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Bibliography: pages 133-155. / Insulin regulates cellular gene expression and modulates specific mRNA levels in liver cells. As yet, the mechanism of this control is still unclear. The effects are initiated following the binding of insulin to the plasma membrane receptor. Although several mediators of the signal from the plasma membrane to the nucleus have been proposed, none has proved capable of eliciting all of the effects of insulin on gene expression. Therefore, the possibility that insulin itself may directly regulate transcription at the level of the nucleus, was investigated.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xiv, 200 p.; also includes graphics (some col.). Includes bibliographical references (p. 167-197). Available online via OhioLINK's ETD Center

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

SUMO-1/2/3 are members of the ubiquitin-like family of protein modifiers. These proteins are covalently attached to numerous proteins in a directed and controlled manner. SUMO conjugation primarily occurs to proteins containing an exposed SUMO conjugation motif, (I, V, L, F)KxE, where x represents any amino acid. SUMO conjugation is controlled by key enzymes, a SUMO activating enzyme, SAE1/2 and a SUMO conjugating enzyme, Ubc9, which is responsible for substrate recognition, and the efficiency of this pathway can be increased by one of many SUMO ligase enzymes. This modification alters the substrate's characteristics and results in a change of state, such as stability, localisation, or activity. p300, a transcriptional co-activator, contains an evolutionary conserved tandem SUMO modification motif, located in a transcriptional repression domain. p300 was efficiently conjugated, both in vitro and in vivo, by SUMO-1/2/3, within this repression domain to both SUMO conjugation motifs. The SUMO conjugation to p300 correlated with p300 ability to repress transcription, requiring both SUMO conjugation motifs for full transcription repression activity. This repression activity was mediated through SUMO recruitment of histone deacetylase 6. Repression could be alleviated through co-expression of a SUMO-specific protease thereby suggesting a potential regulatory mechanism for transcription control of SUMO modified substrates. Despite utilising the same conjugation machinery, there remained the potential for distinct roles for the SUMO isoforms. SUMO -2/3, which form a distinct group from SUMO-1, were shown to preferentially mediate the transcription repression abilities of a number of known SUMO modifiable substrates: p300, Elk-1, and SP3. Further differences were observed in the ability of SUMO-1 and SUMO-2/3 to influence the nuclear organisation of p80 coilin.

Tissue-resident memory T cells (TRM) are a non-circulating subset of memory that are maintained at sites of pathogen entry and mediate optimal protection against reinfection. Lung TRM can be generated in response to respiratory infection or vaccination, however, the molecular pathways involved in CD4+TRM establishment have not been defined. Here, we performed transcriptional profiling of influenza-specific lung CD4+TRM following influenza infection to identify pathways implicated in CD4+TRM generation and homeostasis. Lung CD4+TRM displayed a unique transcriptional profile distinct from spleen memory, including up-regulation of a gene network induced by the transcription factor IRF4, a known regulator of effector T cell differentiation. In addition, the gene expression profile of lung CD4+TRM was enriched in gene sets previously described in tissue-resident regulatory T cells. Up-regulation of immunomodulatory molecules such as CTLA-4, PD-1, and ICOS, suggested a potential regulatory role for CD4+TRM in tissues. Using loss-of-function genetic experiments in mice, we demonstrate that IRF4 is required for the generation of lung-localized pathogen-specific effector CD4+T cells during acute influenza infection. Influenza-specific IRF4−/− T cells failed to fully express CD44, and maintained high levels of CD62L compared to wild type, suggesting a defect in complete differentiation into lung-tropic effector T cells. This finding identifies IRF4 as an important regulator of CD4+TRM generation in response to respiratory infection. Furthermore, comparing whole transcriptome profiling of mouse and human lung memory T cell subsets, we define a lung CD4+TRM gene signature common to mice and humans. IRF4 protein was specifically up-regulated in lung CD4+TRM but not in circulating memory subsets, in both humans and mice previously infected with influenza. This result suggest that high expression of IRF4 contributes to a cross-species conserved molecular pathway of long term maintenance of CD4+TRM in the lung. Overall, our findings confirm lung CD4+TRM as a unique memory T cell subset regulated by tissue-specific transcription factors. These results have important implications in focusing future studies of tissue resident memory T cells to factors with translational potential. Importantly, by determining the lung CD4+TRM gene signature common to mice and humans, we motivate future genetic studies that could lead to the complete identification of the mechanisms of TRM maintenance in humans.

In the past decade, technologies such as the DNA microarray and ChIP-on-chip have generated a large amount of high-throughput data for biologists. Although these data has provided us systems-level information about gene regulation, a major challenge in systems biology is to derive methodologies that will infer the underlying dynamics and mechanisms of gene regulation. This thesis research is focused on understanding these mechanisms of transcriptional regulation using systems biology approaches. Transcription regulatory networks play an important role in mediating external stimuli and coordinating responses to changing environments. Different methods that infer regulatory interactions directly from microarray data have been developed in the recent past. However, the implicit assumption in these methods that the transcription factor (TF) mRNA expression can be used as a proxy of its activity at protein level is not always correct, due to post-transcriptional and post-translational modifications of TFs. In this study, a method named iARACNe was developed. It uses the inferred TF activities to estimate the regulatory activity between TFs and their targets. The study demonstrated that the accuracy of the inferred networks using this method was greatly improved. Two additional methods, OmniMiner and coEDGi, which allow a better understanding of the physical interactions between TFs and target genes, were developed in this thesis research. OmniMiner detects and predicts the potential binding sites for the TFs of interest, while coEDGi enables identification of common enhancers upstream of co-regulated genes. Compared to other approaches which only allow isolated analyses, the systems biology approaches developed in this research provide an opportunity for biologists to study transcriptional regulations from both functional genomics and regulatory sequence perspectives simultaneously.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 56-68).

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 128-143). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / On t.p. "Trp" is superscript. Includes bibliographical references (leaves 152-172). Also available in electronic version. Access restricted to campus users.