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A molecule that is not identical to its mirror image is said to be chiral. A racemic mixture, or a racemate, is one that has equal amounts of S- and R-enantiomers of a chiral molecule. Two examples of frequently prescribed racemic drugs are the antidepressants venlafaxine (VEN) and citalopram (CIT). The R-enantiomer of VEN is a potent inhibitor of serotonin and noradrenaline reuptake, while the S-enantiomer is more selective in inhibiting serotonin reuptake. CIT is a selective serotonin reuptake inhibitor and the S-enantiomer is responsible for this effect. The R-enantiomer of CIT is therapeutically inactive, but displays other effects or side-effects. Due to the potential of different pharmacological and toxicological activities of the VEN and CIT enantiomers, it is of great interest to investigate the individual enantiomers of these drugs, concerning both pharmacokinetics and pharmacodynamics. For this purpose, it is necessary to develop stereoselective bioanalytical methods. A major clinical problem in the use of many drugs is the inter-individual variability in drug metabolism and response. Genetic variations contribute to this variability, including e.g. polymorphisms in the cytochrome P450 (CYP) enzymes. Approximately 7% of all Caucasians lack the polymorphic isoenzyme CYP2D6 and these individuals are classified as poor metabolisers. Both VEN and CIT are partly metabolised by CYP2D6. However, it is not completely known how CYP2D6 deficiency may influence the in vivo pharmacokinetics of these drugs, especially regarding the enantiomeric disposition. The overall aim of this thesis was to study the relationship between pharmacokinetics and pharmacogenetics for VEN and CIT, with emphasis on enantiomeric drug disposition in different biomatrices. In Paper I, a validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for enantioselective determination of VEN and its three major metabolites was developed and applied in plasma from patients and whole blood samples from forensic autopsy cases. In Papers II and III, the genetic influence on enantiomeric drug disposition in serum and brain following administration of racemic CIT and VEN to Sprague-Dawley and Dark Agouti rats was studied. The female Sprague-Dawley and Dark Agouti rats are considered the animal counterparts of the human extensive and poor metaboliser CYP2D6 phenotypes, respectively. Significant quantitative strain-related differences in the pharmacokinetics of CIT and VEN, and their metabolites, were observed. The results indicate that the CYP2D enzymes display a significant impact on the stereoselective metabolism of these drugs. The findings also highlight the importance of comparing different rat strains when conducting experimental pharmacokinetic studies. In Paper IV, the relation between CYP2D6 genotype and the disposition of the enantiomers of VEN and its metabolites in femoral blood from forensic autopsy cases was studied. A substantial variation in the relationship between the S- and R-enantiomers of VEN, and metabolites, was found. In individuals lacking two functional CYP2D6 alleles, a low enantiomeric S/R VEN ratio was strongly related to a high S/R ratio for the main metabolite O-desmethylvenlafaxine. Hence, by using enantioselective analysis of VEN and O-desmethylvenlafaxine, it is possible to predict if a person is a poor metaboliser genotype/phenotype for CYP2D6.

The proper folding of a protein into its defined three--‐dimensional structure is one of the many fundamental challenges a cell encounters. A number of tightly controlled pathways have evolved to assist in the proper folding of a protein, but also to aid in the removal of misfolded proteins. Despite the presence of these pathways accumulation of misfolded proteins can still occur. Amyloid deposits consist of misfolded proteins with a characteristic highly ordered fibrillar structure that will exert affinity for the amyloid dye Congo red and has a unique X-ray diffraction pattern. Currently 27 different proteins have been identified as amyloid forming proteins in human, however the exact role of amyloid in the pathogenesis of the connected disease is most often unclear. Islet amyloid is made up of the beta cell derived hormone islet amyloid polypeptide (IAPP) and is associated with the development of type 2 diabetes. Propagation of IAPP-fibrils is believed to be one important cause of the pancreatic beta cell death detected in patients with type 2 diabetes. IAPP is a naturally occurring polypeptide hormone stored and secreted together with insulin. IAPP and insulin arise from posttranslational processing of their biological inactive precursors proIAPP and proinsulin. In addition to human, cat and monkey IAPP will form amyloid deposits in conditions resembling human type 2 diabetes. However, IAPP from mouse and rat do not form amyloid as a result of the differences in amino acid sequence. My main research goal was to establish a unique model system suitable to study the effects of proIAPP and IAPP aggregation. I selected Drosophila melanogaster due to its many suitable characteristics as a model organism and its superior genetic toolbox. I have demonstrated that over--‐expression of hproIAPP and hIAPP in the central nervous system (CNS) results in aggregate formation in the brain and neighbouring fat body. Consistent with previous studies, expression of mIAPP does not result in the formation of aggregates. To investigate the intracellular effects of hproIAPP and hIAPP aggregation on a specific population of neurons, we targeted the expression of these peptides specifically to 16 neurons in the brain, the pdf- neurons. These pdf-neurons are divided into 2 clusters of 8 cells per brain hemisphere. First I showed that expression of aggregation prone hIAPP and hproIAPP resulted in significant death of the 8 cells, whereas expression of mIAPP had no such effect. In efforts to pinpoint the mechanisms behind the observed cell death I demonstrated that hproIAPP and hIAPP both pass the ERs quality control for protein folding and that the initiated cell death does not occur through classical apoptosis. Instead, selective autophagy is activated by hIAPP and hproIAPP. This activation counteracts the usually neuro-protective effects of autophagy and contributes to cell death. Strikingly, I also showed that Aâ, the amyloid protein implicated in Alzheimer’s disease, does not exhibit any intracellular toxicity when expressed in pdf-cells. This supports the existence of separate toxic pathways for different amyloid proteins.

Cardiovascular disease (CVD) is the major cause of death worldwide. Underlying causes, such as atherosclerosis and hypertension, are associated with remodeling of the vessel wall ultimately leading to loss of structural integrity. There are a number of factors that can influence vascular remodeling and hence structural integrity. The overall aim of this thesis was to investigate aortic wall integrity in relation to genetics and blood flow. The influence of SNPs within the currently most robust susceptibility locus identified for CVD (chromosome 9p21.3) on abdominal aortic integrity was studied in elderly individuals. In men, risk-variants were associated with a decreased abdominal aortic stiffness, independent of other factors related to arterial stiffness. Impaired mechanical properties of the abdominal aortic wall may explain the association between chromosome 9p21.3 and vascular disease. Plasminogen activator inhibitor 1 (PAI-1) is the key inhibitor of fibrinolysis, and involved in several processes associated with vascular remodeling. We investigated the impact of the PAI-1 4G/5G polymorphism on central aortic blood pressure as this pressure more strongly relates to cardiovascular morbidity and mortality than the peripheral pressure. Elderly women carrying the 4G/4G genotype had higher central aortic blood pressure than women carrying the 5G/5G genotype. The association was regardless of other risk factors related to hypertension, suggesting that an impaired fibrinolytic potential may play an important role in the development of hypertension in women. Blood flow is a strong determinant of arterial growth and vascular function. We investigated flow-dependent gene expression and vessel wall morphology in the rat aorta under physiological conditions. Microarray analysis revealed a strong differential gene expression between disturbed and uniform flow pattern regions, particularly associated with transcriptional regulation. Moreover, several genes related to Ca2+ signalling were among the most highly differentially expressed. Up-regulation of Ca2+-related genes may be due to endothelial response to disturbed flow and assembly of cilia, consequently leading to functional and structural modifications of the vessel wall. Bicuspid aortic valve (BAV) is a congenital disorder associated with disturbed ascending aortic blood flow. Using a new strategy to dissect flow-mediated gene expression we identified several novel flow-associated genes, particularly related to angiogenesis, wound healing and mechanosensing, showing differential expression in the ascending aorta between BAV and tricuspid aortic valve patients. Fifty-five percent of the identified genes were confirmed to be flowresponsive in the rat aorta. A disturbed flow, and consequently an altered gene expression, may contribute to the increased aneurysm susceptibility associated with BAV morphology.

The size of a myocardial infarction (MI) and the concurrent effect on left ventricular (LV) function are essential for decisions regarding patient care and treatment. Images produced with the late gadolinium enhancement (LGE) technique visualize the scar with high spatial resolution. The general aim of this thesis was to study methods to assess scar size in chronic MI, primarily with the use of LGE, and to relate area‐at‐risk and LV function to scar size. Myocardial perfusion single photon emission computed tomography (MPS) is a well established technique for the assessment of MI size. Our study showed that there is a fairly good agreement between MPS and LGE in the determination of scar size. Wall motion score index (WMSI) correlated moderately with both infarct size and infarct extent determined with LGE. Manual delineation of myocardium and scar is time consuming and subjective and there is a need for help in objective assessment. We showed that the semi‐automatic computer software, Segment, reduced the evaluation time ≥50% with maintained clinical accuracy. The segmented scar sequence ‐ inversion recovery fast gradient echo, IR_FGRE, is a well documented sequence for scar determination, however, the sequence requires regular heart rhythm and breath holding for good imaging. We showed that a single shot scar sequence ‐ steady state free precession, SS_SSFP ‐ acquired under free breathing in patients with ongoing atrial fibrillation, had significantly better image quality than IR_FGRE. The scar size and the error of determination were equal for both sequences and the examination time was shorter with SS_SSFP. In an acute MI it is essential to know the myocardial area‐at‐risk. WMSI is clinically the most common way of assessing LV function, but is highly subjective. Tissue Doppler imaging with strain measurements is considered objective and quantitative in assessing both global and regional LV function compared to WMSI. Our results showed that WMSI is superior to strain for the detection of scar with transmurality ≥50% in patients with acute MI. Also WMSI correlated better than strain on all levels (global, regional, segmental) with final scar size determined with LGE. LGE images visualize myocardial scar much more distinctly than any other modality. This new technique needs clinical validation but promises intense competition with existing modalities such as myocardial scintigraphy and echocardiography. However, in individual patient care all modalities should be used according to their own advantages and limitation.

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