Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves : 325-355). Also available on the Internet.

Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules. In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase. I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

The error free repair of DNA double strand breaks through the homologous recombinational repair pathway is essential for organisms of all types to sustain life. A detailed structural and mechanistic understanding of this pathway has been the target of intense study since the identification of bacterial recA, the gene whose product is responsible for the catalysis of DNA strand exchange, in 1965. The work presented here began with defining residues that are important for the assembly and stability of the RecA filament, and progressed to the identification of residues critical for the transfer of ATP-mediated allosteric information between subunits in the protein's helical filament structure. My work then evolved to investigate similar mechanistic details concerning the role of ATP in the human RecA homolog, Rad51. Results from non-conservative mutagenesis studies of the N-terminal region of one subunit and the corresponding interacting surface on the neighboring subunit within the RecA protein, led to the identification of residues critical for the formation of the inactive RecA filament but not the active nucleoprotein filament. Through the use of specifically engineered cysteine substitutions we observed an ATP-induced change in the efficiency of cross subunit disulfide bond formation and concluded that the position of residues in this region as defined by the current crystal structure may not accurately reflect the active form of the protein. These ATP induced changes in positioning led to the further investigation of the allosteric mechanism resulting in the identification of residue Phe217 as the key mediator for ATP-induced information transfer from one subunit to the next. In transitioning to investigate homologous mechanisms in the human pathway I designed a system whereby we can now analyze mutant human proteins in human cells. This was accomplished through the use of RNA interference, fluorescent transgenes, confocal microscopy and measurements of DNA repair. In the process of establishing the system, I made the first reported observation of the cellular localization of one of the Rad51 paralogs, Xrcc3, before and after DNA damage. In addition we found that a damage induced reorganization of the protein does not require the presence of Rad51 and the localization to DNA breaks occurs within 10 minutes. In efforts to characterize the role of ATP in human Rad51 mediated homologous repair of double strand breaks we analyzed two mutations in Rad51 specifically affecting ATP hydrolysis, K133A and K133R. Data presented here suggests that, in the case of human cells, ATP hydrolysis and therefore binding, by Rad51 is essential for successful repair of induced damage.

Aside from an indirect effect of PPARgamma activation to reduce insulin resistance and to facilitate adiponectin release, PPARgamma agonist could also exert direct effects on blood vessels. I provided a first line of experimental evidence demonstrating that PPARgamma agonist rosiglitazone up-regulates the endothelin B receptor (ETBR) expression in mouse aortas and attenuates endothelin-1-induced vasoconstriction through an endothelial ET BR-dependent NO-related mechanism. ETBR up-regulation inhibits endothelin-1-induced endothelin A receptor (ETAR)-mediated constriction in aortas and mesenteric resistance arteries, while selective ETBR agonist produces endothelium-dependent relaxations in mesenteric resistance arteries. Chronic treatment with rosiglitazone in vivo or acute exposure to rosiglitazone in vitro up-regulate the ETsR expression without affecting ETAR expression. These results support a significant role of ETBR in contributing to the increased nitric oxide generation upon stimulation with PPARgamma agonist. This study provides additional explanation for how PPARgamma activation improves endothelial function. / Firstly, I demonstrated that adipocyte-derived adiponectin serves as a key link in PPARgamma-mediated amelioration of endothelial dysfunction in diabetes. Results from ex vivo fat explant culture with isolated arteries showed that PPARgamma expression and adiponectin synthesis in adipose tissues correlate with the degree of improvement of endothelium-dependent relaxation in aortas from diabetic db/db mice. PPARgamma agonist rosiglitazone elevates the adiponectin release and restores the impaired endothelium-dependent relaxation ex vivo and in vivo, in arteries from both genetic and diet-induced diabetic mice. The effect of PPARgamma activation on endothelial function that is mediated through the adiponectin- AMP-activated protein kinase (AMPK) cascade is confirmed with the use of selective pharmacological inhibitors and adiponectin -/- or PPARgamma+/- mice. In addition, the benefit of PPARgamma activation in vivo can be transferred by transplanting subcutaneous adipose tissue from rosiglitazone-treated diabetic mouse to control diabetic mouse. I also revealed a direct effect of adiponectin to rescue endothelium-dependent relaxation in diabetic mouse aortas, which involves both AMPK and cyclic AMP-dependent protein kinase signaling pathways to enhance nitric oxide formation accompanied with inhibition of oxidative stress. These novel findings clearly demonstrate that adipocyte-derived adiponectin is prerequisite for PPARgamma-mediated improvement of endothelial function in diabetes, and thus highlight the prospective of subcutaneous adipose tissue as a potentially important intervention target for newly developed PPARgamma agonists in the alleviation of diabetic vasculopathy. / To summarize, the present investigation has provided a few lines of novel mechanistic evidence in support for the positive roles of PPARgamma and PPARdelta activation as potentially therapeutic targets to combat against diabetic vasculopathy. / Type 2 diabetes mellitus and obesity represent a global health problem worldwide. Most diabetics die of cardiovascular and renal causes, thus increasing the urgency in developing effective strategies for improving cardiovascular outcomes, particularly in obesity-related diabetes. Recent evidence highlights the therapeutic potential of peroxisome proliferators activated receptor (PPAR) agonists in improving insulin sensitivity in diabetes. / While agonists of PPARalpha and PPARgamma are clinically used, PPARdelta is the remaining subtype that is yet to be a target for current therapeutic drugs. Little is available in literature about the role of PPARdelta in the regulation of cardiovascular function. The third part of my thesis focused on elucidating cellular mechanisms underlying the beneficial effect of PPARdelta activation in the modulation of endothelial function in diabetes. PPARdelta agonists restore the impaired endothelium-dependent relaxation in high glucose-treated aortas and in aortas from diabetic db/db mice through activation of a cascade involving PPARdelta, phosphatidylinositol 3-kinase, and Akt. PPARdelta activation increases Akt and endothelial nitric oxide synthase and nitric oxide production in endothelial cells. The crucial role of Akt is confirmed by selective pharmacological inhibitors and transient transfection of dominant negative Akt plasmid in these cells. Treatment with PPARdelta agonist GW501516 in vivo augments endothelial function in diabetic db/db and diet-induced obese mice. The specificity of GW501516 for PPARdelta is proven with the loss of its effect against high glucose-induced impairment of endothelium-dependent relaxation in aortas from PPARdelta knockout mice. In addition, oral administration of GW501516 in vivo fails to improve endothelial function in diet-induced obese PPARdelta deficient mice. / Tian, Xiaoyu. / Adviser: Huang Yu. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Hepatocellular carcmoma (HCC) is the third most common cause of cancer-related deaths worldwide, with a gender prevalence observed in men. Recent studies have suggested that elevated activity of the androgen axis is one major host factor underlying this disparity between genders. The androgen receptor (AR) mediates function of androgen in vital developmental and oncogenic pathways by binding to genomic androgen response elements, which influence the transcription of downstream target genes. AR is overexpressed in 60-80% of human HCCs. Genetic studies further established the pivotal role ofAR in hepatocarcinogenesis, where liver-specific knockout of AR significantly reduced tumorigenicity in carcinogen- and HBV-induced HCC mouse models. However, AR-inducedhepatocarcinogenesis is far from fully understood, in part because little is known about the identity and role of direct AR-dependent targeted genes in hepatocytes. / In this study, we used genome-wide location and functional analyses to identify a critical mediator of AR signaling, cell cycle-related kinase (CCRK), in driving beta-cateninl T-cell factor (TCF)-dependent hepatocarcinogenesis. Using chromatin immunoprecipitation followed by promoter array analysis of AR-overexpressing HCC cell lines, we found a number of cell cycle-related genes that are likely under the direct modulation of AR. Cell cycle-related kinase (CCRK), previously shown to promote glioblastoma tumorigenesis, was found to be the most significantly-bound AR target ( p<0.0001). CCRK was directly up-regulated by ligand-activated AR through promoter binding and required for AR-induced G1-S cell cycle progression because (1) CCRK overexpression attenuated cell cycle blockage by AR knockdown and (2) CCRK inhibition counteracted AR-mediated cell cycle progression. Ectopic CCRK expression induced immortalized liver cell proliferation, malignant transformation and tumor formation in immunodeficient mice, whereas CCRK inhibition decreased HCC cell growth in vitro and in vivo. These functional assays demonstrated that CCRK is a potential oncogene in HCC. Mechanistically, CCRK activated beta-catenin/TCF-dependent transcription through phosphorylation of glycogen synthase kinase-3beta and induced the expressions of beta-catenin target genes, cyclin D1 (CCND1) and epidermal growth factor receptor (EGFR). Inhibition of beta-catenin/TCF signaling attenuated CCRK-induced cell cycle progression, colony formation and tumorigenicity. Conversely, HCC cell growth inhibition by CCRK knockdown was rescued by constitutively active beta-catenin or TCF. In agreement with these findings, activation of the AR/CCRK/beta-catenin axis was frequently observed in primary HCCs. More importantly, CCRK over-expression was correlated with tumor staging and poor overall survival in a cohort ofhuman HCC tissues. / Together, our data reveal a new cascade for AR function in hepatocarcinogenesis via the activation of beta-catenin/TCF signaling. This study also reveals that CCRK is a novel focal link between two prominent signaling pathways vital for HCC growth and thus represents a new therapeutic target for HCC treatment. / Feng, Hai. / Adviser: Sung Jao Yiu. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 161-177). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ng, Chor Fung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.