Delivery of drugs to the brain requires passage across the blood-brain barrier (BBB). Both for drugs already on the market and for new drugs under development, it is important to know to what extent a drug enters the CNS. Many drugs used clinically are racemic mixtures, i.e. equal parts of the (S)- and (R)-enantiomers. The present studies focus on the enantiomers and racemates of a number of 5-HT1A receptor agonists and antagonists (pindolol, propranolol, 8-OH-DPAT and other 8-substituted-2-(di-n-propylamino)tetralin derivatives) and BBB transport in vitro and distribution to the brain in vivo. Assays (HPLC-based) were set up or developed for determination of the racemates and the pure enantiomers (chiral column) of drugs in plasma and brain tissue. BBB transport was assessed in vitro using bovine brain endothelial cells cocultured with rat astrocytes. The physicochemical constants (log P, pKa) and plasma protein binding were determined. Pindolol, propranolol and several tetralines accumulated over time in brain tissue. For pindolol and propranolol, but not for most tetralins, the distribution to the brain was stereoselective, (S)>(R). Pretreatment with verapamil, an inhibitor of drug efflux via P-glycoprotein, differentially decreased the brain/plasma ratios of the enantiomers of pindolol and propranolol, indicating that verapamil may also inhibit an influx transport mechanism. In vitro results with racemic pindolol, propranolol and tetralins showed no differences in BBB transport between the enantiomers. A more rapid apical to basolateral transport (influx) vs. the basolateral to apical (efflux) transport of propranolol (not pindolol) and most tetralins in vitro indicated active transport across the BBB. In conclusion, the combined in vivo and in vitro results are consistent with active transport of the studied compounds across the BBB rather than passive diffusion due to their lipophilicity. Some, but not all, chiral drugs are stereoselectively distributed to the brain. Stereoselective plasma protein binding or stereoselective transport across brain endothelial cells does not seem to explain the stereoselective accumulation of pindolol and propranolol. The stereochemical configuration of compounds contributes to their pharmacokinetic as well as their pharmacodynamic uniqueness. The characteristics of the enantiomers of chiral compounds need to be determined empirically rather than based on generalizations from structural or physicochemical information.

Social phobia is a disabling, lifelong disorder characterised by fear in social settings. The aim of the present study was to gain more knowledge about diagnostic, neurobiologic and epidemiologic aspects of social phobia. Thirty-two individuals were assessed by the Structured Clinical Interview for DSM-IV Axis I and II psychiatric disorders, the Karolinska Scales of Personality and the Temperament and Character Inventory. Social phobia was accompanied by concurrent axis I disorders in about 28% of individuals, lifetime axis I disorders in 54%, personality disorders in 60%, and avoidant personality disorder (APD) in 47%. This suggests that there is a high comorbidity between social phobia and APD according to the DSM-IV criteria. The personality profiles associated with social phobia were dominated by anxiety-related traits that were primarily related to social phobia itself and not to the presence of concurrent personality disorders. Eighteen subjects with social phobia and eighteen controls were investigated with positron emission tomography and the radiolabeled serotonin precursor, [3 -11C]–5-HTP (5-HTP). Individuals with social phobia demonstrated proportionally lower regional relative whole brain accumulation of 5-HTP in areas of the frontal and temporal cortices as well as the striatum, but higher accumulation in the cerebellum. This suggests that there are imbalances in presynaptic serotonin function in individuals with social phobia, although this could only be confirmed in men, and not in women. By means of a postal survey, distributed to 2000 randomly selected individuals, social phobia in Sweden was found to be common, with a point prevalence of 15.6%.

The combination of a severe systemic illness, corticosteroids, and neuromuscular blocking agents in patients on the mechanical ventilator often results in a condition known as Acute Quadriplegic Myopathy (AQM). While severe weakness of all spinal nerve innervated muscles is known to be a significant clinical characteristic of the disease, this symptom is typically not recognized until the disease has progressed to an advanced stage. End result effects have been classified, which include the loss of the thick filament, or myosin heavy chain, an in-excitable muscle membrane, and an up-regulation of protein degradation; however, there is little known about the acute stage of AQM. This project has focused on understanding the underlying mechanisms of AQM, specifically in regard to protein synthesis, both at the mRNA and nuclear transcription levels. To study the early stages of the disease two animal models have been developed: rat and pig. Further, we have examined AQM muscle tissue, to investigate the similarities of our animal models to patients, as well as to study the recovery process. Particular interest was directed on the myofibrillar proteins myosin (MyHC) and actin, as they are the primary proteins involved in muscle contraction, as well as the myosin associated proteins, myosin binding protein C and H. At the mRNA level, MyHC and actin are both down-regulated in response to AQM. The myosin binding proteins are affected differently, with H protein increasing during severe atrophy and C protein either being slightly down-regulated or unchanged. Nuclear transcription factors were also affected, with such factors as MuRF1 and MAFbx up-regulated. Thus far results have shown that protein synthesis is altered in AQM and largely contributes to both the development and recovery of the disease. The pathways of protein synthesis may prove to be an ideal target for the prevention of AQM and/or symptom alleviation.

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder that causes fluctuating muscle weakness. MG may be divided into an ocular form and a generalized form based on the involved muscles. Treatment differs between these different MG forms. The majority (80%) of patients with generalized MG are seropositive for antibodies against the acetylcholine receptor (AChR-Ab). Recently a new antibody was detected against muscle specific tyrosine kinase (MuSK) in about 40% of patients who are AChR-Ab seronegative. A few patients with MuSK-Abs have muscular atrophies, as well as electrophysiological myopathy. In this thesis we have characterized MuSK-Ab seropositive [MuSK(+)] patients using clinical parameters, including health-related quality of life (hrQoL), neurophysiology and muscle morphology, and compared them to patients with and without AChR-Abs. The question concerned which factors contribute to their muscle weakness. Additionally, we wanted to determine if single-fiber electromyography (SFEMG) in a limb muscle has any predictive value for generalization of ocular MG. Our results suggest that MuSK(+) patients more often have a myopathic electromyography pattern, although this pattern is found also in other immunological subtypes of MG. The myopathic pattern may be associated with the frequently found mitochondrial abnormalities. However, disturbed neuromuscular transmission is the primary cause of muscle weakness in the majority of MuSK(+) patients, as well as in AChR-Ab seropositive patients. The disease-specific hrQoL MG questionnaire was successfully validated into Swedish and these scores correlated with disturbed neuromuscular transmission in a proximal arm muscle. Abnormal SFEMG findings occur also in muscles outside of the facial area in ocular MG, although this is not predictive of subsequent generalization. MuSK (+) patients have little or no beneficial effect of acetylcholine esterase inhibitors (AChEI). On the contrary AChEI may produce profound adverse effects. We present the hypothesis that this effect of AChEI is due to abnormal receptor morphology in MuSK(+) patients.

Paralogs arise by duplications and belong to families. Ten paralogs (insulin; IGF-1 and -2; INSL3-6 and 3-relaxins) constitute the human insulin-relaxin family. The aim of this study was to outline the duplications that gave rise to the vertebrate insulin-relaxin genes and the chromosomal regions in which they reside. Neurotrophin and Trk-receptor families with more than 300, otherwise unrelated, families had paralogs in the regions hosting insulin/relaxin genes, defining two quadruplicate paralogy-regions, namely: insulin/IGF and INSL/relaxin paralogons. Thereby, the localization of insulin/relaxins in human shows that these regions were formed during two genome duplications at the stem of the vertebrates. We characterized insulin-like genes (INS-L1, -L2 and -L3) in the Ciona intestinalis genome, a species that split from the chordate lineage before the genome duplications. Conserved synteny between the Ciona region hosting the INS-Ls and two human paralogons as well as linkage of the actual paralogons, suggest that a segmental duplication gave rise to the entire region prior to the genome duplications. Synteny together with gene and protein structures demonstrate that INS-L1 is orthologous to the vertebrate INSLs/relaxins, INS-L2 to insulins and INS-L3 to IGFs. This indicates that pro-orthologs of the insulin-relaxin family were formed before Ciona. Our analysis also implies that the INSL/relaxin ancestor switched receptor from tyrosine kinase- to GPCR-type. This probably occurred after the Ciona-stage, but before the genome duplications. Using genes residing within the analyzed human paralogons that were present in a chromosomal region in the Ciona-human ancestor, we identified 37 segments with conserved synteny between the Drosophila melanogaster and human genomes. Orthologs residing in Ciona-, sea urchin- and the fly syntenic segments imply that such segments approximate an ancestral region from which the human paralogons originated. To conclude, the human paralogons are remnants of genome duplications that in addition to segmental- and single duplications, shaped the extant vertebrate genomes. Using the quadruplicate paralogy-regions we were able to deduce duplication events of the insulin-relaxin genes and their chromosomal regions.

During brain activity neurons release the major excitatory transmitter glutamate, which is taken up by astrocytes and converted to glutamine. Glutamine returns to neurons for re-conversion to glutamate. This glutamate-glutamine cycle is energy demanding. Glutamate turnover in injured brain was studied using an animal iron-induced posttraumatic epilepsy model and using neurointensive care data from 33 patients with spontaneous subarachnoid hemorrhage (SAH). Immunoblotting revealed that the functional form of the major astrocytic glutamate uptake protein GLT-1 was decreased 1-5 days following a cortical epileptogenic iron-injection, presumably due to oxidation-induced aggregation. Using microdialysis it was shown that the GLT-1 decrease was associated with increased interstitial glutamate levels and decreased interstitial glutamine levels. The results indicate a possible posttraumatic and post-stroke epileptogenic mechanism. Analysing 3600 microdialysis hours from patients it was found that the interstitial lactate/pyruvate (L/P) ratio correlate with the glutamine/glutamate ratio (r =-0.66). This correlation was as strong as the correlation between L/P and glutamate (r=0.68) and between lactate and glutamate (r=0.65). Pyruvate and glutamine correlated linearly (r=0.52). Energy failure periods, defined as L/P>40, were associated with high interstitial glutamate levels. Glutamine increased or decreased during energy failure periods depending on pyruvate. Energy failure periods were clinically associated with delayed ischemic neurological deficits (DIND) or development of radiologically verified infarcts, confirming that L/P>40 is a pathological microdialysis pattern that can predict ischemic deterioration after SAH. DIND-associated microdialysis patterns were L/P elevations and surges in interstitial glutamine. Glutamine and pyruvate correlated with the cerebral perfusion pressure (r=0.25, r=0.24). Glutamine and the glutamine/glutamate ratio correlated with the intracranial pressure (r=-0.29, r=0.40). Glutamine surges appeared upon substantial lowering of the intracranial pressure by increased cerebrospinal fluid drainage. Increased interstitial glutamine and pyruvate levels may reflect augmented astrocytic glycolysis in recovering brain tissue with increased energy demand due to a high glutamate-glutamine turnover.

Aromatase is an enzyme that facilitates the conversion of androgens to estrogens and may play a role in mood and mental status. The main theme of this thesis is the imaging of brain aromatase by use of the PET technique. The PET tracer for aromatase, 11C-labeled vorozole (VOZ) was developed and evaluated by with in vitro and in vivo methods. In vitro experiments using rat brain showed that VOZ was distributed in the medial amygdala, bed nucleus of the stria terminalis and medial preoptic area, regions of the brain known to be rich in aromatase and the KD value was determined to be 0.60 nM. The in vivo PET study in rhesus monkey brain revealed that VOZ penetrated the blood-brain barrier and accumulated in the amygdala and hypothalamus. Taken together, VOZ is a good PET tracer for in vivo aromatase imaging with high affinity and high sensitivity. This technique was applied to an investigation of brain aromatase under the physiological conditions simulating anabolic-androgenic steroid abuse. A significant increase in VOZ binding by anabolic-androgenic steroids was observed in the bed nucleus of stria terminalis and medial preoptic area in the rat brain. In contrast, no significant change in binding was observed in the medial amygdala. These results indicate that the manner of regulation of aromatase expression might be different in the bed nucleus of stria terminalis and medial preoptic area compared with that in the medial amygdala. The aromatase expression was suggested to be regulated through androgen receptors, as indicated in a study with flutamide treatment. The increased aromatase expression was seen in neurons. The PET study with anabolic steroid-treated rhesus monkeys also showed increased VOZ binding in the hypothalamus but not in the amygdala. The alteration of density of aromatase binding in the hypothalamic area could explain some psychological features of anabolic-androgenic steroid abusers. Novel PET tracers for aromatase were developed and examined. The two newly synthesized 18F-labeled vorozole analogs, [18F]FVOZ and [18F]FVOO, displayed different characteristics. Both tracers showed similar binding pattern as VOZ; however, [18F]FVOO was metabolized very quickly, meaning that this tracer is not suitable as a PET tracer. On the other hand, [18F]FVOZ can be an appropriate PET tracer. The role of aromatase in the human brain has not been clarified yet. To approach this problem by in vivo methods, we have just started PET studies to explore aromatase expression in humans.

Retinal ganglion cells play a crucial role in the relay of visual signals from the eye to the brain. This cell type is affected and eventually lost in the eye disease glaucoma, resulting in progressive and irreversible loss of vision. Studies of the molecular mechanisms leading to retinal ganglion cell death are important for the understanding of the disease and for designing future treatments. This thesis addresses and studies these molecular mechanisms, including alterations in gene expression after experimental retinal injuries. The effects of a neuroprotective drug, brimonidine, after transient retinal ischemia were also studied in order to help explain the mechanisms behind the protective properties of this drug. Several methods, including quantitative reverse transcriptase PCR, micro-arrays, western blot and immunohistochemistry, were used. The results showed that transient retinal ischemia triggers cell division in Müller cells and alters the gene expression of growth factors, their receptors, and intermediate filaments in the retina. Several genes related to the apoptosis process were less affected. Pre-treatment with brimonidine increased the levels of certain growth factors (BDNF, NT3, CNTF, FGF9) compared with vehicle. Brimonidine also had marked effects on genes related to progenitor cells, among them the recognized neural stem cell marker nestin. The increase in levels of nestin after ischemia was countered by brimonidine treatment. Moreover, retinal ganglion cell death following either optic nerve transection or optic nerve crush appears to involve the extrinsic apoptotic pathway although the gene expression response appears to differ between these injuries. The results obtained in this work contribute to an increased understanding of retinal injuries and highlight the importance of Müller cells in the endogenous defense against retinal injuries.

This study is about how to develop younger teens problem solving ability and their social competence. The study carried through by using a qualitative method, two observations and ten interviews. The observations where preparatory for the structured interviews. The respondents where pupils in a class were it is an ongoing project which purpose is to develop childrens problem solving ability and social skills with cognitive training and an empowerment based reasoning model that is called rahyab. Rahyab is a Persian word and it means path- finding. Focus has been on the pupils experiences of working with the model and the school project. The studies approach is to examine how younger teens problem solving ability can be developed by cognitive training and social pedagogics? Other questions that we’ve been trying to answer are: what have the younger teens learned from rahyab and it’s steps? How has the younger teens experienced working with rahyab? and do the younger teens feel that they have and will have use of rahyab? The results and analysis shows that the younger teens can develop their analytic thinking and thereby also their problem solving ability. They feel that they now have a better understanding for how other people think. This means that they appears to have experienced a development in their social competence. The results shows, with the support of prior knowledge, that younger teens problem solving ability can be developed by cognitive training and social pedagogics. / Denna studie handlar om hur man kan utveckla yngre tonåringars problemlösningsförmåga och därigenom deras sociala kompetens. Studien genomfördes med kvalitativ metod i form av två observationer och tio intervjuer. Observationerna var öppna och förberedande inför de strukturerade intervjuerna. Respondenterna var elever i en klass där det pågår ett projekt som syftar till att utveckla barns problemlösningsförmåga och sociala kompetens utifrån kognitiv träning och en empowerment baserad resoneringsmodell, som kallas rahyab. Rahyab är ett persiskt ord som betyder väg- sökning. Fokus har varit på elevernas upplevelser av arbetet med modellen och skolprojektet. Studiens syfte är att undersöka hur yngre tonåringars problemlösningsförmåga kan utvecklas av kognitiv träning och socialpedagogik? Andra frågeställningar som vi har sökt att besvara är: vad har de yngre tonåringarna lärt sig av rahyab och dess olika steg? Hur har de yngre tonåringarna upplevt att arbeta med rahyab? och upplever de yngre tonåringarna att de har och kommer ha användning av rahyab? I resultat och analys framkommer att de yngre tonåringarna kan utveckla sitt analytiska tänkande och därmed också deras problemlösningsförmåga. De upplever att de nu bättre förstår hur andra tänker. De förefaller ha utvecklat sin sociala kompetens. Resultaten visar med stöd av tidigare kunskap att yngre tonåringars problemlösningsförmåga kan utvecklas av kognitiv träning och social pedagogik.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons in the spinal cord, brain stem and motor cortex, leading to paralysis, respiratory failure and death. In about 5% of ALS cases, the disease is associated with mutations in the CuZn-superoxide dismutase (hSOD1) gene. As a rule, ALS caused by hSOD1 mutations is inherited dominantly and the mutant hSOD1s cause ALS by the gain of a noxious property. The present study focused on two hSOD1 mutations with widely differing characters. In Scandinavia, ALS caused by the D90A mutation is inherited in a recessive pattern. Elsewhere, families with dominant inheritance have been found. The properties of D90A mutant hSOD1 are very similar to those of the wild-type protein. The G127insTGGG (G127X) mutation causes a 21 amino acid C-terminal truncation which probably results in an unstable protein. The aim of this thesis was to generate transgenic mice expressing D90A and G127X mutant hSOD1s and to compare these mice with each other and with mice expressing other mutant hSOD1s, in search of a common noxious property. The findings were also compared with the results from studies of human CNS tissue. The cause of the different inheritance patterns associated with D90A mutant hSOD1 was investigated by analyzing erythrocytes from heterozygous individuals from dominant and recessive pedigrees. There was no evidence that a putative protective factor in recessive pedigrees acts by down-regulating the synthesis of D90A mutant hSOD1. In cerebrospinal fluid, there was no difference in hSOD1 content between homozygous D90A patients, ALS patients without hSOD1 mutations and controls. hSOD1 cleaved at the N-terminal end was found in both controls and D90A patients, but the proportion was significantly larger in the latter group. This indicates a difference in degradation routes between mutant and wild-type hSOD1. Both D90A and G127X transgenic mice develop an ALS-like phenotype. Similar to humans, the levels of D90A protein were high. The levels of G127X hSOD1 were very low in the tissues but enriched in the CNS. Similarly, in an ALS patient heterozygous for G127X hSOD1, the levels of the mutant protein were overall very low, but highest in affected CNS areas. Despite the very different levels of mutant hSOD1, both D90A and G127X transgenic mice developed similar levels of detergent-resistant aggregates in the spinal cord when terminally ill. Surprisingly, mice overexpressing wild-type hSOD1 also developed detergent-resistant aggregates, although less and later. Most of the hSOD1 in the CNS of transgenic mice was inactive due to deficient copper charging or because of reduced affinity for the metal. The stabilizing intrasubunit disulfide bond of hSOD1 was partially or completely absent in the different hSOD1s. Both these alterations could increase the propensity of mutant hSOD1s to misfold and form aggregates. The results presented here suggest that the motor neuron degeneration caused by mutant hSOD1s may be attributable to long-term exposure to misfolded, aggregation-prone, disulfide-reduced hSOD1s and that the capacity to degrade such hSOD1s is lower in susceptible CNS areas compared with other tissues. The data also suggest that wild-type hSOD1 has the potential to participate in the pathogenesis of sporadic ALS.