The Doublecortin-Like Kinase (DCLK) gene is involved in neuronal migration during development. Through alternative splicing the DCLK gene also produces a transcript called Ca2+/calmodulin dependent... Show moreThe Doublecortin-Like Kinase (DCLK) gene is involved in neuronal migration during development. Through alternative splicing the DCLK gene also produces a transcript called Ca2+/calmodulin dependent protein kinase (CaMK)-related peptide (CARP) that is expressed exclusively during adulthood in response to neuronal activity. The function of CARP, however, is poorly understood. To study CARP function, we have generated transgenic mice with over-expression of the CARP transcript in, amongst other brain areas, the hippocampus. We aimed to characterize possible behavioral adaptations of these mice by using a Pavlovian fear conditioning approach. This type of fear conditioning, in which both the hippocampus and amygdala are critically involved, allows studying the formation and extinction of fear related memories. We here report on the behavioral adaptations of two distinct transgenic lines: one with high levels of CARP in the hippocampus and amygdala, whilst the other has high levels of CARP in the hippocampal formation, but not in the amygdala. We tested both mouse lines separately by comparing them to their wild-type littermate controls. We provide evidence suggesting consolidation of contextual fear memories is strengthened in mice of both transgenic lines. (C) 2010 Elsevier Inc. All rights reserved. Show less
Doublecortin (DCX) and DCX-domain containing Doublecortin-Like Kinase (DCLK) gene splice variants function during embryonic development, where they play a role in microtubule binding. Although a... Show moreDoublecortin (DCX) and DCX-domain containing Doublecortin-Like Kinase (DCLK) gene splice variants function during embryonic development, where they play a role in microtubule binding. Although a role for the DCLK gene during embryogenesis is clearly established, it encodes multiple, different transcripts, some of which are expressed in the adult brain or in response to neuronal activity. This suggests that the DCLK gene may have additional functions beyond neuronal development. Strikingly, the roles of two DCLK gene products, DCLK-short and CaMK-related peptide (CARP), remain largely elusive. Therefore, we have generated transgenic mice with over-expression of either CARP or a constitutively active form of DCLK-short, called _C-DCLK-short, in the brain. This has opened up the possibility to study the effect of over-expression of these DCLK transcripts in the brain during adulthood. To gain more insight in DCLK gene function in the adult brain we aimed to study and describe the phenotypes of these transgenic mice at different functional levels, such as the genetic, network and behavioural level. Show less
Following antigen encounter, activated T cells can give rise to functionally distinct T cell subsets. Understanding how different T cell subsets arise requires technologies that can monitor the... Show moreFollowing antigen encounter, activated T cells can give rise to functionally distinct T cell subsets. Understanding how different T cell subsets arise requires technologies that can monitor the developmental potential of single precursor cells (chapter 2). This thesis describes the development and use of two novel genetic tagging strategies aimed at following cell differentiation in vivo. These strategies are based on the marking of precursor cells with unique DNA sequences (barcodes), following which cell fate is analyzed by barcode comparison of different daughter populations. The first technology, termed cellular barcoding, makes use of a retroviral barcode library to provide T cells with unique genetic tags via in vitro transduction. Cellular barcoding was used to analyze the kinship of diverse T cell populations (chapter 3-5) as well as to measure the clonality of antigen-specific T cell responses under varying conditions of infection (chapter 6). The second technology, termed in vivo barcoding, makes use of a transgenic mouse model in which unique DNA sequences are introduced via inducible VDJ recombination. The feasibility of in vivo barcoding was demonstrated by conditionally labeling lung and liver cells with different barcodes (chapter 7). Together, these studies have yielded important new insights for vaccine optimization. Show less
The thesis describes the application of several different magnetic resonance (MR) techniques to study the effects of the progression of disease in a transgenic mouse model of Alzheimer's. Using MR... Show moreThe thesis describes the application of several different magnetic resonance (MR) techniques to study the effects of the progression of disease in a transgenic mouse model of Alzheimer's. Using MR imaging, the amyloid plaque deposition was visualized and the plaque load quantified in the same mice as they aged. Concurrently the transverse relaxation time (T2) was measured in affected brain regions and shown to decrease over time as plaque-load increased. To study the neurochemical profile in the mouse brain brain both one- (1D) and two-dimensional (2D) MR spectroscopic techniques were employed. 1D MRS is widely used in similar research, but has limited spectral resolution. To overcome this limitation, a 2D MRS technique was implemented and optimized for use in mouse brain. This technique, L-COSY, allowed the detection of several metabolites which were not visible using standard 1D MRS techniques. This technique was subsequently used to study the effects of Alzheimer's on the neurochemical profile. Observed changes were correlated with plaque deposition. Show less
The research described in this thesis focussed on the role of apolipoproteins in lipid metabolism, inflammation and bacterial sepsis, with specific emphasis on apoCI. From studies in human APOC1_... Show moreThe research described in this thesis focussed on the role of apolipoproteins in lipid metabolism, inflammation and bacterial sepsis, with specific emphasis on apoCI. From studies in human APOC1_-transgenic and apoc1-/- mice, we were able to identify apoCI as a potent inhibitor of triglyceride hydrolysis by inhibiting lipoprotein lipase. Since APOC1 mice have thus increased VLDL levels, and VLDL protects against bacterial infection, we studied whether apoCI could play a role in inflammation and infection. We found that apoCI was able to bind lipopolysaccharide (LPS), the main toxic component of Gram-negative bacteria. Interestingly, although other apolipoproteins which have been studied have anti-inflammatory properties, we found that apoCI is a pro-inflammatory protein. By enhancing the biological response towards LPS and Gram-negative bacteria, apoCI dose-dependently improved the anti-bacterial attack, and protected against intrapulmonal Klebsiella pneumoniae-induced sepsis. Consistent with these experimental findings we also found that subjects with high plasma apoCI levels were less prone to infection-related mortality during follow-up, independent of plasma lipid levels. Likewise, survivors of severe sepsis showed higher plasma apoCI levels as compared to non-survivors, again independent of lipid levels. Taken together, our findings indicate that apoCI is an important determinant of the inflammatory response in mice and humans. Show less
The presence of calcium deposits in the vessel wall is indicative of advanced atherosclerosis, and the extent of coronary calcification has been found to add prognostic significance to conventional... Show moreThe presence of calcium deposits in the vessel wall is indicative of advanced atherosclerosis, and the extent of coronary calcification has been found to add prognostic significance to conventional risk factors of coronary artery disease. However, the mechanisms underlying vascular calcification are still obscure. The major objective of the work described in the first part of this thesis was to elucidate the mechanisms involved in atherosclerotic calcification. To study the process of VSMC calcification we developed and characterized an in vitro model of neonatal rat VSMC calcification. To investigate whether pharmacotherapy may affect vascular calcifications, we have studied the effect of a calcium antagonist (amlodipine) and a statin (atorvastatin) and their combination on this process. Inflammation is an important mechanism in the atherosclerotic process, and prospective and cross-sectional clinical and epidemiological studies have shown that CRP is consistently associated with CVD. The causality of CRP in atherosclerosis is discussed. To enable the study of the effect of CRP on atherosclerosis development in vivo, ApoE*3-Leiden/hCRP transgenic mice were generated and studied. The effects of a calcium antagonist (amlodipine), administered either alone or in combination with a statin (atorvastatin), on early atherosclerosis development in ApoE*3-Leiden/hCRP was investigated. Show less
