The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

Pathophysiology of Congenital Nephrotic Syndrome of the Finnish type

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disease which is highly enriched in the Finnish population. It is caused by mutations in the NPHS1 gene encoding for nephrin, which is a major component of the glomerular filtration barrier in the kidney. Patients with NPHS1 have heavy proteinuria and nephrotic syndrome (NS) from birth and develop renal fibrosis in early childhood. Renal transplantation (TX) is the only curative treatment for NPHS1. These patients form the largest group of pediatric kidney transplant children in our country. The NPHS1 kidneys are removed in infancy and they serve as an excellent human material for studies of the pathophysiology of proteinuric kidney diseases. Sustained proteinuria is a major factor leading to end-stage renal failure and understanding this process is crucial for nephrology. In this study we investigated the glomerular and tubulointerstitial changes that occur in the NPHS1 kidneys during infancy as well as the expression of nephrin in non-renal tissues. We also studied the pathology and management of recurrent proteinuria in kidney grafts transplanted to NPHS1 children. Severe renal lesions evolved in patients with NPHS1 during the first months of life. Glomerular sclerosis developed through progressive mesangial sclerosis, and capillary obliteration was an early consequence of this process. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. Few inflammatory cells were detected in the mesangial area. The glomerular epithelial cells (podocytes) showed severe ultrastructural changes and hypertrophy. Podocyte proliferation and apoptosis were rare, but moderate amounts of podocytes were detached and ended up in the urine. The results showed that endocapillary lesions not extracapillary lesions, as generally believed were important for the sclerotic process in the NPHS1 glomeruli. In the tubulointerstitium, severe lesions developed in NPHS1 kidneys during infancy. Despite heavy proteinuria, tubular epithelial cells (TECs) did not show transition into myofibroblasts. The most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1). Interstitial inflammation and fibrosis were first detected in the paraglomerular areas and the most abundant inflammatory cells were monocytes/macrophages. Arteries and arterioles showed intimal hypertrophy, but the pericapillary microvasculature remained quite normal. However, excessive oxidative stress was evident in NPHS1 kidneys. The results indicated that TECs were relatively resistant to the heavy tubular protein load. Nephrin was at first thought to be podocyte specific, but some studies especially in experimental animals have suggested that nephrin might also be expressed in non-renal tissues such as pancreas and central nervous system. The knowledge of nephrin biology is important for the evaluation of nephrin related diseases. In our study, no significant amounts of nephrin protein or mRNA were detected in non-renal tissues of man and pig as studied by immunohistochemistry and in situ hybridization. The phenotype analysis of NPHS1 children, who totally lack nephrin, revealed no marked impairment in the neurological, testicular, or pancreatic function speaking against the idea that nephrin would play an important functional role outside the kidney. The NPHS1 kidneys do not express nephrin and antibodies against this major glomerular filter protein have been observed in NPHS1 children after renal TX most likely as an immune reaction against a novel antigen. These antibodies have been associated with the development of recurrent NS in the kidney graft of NPHS1 patients. In our study, a third of the NPHS1 patients homozygous for Fin-Major mutation developed recurrent NS in the transplanted graft. Re-transplantations were performed to patients who lost their graft due to recurrent NS and heavy proteinuria immediately developed in all cases. While 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of plasma exchange (PE) to the treatment of recurrent nephroses increased the remission rate from 54% to 89%. If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function. In conclusion, the results show that the lack of nephrin in podocyte slit diaphragm in NPHS1 kidneys induces progressive mesangial expansion and glomerular capillary obliteration and inflicts interstitial fibrosis, inflammation, and oxidative stress with surprisingly little involvement of the TECs in this process. Nephrin appears to have no clinical significance outside the kidney. Development of antibodies against nephrin seems to be a major cause of recurrent NS in kidney grafts of NPHS1 patients and combined use of PE and cyclophosphamide markedly improved remission rates.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Neurochemical regulation of auditory information processing studied with EEG/MEG: application to schizophrenia

Cognitive impairments of attention, memory and executive functions are a fundamental feature of the pathophysiology of schizophrenia. The neurophysiological and neurochemical changes in the auditory cortex are shown to underlie cognitive impairmentsin schizophrenia patients. Functional state of the neural substrate of auditory information processing could be objectively and non-invasively probed with auditory event-related potentials (ERPs) and event- related fields (ERFs). In the current work, we explored the neurochemical effect on the neural origins of auditory information processing in relation to schizophrenia. By means of ERPs/ERFs we aimed to determine how neural substrates of auditory information processing are modulated by antipsychotic medication in schizophrenia spectrum patients (Studies I, II) and by neuropharmacological challenges in healthy human subjects (Studies III, IV). First, with auditory ERPs we investigated the effects of olanzapine (Study I) and risperidone (Study II) in a group of patients with schizophrenia spectrum disorders. After 2 and 4 weeks of treatment, olanzapine has no significant effects on mismatch negativity(MMN) and P300, which, as it has been suggested, respectively reflect preattentive and attention-dependent information processing. After 2 weeks of treatment, risperidone has no significant effect on P300, however risperidone reduces P200 amplitude. This latter effect of risperidone on neural resources responsible for P200 generation could be partly explained through the action of dopamine. Subsequently, we used simultaneous EEG/MEG to investigate the effects of memantine (Study III) and methylphenidate (Study IV) in healthy subjects. We found that memantine modulates MMN response without changing other ERP components. This could be interpreted as being due to the possible influence of memantine through the NMDA receptors on auditory change- detection mechanism, with processing of auditory stimuli remaining otherwise unchanged. Further, we found that methylphenidate does not modulate the MMN response. This finding could indicate no association between catecholaminergic activities and electrophysiological measures of preattentive auditory discrimination processes reflected in the MMN. However, methylphenidate decreases the P200 amplitudes. This could be interpreted as a modulation of auditory information processing reflected in P200 by dopaminergic and noradrenergic systems. Taken together, our set of studies indicates a complex pattern of neurochemical influences produced by the antipsychotic drugs in the neural substrate of auditory information processing in patients with schizophrenia spectrum disorders and by the pharmacological challenges in healthy subjects studied with ERPs and ERFs.

Candidate gene studies on cardiovascular traits

Cardiovascular diseases (CVD) are a major cause of death and disability in Western countries and a growing health problem in the developing world. The genetic component of both coronary heart disease (CHD) and ischemic stroke events has been established in twin studies, and the traits predisposing to CVD, such as hypertension, dyslipidemias, obesity, diabetes, and smoking behavior, are all partly hereditary. Better understanding of the pathophysiology of CVD-related traits could help to target disease prevention and clinical treatment to individuals at an especially high disease risk and provide novel pharmaceutical interventions. This thesis aimed to clarify the genetic background of CVD at a population level using large Nordic population cohorts and a candidate gene approach. The first study concentrated on the allelic diversity of the thrombomodulin (THBD) gene in two Finnish cohorts, FINRISK-92 and FINRISK-97. The results from this study implied that THBD variants do not substantially contribute to CVD risk. In the second study, three other candidate genes were added to the analyses. The study investigated the epistatic effects of coagulation factor V (F5), intercellular adhesion molecule -1 (ICAM1), protein C (PROC), and THBD in the same FINRISK cohorts. The results were encouraging; we were able to identify several single SNPs and SNP combinations associating with CVD and mortality. Interestingly, THBD variants appeared in the associating SNP combinations despite the negative results from Study I, suggesting that THBD contributes to CVD through gene-gene interactions. In the third study, upstream transcription factor -1 (USF1) was analyzed in a cohort of Swedish men. USF1 was associated with metabolic syndrome, characterized by accumulation of different CVD risk factors. A putative protective and a putative risk variant were identified. A direct association with CVD was not observed. The longitudinal nature of the study also clarified the effect of USF1 variants on CVD risk factors followed in four examinations throughout adulthood. The three studies provided valuable information on the study of complex traits, highlighting the use of large study samples, the importance of replication, and the full coverage of the major allelic variants of the target genes to assure reliable findings. Although the genetic basis of coronary heart disease and ischemic stroke remains unknown, single genetic findings may facilitate the recognition of high-risk subgroups.

Pathophysiological factors of irritable bowel syndrome, and the effects of probiotic supplementation

Gastrointestinal symptoms and impaired quality of life caused by irritable bowel syndrome (IBS) affect up to 20% of the adult population worldwide. The exact aetiology and pathophysiology of IBS are incompletely understood. Clinical studies suggest that supplementation with certain probiotics may be beneficial in IBS, but there is not enough evidence to make general recommendations. The aim of this thesis was to investigate microbiota- and mucosa-associated pathophysiological factors of IBS, and to evaluate the long-term effects of multispecies probiotic supplementation on symptoms, quality of life, intestinal microbiota and systemic inflammatory markers in IBS. The intestinal microbiota composition in IBS patients and healthy control subjects was analysed by quantitative polymerase chain reaction (qPCR). Significantly lower counts for the Clostridium coccoides and the Bifidobacterium catenulatum groups were found in IBS compared to controls. Quantitative differences also appeared in subgroup analysis based on the predominant bowel habit: diarrhoea patients harboured significantly lower numbers of Lactobacillus spp. than the constipation-predominant patients, while higher counts for Veillonella spp. were detected in constipation-predominant patients compared to healthy controls. Analysis of mucosal biopsies by a metabolomic approach revealed multiple differences between patients and controls. The most prominent finding was an upregulation of specific lipid species, principally lysophosphatidylcholines and ceramides, in IBS. The effects of multispecies probiotic supplementation with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium breve Bb99 or Bifidobacterium animalis subsp. lactis Bb12 was evaluated in two, randomised, double-blind, placebo-controlled trials. Compared to placebo, the probiotic supplementation significantly reduced the total symptoms of IBS. No effects on bowel habit were seen. Health-related quality of life (HRQOL) is reduced in patients with IBS in comparison with the Finnish population on the whole. The probiotic supplementation improved one IBS-specific domain of quality of life (bowel symptoms), whereas no other effects on HRQOL were seen. The probiotics had no major effects on the predominant microbiota as measured by qPCR, but a microarray-based analysis suggested that the probiotic consumption stabilised the microbiota. No effects on serum sensitive-CRP or cytokines were detected. In conclusion, alterations in the microbiota composition and in the mucosal metabolite profile are potential pathophysiological factors of IBS. Multispecies probiotic supplementation alleviates the gastrointestinal symptoms of IBS, and improves the bowel symptoms domain of HRQOL. Probiotic supplementation in IBS is associated with a stabilisation of microbiota, but it does not influence systemic inflammatory markers.

Immunodeficiencies, pathogens and sex in upper respiratory diseases

In the first part of this thesis the association of different forms of sinonasal diseases and plasma concentrations of C3, C4, immunoglobulins, immunoglobulin G subclasses, C4A and C4B gene numbers were studied in 287 adult patients and 150 sex-matched adult controls. Patients were well characterized and stratified into groups using strict clinical criteria and females and males were also studied as separate groups. Severe primary antibody antibody deficiencies were rare in patients coming to sinonasal operations. Female patients had more recurrent sinusitis and other mucosal infections and males had more nasal polyposis. Upregulation of complement activity was seen in acute rhinosinusitis patients (high levels of plasma C3, C4, and complement classical pathway activity CH50) and male patients coming to sinonasal operations (high levels of plasma C3 and C4). In females, total and partial C4B deficiencies and lower levels of IgG1 and IgG3 were associated with rhinosinusitis leading to sinonasal operations. C4A deficiencies were found to predispose to severe chronic rhinosinusitis in females and males. In female patients with chronic or recurrent rhinosinusitis with nasal polyposis C4B deficiencies seem to predispose to the disease, but in males with a similar disease C4B deficiencies seem to be protective. This suggests a different pathophysiology between sexes in this form of sinonasal disease. In the second part of this thesis work 213 children coming to elective tonsillectomy were studied and compared with 155 randomly selected school children. An association with recurrent upper respiratory tract infections and hypersensitivity disorders was seen especially in children under 7 years of age. However, this association was not seen in levels of specific IgE to respiratory allergens in the same age group. Both symptomatic respiratory allergy and specific IgE to respiratory allergens became more common in boys than girls over 7 years of age. We were able to show that although both rhinoviruses and bacterial pathogens were found in the tonsils, no association between their presence and clinical forms of tonsillar disease was seen. The ability of GAS to bind complement regulators FH and C4BP did not differ between strains causing tonsillar diseases or septicemia, suggesting that other virulence mechanisms of the bacteria are more important.

Animal model and molecular interactions of Cln5

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited pediatric neurodegenerative disorders, leading to retinal degeneration, death of selective neuronal populations and accumulation of autofluorscent ceroid-lipopigments. The clinical manifestations are generally similar in all forms. The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin) is a form of NCL, especially enriched in the Finnish population. The aim of this thesis was to analyse the brain pathology of vLINCLFin utilising the novel Cln5-/- mouse model. Gene expression profiling of the brains of already symptomatic Cln5-/- mice revealed that inflammation, neurodegeneration and defects in myelinization are the major characteristics of the later stages of the disease. Histological characterization of the brain pathology confirmed that the thalamocortical system is affected in Cln5-/- mice, similarly to the other NCL mouse models. However, whereas the brain pathology in all other analyzed NCL mice initiate in the thalamus and spread only months later to the cortex, we observed that the sequence of events is uniquely reversed in Cln5-/- mice; beginning in the cortex and spreading to the thalamus only months later. We could also show that even though neurodegeneration is inititated in the cortex, reactive gliosis and loss of myelin are evident in specific nuclei of the thalamus already in the 1 month old brain. To obtain a deeper insight into the disturbed metabolic pathways, we performed gene expression profiling of presymptomatic mouse brains. We validated these findings with immunohistological analyses, and could show that cytoskeleton and myelin were affected in Cln5-/- mice. Comparison of gene expression profiling results of Cln5-/- and Cln1-/- mice, further highlighted that these two NCL models share a common defective pathway, leading to disturbances in the neuronal growth cone and cytoskeleton. Encouraged by the evidence of this defected pathway, we analyzed the molecular interactions of NCL-proteins and observed that Cln5 and Cln1/Ppt1 proteins interact with each other. Furthermore, we demonstrated that Cln5 and Cln1/Ppt1 share an interaction partner, the F1-ATP synthase, potentially linking both vLINCLFIN and INCL diseases to disturbed lipid metabolism. In addition, Cln5 was shown to interact with other NCL proteins; Cln2, Cln3, Cln6 and Cln8, implicating a central role for Cln5 in the NCL pathophysiology. This study is the first to describe the brain pathology and gene expression changes in the Cln5-/- mouse. Together the findings presented in this thesis represent novel information of the disease processes and the molecular mechanisms behind vLINCLFin and have highlighted that vLINCLFin forms a very important model to analyze the pathophysiology of NCL diseases.

Unravelling Molecular and Cellular Disease Mechanisms in Infantile Neuronal Ceroid Lipofuscinosis (INCL)

Studying neurodegeneration provides an opportunity to gain insights into normal cell physiology, and not just pathophysiology. In this thesis work the focus is on Infantile Neuronal Ceroid Lipofuscinosis (INCL). It is a recessively inherited lysosomal storage disorder. The disease belongs to the neuronal ceroid lipofuscinoses (NCLs), a group of common progressive neurodegenerative diseases of the childhood. Characteristic accumulation of autofluorescent storage material is seen in most tissues but only neurons of the central nervous system are damaged and eventually lost during the course of the disease leaving most other cell types unaffected. The disease is caused by mutations in the CLN1 gene, but the physiological function of the corresponding protein the palmitoyl protein thioesterase (PPT1) has remained elusive. The aim of this thesis work was to shed light on the molecular and cell biological mechanisms behind INCL. This study pinpointed the localization of PPT1 in axonal presynapses of neurons. It also established the role of PPT1 in early neuronal maturation as well as importance in mature neuronal synapses. This study revealed an endocytic defect in INCL patient cells manifesting itself as delayed trafficking of receptor and non-receptor mediated endocytic markers. Furthermore, this study was the first to connect the INCL storage proteins the sphingolipid activator proteins (SAPs) A and D to pathological events on the cellular level. Abnormal endocytic processing and intracellular re-localization was demonstrated in patient cells and disease model knock-out mouse neurons. To identify early affected cellular and metabolic pathways in INCL, knock-out mouse neurons were studied by global transcript profiling and functional analysis. The gene expression analysis revealed changes in neuronal maturation and cell communication strongly associated with the regulated secretory system. Furthermore, cholesterol metabolic pathways were found to be affected. Functional studies with the knock-out mouse model revealed abnormalities in neuronal maturation as well as key neuronal functions including abnormalities in intracellular calcium homeostasis and cholesterol metabolism. Together the findings, introduced in this thesis work, support the essential role of PPT1 in developing neurons as well as synaptic sites of mature neurons. Results of this thesis also elucidate early events in INCL pathogenesis revealing defective pathways ultimately leading to the neurodegenerative process. These results contribute to the understanding of the vital physiological function of PPT1 and broader knowledge of common cellular mechanisms behind neurodegeneration. These results add to the knowledge of these severe diseases offering basis for new approaches in treatment strategies.

Transforming growth factor -beta signaling through Smad3 in allergy : Studies in the mechanisms of asthma, atopic dermatitis and allergic contact reactions

Transforming growth factor β signalling through Smad3 in allergy Allergic diseases, such as atopic dermatitis, asthma, and contact dermatitis are complex diseases influenced by both genetic and environmental factors. It is still unclear why allergy and subsequent allergic disease occur in some individuals but not in others. Transforming growth factor (TGF)-β is an important immunomodulatory and fibrogenic factor that regulates cellular processes in injured and inflamed skin. TGF-β has a significant role in the regulation of the allergen-induced immune response participating in the development of allergic and asthmatic inflammation. TGF-β is known to be an immunomodulatory factor in the progression of delayed type hypersensitivity reactions and allergic contact dermatitis. TGF-β is crucial in regulating the cellular responses involved in allergy, such as differentiation, proliferation and migration. TGF-β signals are delivered from the cytoplasm to the nucleus by TGF-β signal transducers called Smads. Smad3 is a major signal transducer in TGF-β -signalling that controls the expression of target genes in the nucleus in a cell-type specific manner. The role of TGF-β-Smad3 -signalling in the immunoregulation and pathophysiology of allergic disorders is still poorly understood. In this thesis, the role of TGF-β-Smad -signalling pathway using Smad3 -deficient knock out mice in the murine models of allergic diseases; atopic dermatitis, asthma and allergic contact reactions, was examined. Smad3-pathway regulates allergen induced skin inflammation and systemic IgE antibody production in a murine model atopic dermatitis. The defect in Smad3 -signalling decreased Th2 cytokine (IL-13 and IL-5) mRNA expression in the lung, modulated allergen induced specific IgG1 response, and affected mucus production in the lung in a murine model of asthma. TGF-β / Smad3 -signalling contributed to inflammatory hypersensitivity reactions and disease progression via modulation of chemokine and cytokine expression and inflammatory cell recruitment, cell proliferation and regulation of the specific antibody response in a murine model of contact hypersensitivity. TGF-β modulates inflammatory responses - at least partly through the Smad3 pathway - but also through other compensatory, non-Smad-dependent pathways. Understanding the effects of the TGF-β signalling pathway in the immune system and in disease models can help in elucidating the multilevel effects of TGF-β. Unravelling the mechanisms of Smad3 may open new possibilities for treating and preventing allergic responses, which may lead to severe illness and loss of work ability. In the future the Smad3 signalling pathway might be a potential target in the therapy of allergic diseases.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Genetic background of bipolar disorder and related cognitive impairments

Bipolar disorder (BP) is a complex psychiatric disorder characterized by episodes of mania and depression. BP affects approximately 1% of the world’s population and shows no difference in lifetime prevalence between males and females. BP arises from complex interactions among genetic, developmental and environmental factors, and it is likely that several predisposing genes are involved in BP. The genetic background of BP is still poorly understood, although intensive and long-lasting research has identified several chromosomal regions and genes involved in susceptibility to BP. This thesis work aims to identify the genetic variants that influence bipolar disorder in the Finnish population by candidate gene and genome-wide linkage analyses in families with many BP cases. In addition to diagnosis-based phenotypes, neuropsychological traits that can be seen as potential endophenotypes or intermediate traits for BP were analyzed. In the first part of the thesis, we examined the role of the allelic variants of the TSNAX/DISC1 gene cluster to psychotic and bipolar spectrum disorders and found association of distinct allelic haplotypes with these two groups of disorders. The haplotype at the 5’ end of the Disrupted-in-Schizophrenia-1 gene (DISC1) was over-transmitted to males with psychotic disorder (p = 0.008; for an extended haplotype p = 0.0007 with both genders), whereas haplotypes at the 3’ end of DISC1 associated with bipolar spectrum disorder (p = 0.0002; for an extended haplotype p = 0.0001). The variants of these haplotypes also showed association with different cognitive traits. The haplotypes at the 5’ end associated with perseverations and auditory attention, while the variants at the 3’ end associated with several cognitive traits including verbal fluency and psychomotor processing speed. Second, in our complete set of BP families with 723 individuals we studied six functional candidate genes from three distinct signalling systems: serotonin-related genes (SLC6A4 and TPH2), BDNF -related genes (BDNF, CREB1 and NTRK2) and one gene related to the inflammation and cytokine system (P2RX7). We replicated association of the functional variant Val66Met of BDNF with BP and better performance in retention. The variants at the 5’ end of SLC6A4 also showed some evidence of association among males (p = 0.004), but the widely studied functional variants did not yield any significant results. A protective four-variant haplotype on P2RX7 showed evidence of association with BP and executive functions: semantic and phonemic fluency (p = 0.006 and p = 0.0003, respectively). Third, we analyzed 23 bipolar families originating from the North-Eastern region of Finland. A genome-wide scan was performed using the 6K single nucleotide polymorphism (SNP) array. We identified susceptibility loci at chromosomes 7q31 with a LOD score of 3.20 and at 9p13.1 with a LOD score of 4.02. We followed up both linkage findings in the complete set of 179 Finnish bipolar families. The finding on chromosome 9p13 was supported (maximum LOD score of 3.02), but the susceptibility gene itself remains unclarified. In the fourth part of the thesis, we wanted to test the role of the allelic variants that have associated with bipolar disorder in recent genome-wide association studies (GWAS). We could confirm findings for the DFNB31, SORCS2, SCL39A3, and DGKH genes. The best signal in this study comes from DFNB31, which remained significant after multiple testing corrections. Two variants of SORCS2 were allelic replications and presented the same signal as the haplotype analysis. However, no association was detected with the PALB2 gene, which was the most significantly associated region in the previous GWAS. Our results indicate that BP is heterogeneous and its genetic background may accordingly vary in different populations. In order to fully understand the allelic heterogeneity that underlies common diseases such as BP, complete genome sequencing for many individuals with and without the disease is required. Identification of the specific risk variants will help us better understand the pathophysiology underlying BP and will lead to the development of treatments with specific biochemical targets. In addition, it will further facilitate the identification of environmental factors that alter risk, which will potentially provide improved occupational, social and psychological advice for individuals with high risk of BP.