteoblast-like cells with this hormone and P2Y) agonists resulted in potentiation of P2Y) agonist-induced [Ca2+Ji release by PTH, while PTH alone produced no [Ca2+]j elevation at all. The mechanism of this potentiation was attributed to Gs activation following PTH receptor stimulation, Gq having no involvement. Co-stimulation of these cells by PTH and P2Y) agonists also resulted in synergistic immediate early gene expression. These findings suggested that extracellular nucleotides are able to sensitize osteoblasts to the actions of PTH, providing a mechanism for localizing the response to this systemic hormone in bone, consistent with the discrete pattern of remodelling observed in the skeleton. Finally, the involvement of PTH on osteoclast formation was investigated. PTH was found to inhibit this process via activation of contaminating T lymphocytes present in osteoclast cultures. In conclusion this thesis presents evidence to suggest that extracellular nucleotides are important local signaling molecules in bone, affecting both osteoclast and osteoblast activity, alone and in combination with systemic factors such as PTH. Additionally, a novel action of PTH acting via lymphocytes to affect osteoclast formation is described.

This doctoral dissertation deals generally with the reform of the Chinese (Mainland) foreign banking law in the context of the international convergence of supervisory standards. The starting premise is that the current foreign banking laws are out of line with international supervisory standards and practices in various fundamental respects. Moreover, the Chinese legislators and bank supervisors lack a meaningful appreciation and practical cultivation of commonly accepted supervisory values. These realities have underscored the importance of overhauling the foreign banking laws. The overarching thesis of this dissertation is that for China to develop a viable and modern banking system, it will need to develop and to implant a suitable legal infrastructure consistent with emerging international supervisory standards and with WTO requirements and aspirations for financial sector liberalization. On this vein, I propose a set of reforms that would create a legal environment for competitive equality between foreign banks, while at the same time protecting the "safety and soundness" of the Chinese banking system. I start by looking at the entry of foreign banks into the Chinese market. My major proposal, in this respect, is that the Chinese foreign banking law should clearly specify mandatory and discretionary licensing criteria. Since the licensing of a foreign bank is a process of mutual cooperation between Chinese and foreign supervisors, I recommend that the foreign banking law should incorporate into the licensing process the negotiation of a supervisory agreement between Chinese and foreign supervisors. I then examine the on-going regulation of foreign banks. In this respect, I propose that foreign banks should have autonomy to determine the adequacy of capital, liquidity and provisioning, although some quantitative prescriptions are still necessary. I suggest further that the foreign banking law should introduce risk-focused supervision. I also propose that bank supervisors should play an important role in ensuring that foreign banks establish sound bank management and public disclosure. Finally, I consider foreign bank crisis management. I propose that the Chinese foreign banking law should establish a joint responsibility of China and foreign countries on "lending of last resort" functions. Foreign banks should be required to participate in China's or their own countries' deposit insurance schemes. I also advocate a rule-based approach to foreign bank failure resolution in order to reduce traditional strong political pressure on the Chinese supervisors when they deal with bank failures. In sum, this dissertation conducts a critical examination of the current Chinese foreign banking laws vis-a-vis an analysis of compatibility with international standards and practices. The end result of this research is a number of considered recommendations for legal reform that I think should improve significantly the CUlJ'ent Chinese foreign banking laws.

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published_or_final_version / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

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