Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Konstrukcija mnimosti : K poètike "Voskovoj persony" Ju. Tynjanova

The subject of this work is the poetics of «The Wax Effigy», a short novel or novella by Jurii Tynianov, Russian writer, literary critic, historian of literature and prominent literary theoretician. The plot structure of the novel is based upon a real event, the creation by Bartolomeo Carlo Rastrelli in 1725 of a wax sculpture of the first Russian emperor, Peter the Great. «Construction of the Sham» consists of three chapters, an introduction and a conclusion. Due to the fact that Tynianov was at the same time a prose writer and theoretician of literature it seemed important to consider the reception of his prose and his works on literary theory in relationship to each other. The introduction is devoted to this task. The first chapter is about the history of the creation of the novel and its reception. Tynianov stopped writing one short story in order to write the novel; these two works have some common traits. It seems almost obvious that his work on the first text was a real step toward the creation of the second. In the first story there is an opposition of dead/alive which is semantic prefiguring of a central motif in «The Wax Effigy». An analysis of the reception of the novel demonstrated that almost every critic writing about the novel has described it as nonsense. Critics considered Tynianov's work in terms of «devices» and «content» and could not understand how devices are related to the content of the novel: the novel was thought as a signifier without any signified. Implicitly, critics thought the signified of the novel as a traditional one of the historical novel, as the historiosophical «idea», embodied in the system of literary devices. In this case literature becomes something instrumental, a kind of expression of extraliterary content. In contradistinction to that Tynianov considered literary semantics as an effect of the literary structure. From his point of view the literary sense is immanent to the process of signification accomplished inside the literary text. The second chapter is devoted to a rhetorical analysis of the opposition dead/alive. Tynianov systematically compares both terms of the opposition. As a result of this strategy the wax effigy of the dead emperor becomes «as if» alive and the world of living people «as if» dead. The qualifier «as if» refers to the fact that Tynianov creates an ambiguous semantic system. This rhetoric is related to European Romanticism and his «fantastic literature» (Merimé, Hoffmann, Maupassant etc.). But Tynianov demonstrates a linguistic origin of the strange fantoms created by romantics; he demystifies these idols by parodying the fantastic literature, that is, showing «how it was done». At the same time, the opposition mentioned above refers to his idea of «incongruity» which plays a prominent role in Tynianov s theory but has never been conceptualised. The incongruity is a inner collision of the literary text; from Tynianov's point of view the meaning of the work of literature is always a dynamic collision of semantically heterogeneous elements struggling with each other. In «The Wax Effigy» Tynianov creates a metalevel of the work demonstrating the process of creation of the literary sense. The third chapter is a reconstruction of Tynianov's conception of the historical prose, specifically of the mechanisms by which historical facts are transformed into literary events. Tynianov thought that the task of the historical novelist is to depict his hero as an actor, to demonstrate that as a wearer of many masks he is a creator of appearances, ambiguities. Here, in the «figure of fiction» (Andrei Belyi), the very idea of the historical prose and rhetoric employed in «The Wax Effigy», history and literature meet each other. In his last theoretical work, «On parody» Tynianov writes about the so-called sham structure of parody. In his opinion every parody is a text about other texts and «serious» work which could be read at the same time as a text about «reality». This twofold structure of parody is that of «The Wax Effigy»: that text speaks about ambiguities of the history and about ambiguities of the literary sense, about social reality of the past and - about the working of the literature itself. «The Wax Effigy» is written as a autoreflective text, as an experiment in literary semantics, as a system of literary ambiguities - of hero, rhetoric and the text itself. The meaning of the novel is created not by the embodiment extraliterary idea, but by the process of signification accomplished inside the work of literature. In this sense Tynianov's novel is parody, a break with the tradition of the historical novel preceding «The Wax Effigy».

Idyll eller verklighet? : Albert Edelfelt och Gunnar Berndtson i det moderna genombrottets ambivalens

Idyll or Reality? Albert Edelfelt, Gunnar Berndtson and the ambivalent breakthrough of modernity Albert Edelfelt (1854-1905) and Gunnar Berndtson (1854-1895) have much in common. In this dissertation, I study their paintings of local peasants and fishermen and of the gentry’s summer in the county of Uusimaa in southern Finland, in the context of Nordic history of ideas. The breakthrough of modernity, with its attention on debating social problems, provides a novel angle into the oeuvres of the two artists. My focus is on the paintings which emerge in the collision of the public discussion of social matters and the values of the artists’ manorial milieu. The artists’ relation to the public discussion is scrutinized through two of the main topics: the question of the common people and democracy, and the question of equality between men and women. My dissertation is a contextual study which is based on the analysis of the artworks of Edelfelt and Berndtson, on their letters, and on the study of drama and fiction of their time. The notion “liberté, egalité, fraternité” is linked to the breakthrough of modernity. Both artists were aware of the ideal of equality. They used the means and the themes of contemporary art in their presentations, but their pictures contain the ideal of an earlier epoch: the hardworking, but still complacent common people. This conception of the common people is also reflected in the poems of J. L. Runeberg. Women of the late 19th century challenged woman’s primary role as wife and mother. In Edelfelt’s and Berndtson’s depictions of the gentry enjoying summer, women and children have the main role. Notwithstanding the debate of the breakthrough of modernity they depicted women almost without exception as good mothers. Their paintings often depict lazy days in the sunshine, which were, in reality, rare moments for the mistress of the house. Edelfelt’s and Berndtson’s subjects from the Uusimaa countryside coincide with the topics of the breakthrough of modernity, but both the pictures of the common people and the depictions of the gentry enjoying summer, are a retouched picture of reality, often an idyll, in which the public discussion of social matters is evident only materially or not at all.

Olen maa johon tahdot : Timo K. Mukan maailmankuvan poetiikkaa

The dissertation deals with the prose texts of the Finnish writer Timo K. Mukka, renowned for his depictions of his native Lapland. This research concerns the creation of world view in Mukka s prose, which is approached by studying what Mikhail Bakhtin calls generic change. Such genre change is the most characteristic feature of Mukka s prose. His prose is permeated with two genres in particular and changes between them: the ballad-like, archaistic and romantic prose-poem style and naturalistic, even grotesque expressions. In addition, these genres are associated with sublime and grotesque styles so that generic change tends to involve also stylistic changes in Mukka s prose. This study probes the tension-filled interrelationships between the ballad and naturalistic prose by examining the discourse of Mukka s characters. It is shown that these characters invariably find themselves in what Bakhtin calls the chronotope of the threshold; that is, the plots of Mukka s novels and short stories depict situations in which the characters are faced with decisions and deeds that will profoundly impact their lives. The discourse of the threshold affects the characters speech by filling it with dialogical dimensions. This makes their communication ethically loaded and polyphonic. This study is based on Mikhail Bakhtin s theory of the novel and international Bakhtin s studies. I also take into consideration the theoretical developments of Bakhtin s work; for example, the concept of ressentiment, adapted from the Bakhtin scholar Michel André Bernstein, plays an important role. In order to explicate on the psychology of Mukka s characters such as melancholy, abjection, sadism, and taboo I use the concepts familiar from Freudian psychoanalysis. The corpus of my research consists of the following texts: the long prose texts Maa on syntinen laulu. Balladi (1964), Tabu (1965), Täältä jostakin. Romaani (1965), Laulu Sipirjan lapsista. Romaani (1966), Ja kesän heinä kuolee. Kertomus sairaudesta (1968) ja Kyyhky ja unikko (1970) and the short story collections Koiran kuolema (1967) ja Lumen pelko (1970), and with Tabu published short story Sankarihymni , the short story Katkelma laajemmasta laulelmasta from the collection Rakastaa: Kaksitoista novellia rakkaudesta (1965) and also the short stories which were published in various Finnish journals: Yöt (1965), Liisa (1967), Tyttö (1967) ja Näin hetki sitten ketun (1970). I pay particular attention to the novel Maa on syntinen laulu, because it expresses the generic change characteristic of Mukka s world view in a specifically clear and lively way. The dissertation is in Finnish. Key words: Timo K. Mukka, world view, genre change, ballad, naturalism, grotesque realism, Mikhail Bakhtin, dialogism, polyphony, chronotope, sublime, grotesque, ressentiment, Sigmund Freud, melancholy, taboo, abject, sadism, reduced laughter, modern parody.

Den brændende Fakkel : Om dobbeltgrebet i Knud Lyne Rahbeks liv og revolutionsroman

Professor Knud Lyne Rahbek was a novelist, playwright, poet, magazine editor, journalist, socialite person, host of the Bakkehus , historian, theatre manager, translator, publisher etc., but his versatility either side of 1800 is better known than read and more despised than understood. In terms of methodology, the thesis is based on biographical, historical and philological research, while at the same time making use of formalistic and close reading methods. This study begins and ends with 7th of February 1800, when Kamma and Knud Lyne Rahbek join the exiled P.A. Heiberg at the inn near Frederiksberg Castle. What falls between is an interpretation of Rahbek s works in the service of democracy, human rights and freedom of the press as a pragmatic navigation between activities - both subversive and legitimate. Posterity mistook this range as mere spinelessness, and Rahbek was relegated to the literary and historical margins as an anachronism and as a jack of all trades, who did not know what he really wanted and therefore flitted about in so many fields just to be present. But Rahbek s problem was not one of standpoint, but rather how to find a balance between totalizing attitudes and confrontations between rebellious idealism and deep-rooted absolutism, without foregoing his belief in enlightenment, humanism and tolerance. In this way, and also through his personal conduct, which at that time was seen as jovial bonhommie, he made his contribution to the development of modern democratic Denmark in the full awareness of a popular, peaceful and down-to-earth community. Rahbek s principal work about the event of the French Revolution, which provides the focus for the above, is Camill og Constance. Et Revolutions Skilderie (1799). For today s reader, the novel about the revolution is an obvious example of a historical novel, as it does not only provide fictionalized information about past events placing them in a generally accepted perspective of historical development, but also gives the characters qualities, which, in Rahbek s words, allows the real events to influence the fictional characters. From this point of view, the novel of the revolution has shifted the benchmark for the first real historical novel on the European literary scene back by fifteen years. Lacking the aura so easily foisted on fearless iconoclasts or tragic losers, Rahbek s contribution may seem modest in spite of its enormous volume; but only when it is not evaluated in its full context, which is the development of Denmark towards an international democratic society.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Novel Multiple Sclerosis Predisposing Genetic Variants Outside the HLA Region

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Both environmental factors and several predisposing genes are required to generate MS. Despite intensive research these risk factors are still largely unknown, the pathogenesis of MS demyelination is poorly understood, and no curative treatment exists. Both prevalence and familial occurrence of MS are exceptionally high in a Finnish population subisolate, Southern Ostrobothnia, presumably due to enrichment of predisposing genetic variants within this region. Previous linkage scan on MS pedigrees from Southern Ostrobothnia detected three main MS loci on chromosomes 5p, 6p (HLA) and 17q. Linkage studies in other populations have also provided independent evidence for the location of MS susceptibility genes in these regions. Further, these loci are syntenic to the experimental autoimmune encephalomyelitis (EAE) susceptibility loci of rodents. In this thesis work an effort was made to localize MS predisposing alleles of the linked loci outside the HLA region by studying familial MS cases from the Southern Ostrobothnia isolate. Analysis of the 5p locus revealed one region, flanking the complement component 7 (C7) gene. The identified relatively rare haplotype seems to have a fairly large effect on genetic susceptibility of MS (frequency MS 12%, controls 4%; p=0.000003, OR=2.73). Evidence for association with alleles of the region and MS was seen also in more heterogeneous populations. Convincingly, plasma C7 protein levels and complement activity correlated with the risk haplotype identified. The finding stimulated us to study other complement cascade genes in MS. No evidence for association could be observed with the complement component coding genes outside 5p. A scan of the 17q locus provided evidence for association with variants of the protein kinase C alpha (PRKCA) gene (p=0.0001). Modest evidence for association with PRKCA was observed also in Canadian MS families. Finally we used a candidate gene based approach to identify potential MS loci. Mutations of DAP12 and TREM2 cause a recessively inherited CNS white matter disease PLOSL. Interestingly, DAP12 and TREM2 are located in MS regions on 6p and 19q, and we tested them as potential candidate genes in the Finnish MS sample. No evidence for association with MS was observed. This thesis provides an example of how extended families from special populations can be utilized in fine-mapping of the linked loci. A first relatively rare MS variant was identified utilizing the strength of a Finnish population subisolate. This variant seems to have an effect on activity of the complement system, which has previously been suggested to have an important role in the pathogenesis of MS.