Blood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary... Show moreBlood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary cilium is a component of the endothelial shear sensor. This luminal cell protrusion contains microtubules and is connected to the microtubular cytoskeleton. We identified cilia on endothelial cells of the embryonic heart in areas of low or oscillatory shear stress. This shear-related distribution is reminiscent of the distribution of atherosclerotic lesions in the adult arterial system, as lesions develop at sites of low or oscillating shear (athero-prone flow). Ciliated endothelial cells are exclusively present at these atherosclerotic predilection sites in adult mice. Athero-prone (oscillatory) but not athero-protective (steady or pulsatile) flow induces ciliation of cultured endothelial cells. Moreover, the endothelial shear response is dependent on the microtubular cytoskeleton and primary cilia sensitise the endothelium for shear. Taken together, these data demonstrate that primary cilia are induced by athero-prone flow and that ciliated cells are more sensitive to shear stress. We conclude that the endothelial biosensor for shear stress is the microtubular cytoskeleton and that the attached primary cilium functions as a signal amplifier in areas subjected to athero-prone flow. Show less
Total mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal... Show moreTotal mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal adenomas. Incorrect preoperative staging before TEM is a problem. Therefore the aim of this thesis was to identify molecular differences between rectal tumors of different stages, using gene expression profiling and genomic analysis. First protocols and data analysis algorithms for a new type of SNP array were developed. These studies showed that reliable LOH and copy number changes could be obtained from both frozen and paraffin embedded material. Consequently SNP arrays were used to type groups of TEM and TME treated samples. Five genomic events were found which could make a clear discrimination between adenomas and carcinomas. Early carcinomas treated by TEM, which were not recognized preoperatively as carcinomas, showed already carcinoma-associated aberrations. Analysis of tree core biopsies per patient showed a large degree of intra- tumor heterogeneity; although a good correlation was obtained between the biopsy with the largest number of aberrations and its corresponding tumor fraction. Gene expression array analysis was performed on the same samples as the SNP array series. A high concordance between chromosomal aberrations and changes in gene expression was observed. Finally a clinical application of these data is discussed in the preoperative staging of rectal tumours. Show less
Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore,... Show moreUsing newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene wide expression analysis that would hint to the molecular pathways that are altered upon loss of mitochondrial ATP production as a consequence of A3243G mtDNA mutation. Extensive post-transcriptional adaptation in the form of global translation repression, was however apparent. A comparison between two mtDNA haplotypes indicated, that these presumably neutral sequence variants can affect the nuclear expression program, which tentatively indicates that mtDNA haplotype can affect phenotype. Show less
Arsenic (As) is a notoriously poisonous metalloid with known hazardous effects to human health. The project described in this thesis was aimed at elucidating the probable mechanism of As-induced... Show moreArsenic (As) is a notoriously poisonous metalloid with known hazardous effects to human health. The project described in this thesis was aimed at elucidating the probable mechanism of As-induced neurotoxicity in vivo and in vitro. The animal studies in this thesis were designed to answer questions about the effect of As on the peripheral nervous system after sub-acute and chronic intoxication of laboratory rats. Protein composition analysis showed compositional changes in sciatic nerves proteins. Protein expression of neurofilament heavy (NF-H) and neurofilament medium (NF-M) remained unchanged. Neurofilament protein light (NF-L) expression was reduced, while _- and m-calpain protein expression was increased, both in a dose/time pattern. Furthermore, NF-H protein was hypophosphorylated; while NF-L and microtubule-associated protein tau (MAP-tau) proteins were phosphorylated. In the in vitro studies, effects of As species were tested in various cell culture models and the manner of their hyperphosphorylation was further studied for a better understanding of the disruption of neuroskeletal integrity by As. In vitro studies showed that the compositional changes were not caused by the changes on RNA expression levels, rather a post-translational activity. Cells treated with arsenite showed cleavage of p35 to p25 by calpain, which is mediated by an increase of Ca2+ in the cells. Over expression of calpain results in hyperphosphorylation of NF-L and activated calpain is also responsible for NF-L degradation. Show less
