Freiburg (Breisgau), Universiẗat, Diss., 2004.

Thesis (doctoral)--Utrecht, 1981.

Universiẗat, Diss., 2002--Dortmund.

Proefschrift Maastricht. / Met lit. opg. - Met samenvatting in het Nederlands.

Proefschrift (Med.) Maastricht. / Lit.opg. - Samenvatting in het Nederlands.

The activation of Protein Kinase A (PKA) and of Hyperpolarization-activated and Cyclic Nucleotide-modulated channels (HCN) is controlled by cAMP through cAMP binding domains (CBDs), which serve as cAMP-dependent conformational switches to regulate downstream signaling pathways. The binding of the cAMP allosteric effector removes the auto-inhibition imposed by linkers that are adjacent to the CBDs of PKA and HCN. However, our understanding of how cAMP binding to the structured CBD controls the adjacent inhibitory linkers is currently limited. Herein, we investigate through NMR spectroscopy the interactions between the CBDs of HCN and PKA and the respective adjacent linkers. Chapters 2 and 3 of this thesis focus on the linkers N-terminal to PKA CBD-A and CBD-B, respectively, while Chapter 4 centers on the linker N-terminal to the HCN CBD. We show that in the case of PKA the linker N-terminal to CBD-A is flexible, but is coupled to the CBD-A through state active selective interactions. In the case of the CBD-B of PKA the state selective interactions with the linker N-terminal to it are to a large extent lost and replaced by state-selective inter-CBD interactions, which in turn control the conformational ensemble accessible to the inter-domain linker. Unlike PKA, in the case of HCN, the primary mechanism of cAMP-dependent linker control is through the state-selective destabilization of the structured tetrameric N-terminal linker. Overall, this thesis reports three distinct mechanisms through which linkers in HCN and PKA serve not only as simple covalent threads, but also as integral parts of the allosteric networks underlying auto-inhibition and cAMP dependent activation. / Thesis / Doctor of Philosophy (PhD) / Cyclic adenosine monophosphate or cAMP is a second messenger that is produced by cells to control the internal cellular metabolism in response to external stimuli. The goal of this thesis is to elucidate the structural and dynamical changes that translate the cAMP signal into a specific biological response necessary for the survival of the cell. We used Nuclear Magnetic Resonance (NMR) Spectroscopy to investigate how, under physiological solution conditions, the cAMP interacts with and modifies the cAMP-dependent protein kinase A (PKA) and the hyperpolarization-activated and cyclic nucleotide-gated channels (HCN). Knowledge of both structure and dynamics on both proteins is required in order to fully understand at a molecular level how cAMP works in the human heart. The elucidation of the structural and dynamical changes associated with cAMP-binding is expected to help define general rules applicable to the design of drugs for cardiovascular disorders.

This thesis is part of a large, multi-laboratory project to develop a GPU system-on-chip (SoC) for embedded systems. In support of this project, this current thesis presents the assembler and linker for the overall system. These tools were developed ¡§from scratch¡¨ for this project, because the both the input (to our assembler) and the output (from our linker) have new formats, due to the novelty of our GPU. One of the challenges of the work in this thesis is the problem of memory management. Another is the problem of deciding upon an assembly format. But the largest challenge was in co-design. The assembler has to work with a compiler which is also under development by other students. Also, the machine instructions that we produce have to support the format and functionality of the GPU hardware. To accomplish this, the specific details of this hardware had to be rigorously defined through discussion and negotiation. Furthermore, the memory addresses also required codesign with the benchmark development team, which needs to have access to these memory locations. So codesign issues impacted many of the features of this thesis.

Linker histone H1 associates with nucleosomes, facilitating folding and packaging of DNA into higher order chromatin structure. With 11 variants in mammals, histone H1 is the most divergent histone class. Histone H1 variants are differentially expressed during development and cellular differentiation, and regulate specific gene expression in vivo. Ample studies have established the role of linker histone H1 in chromatin compaction and gene expression regulation; however, its role in diseases, such as cancer, remain understudied. In this study, we explore the role of H1 in ovarian cancer, one of the most devastating gynecological cancers due to its poor prognosis and difficulty in early diagnosis. Although mutations of genes responsible for cell proliferation, differentiation and survival have been found in ovarian cancers, ample evidence also suggests an important role of epigenetic changes in the disease occurrence and progression. Because epigenetic changes do not alter DNA sequence and can be reversed or reprogrammed, they offer an attractive avenue for therapeutic intervention in cancer treatment. Using quantitative RT-PCR assays, we systematically examined the expression of 7 H1 genes in 33 human epithelial ovarian tumors. By clustering analysis, we found that ovarian malignant adenocarcinomas and benign adenomas exhibited characteristic expression patterns. We demonstrate that expression profiling of 7 H1 genes in tumor samples discriminates adenocarcinomas vs. adenomas with high accuracy. These findings indicate that the expression of H1 variants is exquisitely regulated and may serve as potential epigenetic biomarkers for ovarian cancer. To further investigate the role of H1 subtypes in ovarian cancer cells, we employ an over-expression approach to test the function of H1 subtypes in an ovarian cancer cell line OVCAR-3. We found that histone H1.3 over-expression significantly suppresses the growth and colony formation of OVCAR-3 cells. Gene expression arrays identified many genes affected by H1.3 over-expression, and oncogene H19 is among the genes most dramatically repressed by H1.3 over-expression. Over-expression of several other H1 subtypes does not lead to significant reduction of H19 expression, suggesting a specific effect by H1.3. Consistently, knockdown of H1.3 increases H19 expression. Furthermore, increased expression of H1.3 leads to accumulation of H1.3 as well as increased DNA methylation at the regulatory regions of H19. Finally we identified a synergistic effect of H1.3 over-expression and H19 knockdown on inhibition of ovarian cancer cell growth. These results establish oncogene H19 as a direct target of histone H1.3, identify a novel role of H1 variants in ovarian cancer mediated through regulating oncogene H19 expression, and may offer new approaches for ovarian cancer therapeutics.

Proefschrift Rijksuniversiteit Groningen. / CATS (Captopril And Thrombolysis Study). Datum laatste controle: 31-07-1996. Met lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit Maastricht. / Auteursnaam op omslag: Jons Rijcken. Met lit. opg. - Met samenvatting in het Nederlands.