Complement factor H dysfunction in atypical hemolytic uremic syndrome

Alternative pathway (AP) of complement can be activated on any surface, self or non-self. In atypical hemolytic uremic syndrome (aHUS) the AP regulation on self surfaces is insufficient and leads to complement attack against self-cells resulting usually in end-stage renal disease. Factor H (FH) is one of the key regulators of AP activation on the self surfaces. The domains 19 and 20 (FH19-20) are critical for the ability of FH to discriminate between C3b-opsonized self and non-self surfaces and are a hot-spot for mutations that have been described from aHUS patients. FH19-20 contains binding sites for both the C3d part of C3b and self surface polyanions that are needed for efficient C3b inactivation. To study the dysfunction of FH19-20, crystallographic structures of FH19-20 and FH19-20 in complex with C3d (FH19-20:C3d) were solved and aHUS-associated and structurally interesting point mutations were induced to FH19-20. Functional defects caused by these mutations were studied by analyzing binding of the FH19-20 mutant proteins to C3d, C3b, heparin, and mouse glomerular endothelial cells (mGEnCs). The results revealed two independent binding interfaces between FH19-20 and C3d - the FH19 site and the FH20 site. Superimposition of the FH19-20:C3d complex on the previously published C3b and FH1-4:C3b structures showed that the FH20 site on C3d is partially occluded, but the FH19 site is fully available. Furthermore, binding of FH19-20 via the FH19 site to C3b did not block binding of the functionally important FH1-4 domains and kept the FH20 site free to bind heparin or an additional C3d. Binding assays were used to show that FH20 domain can bind to heparin while FH19-20 is bound to C3b via the FH19 site, and that both the FH19 site and FH20 are necessary for recognition of non-activator surfaces. Simultaneous binding of FH19 site to C3b and FH20 to anionic self structures are the key interactions in self-surface recognition by FH and thereby enhanced avidity of FH explains how AP discriminates between self and non-self. The aHUS-associated mutations on FH19-20 were found to disrupt binding of the FH19 or FH20 site to C3d/C3b, or to disrupt binding of FH20 to heparin or mGEnC. Any of these dysfunctions leads to loss of FH avidity to C3b bearing self surfaces explaining the molecular pathogenesis of the aHUS-cases where mutations are found within FH19-20.

Sex steroids in Sjögren's syndrome

Sjögren s syndrome (SS) is a strongly female dominant autoimmune disease. SS targets mainly salivary and lacrimal glands and leads to loss of the secreting acinar cells of these glands. Accordingly, secretion of the affected glands is diminished and the main symptoms of SS, dryness of mouth and eyes, follow. In addition to these sicca symptoms, SS patients suffer from severe fatigue and can have various extraglandular symptoms. To date, the etiology of SS still remains unknown. Female dominance and the late onset of the disease simultaneously with remarkable hormonal changes in the body (menopause, adrenopause) encouraged us to hypothesize that sex steroids, especially androgens, are involved in the onset and progression of SS. We confirmed our hypothesis and showed that patients with SS suffer from androgen depletion both systemically and locally in the target tissue of SS, salivary glands. We especially focused on the local androgen environment in salivary glands and demonstrated that healthy salivary glands contain a complete enzymatic machinery for local synthesis of androgens and estrogens from pro-hormone dehydroepiandrosterone (DHEA). However, in SS salivary glands the enzymes catalyzing the local androgen synthesis are defective and, in a subgroup of patients, practically non-functional. Probably due to this local defect in DHEA processing, therapy with DHEA was found unbeneficial for SS patients in the treatment of fatigue. We also studied the effect of the local androgen depletion on salivary glands. We found that in salivary gland cells and healthy labial salivary glands androgens upregulate integrin subunits α1 and α2, which are important for the communication, differentiation and function of the acinar cells. On the contrary, in SS salivary glands DHEA failed to upregulate these signaling molecules, again probably due to defective processing of DHEA into active androgens. Our finding highlights the importance of the local androgen environment and local DHEA processing for the function and welfare of salivary glands. In conclusion, this study showed that patients with SS are androgen depleted both systemically and locally in salivary glands. SS patients also have a defective local sex steroid synthesizing enzymatic machinery further impairing the local androgen depletion. We also showed that the local androgen defect leads to decreased expression of acinar cell specific integrin molecules, which impairs the signaling between the acinar cells and basement membrane and might thus explain the acinar cell loss seen in SS salivary glands. By showing the importance of the local sex steroid imbalance in SS we have clarified some etiopathogenetic mechanisms of SS, which have thus far remained unknown.

Zur lexikographischen Darstellung der Adjektivsyntax in neueren einsprachigen Wörtbüchern des Deutschen

The main objects of the investigation were the syntactic functions of adjectives. The reason for the interest in these functions are the different modes of use, in which an adjective can occur. All together an adjective can take three different modes of use: attributive (e. g. a fast car), predicative (e. g. the car is fast) and adverbial (e. g. the car drives fast). Since an adjective cannot always take every function, some dictionaries (esp. learner s dictionaries) deliver information within the lexical entry about any restrictions. The purpose of the research consisted of a comparison in relation to the lexical entries of adjectives, which were investigated within four selected monolingual German-speaking dictionaries. The comparison of the syntactical data of adjectives were done to work out the differences and the common characteristics of the lexical entries concerning the different modes of use and to analyse respective to assess them. In the foreground, however, were the differences of the syntactical information. Concerning those differences it had to be worked out, which entry is the grammatically right one respective if one entry is in fact wrong. To find that out an empirical analysis was needed, which based on the question in which way an adjective is used within a context as far as there are no conforming data within the dictionaries. The delivery of the correctness and the homogeneity of lexical entries of German-speaking dictionaries are very important to support people who are learning the German language and to ensure the user friendliness of dictionaries. Throughout the investigations it became clear that in almost half of the cases (over 40 %) syntactical information of adjectives differ from each other within the dictionaries. These differences make it for non-native speakers of course very difficult to understand the correct usage of an adjective. Thus the main aim of the doctoral thesis was it to deliver and to demonstrate the clear syntactical usage of a certain amount of adjectives.

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.

Modification of ventricular repolarization in silent long QT syndrome mutation carriers

Congenital long QT syndrome (LQTS) is a familial disorder characterized by ventricular repolarization that makes carriers vulnerable to malignant ventricular tachycardia and sudden cardiac death. The three main subtypes (LQT1, LQT2 and LQT3) constitute 95% of cases. The disorder is characterized by a prolonged QT interval in electrocardiograms (ECG), but a considerable portion are silent carriers presenting normal (QTc < 440 ms) or borderline (QTc < 470 ms) QT interval. Genetic testing is available only for 60-70% of patients. A number of pharmaceutical compounds also affect ventricular repolarization, causing a clinically similar disorder called acquired long QT syndrome. LQTS carriers - who already have impaired ventricular repolarization - are especially vulnerable. In this thesis, asymptomatic genotyped LQTS mutation carriers with non-diagnostic resting ECG were studied. The body surface potential mapping (BSPM) system was utilized for ECG recording, and signals were analyzed with an automated analysis program. QT interval length, and the end part of the T wave, the Tpe interval, was studied during exercise stress testing and an epinephrine bolus test. In the latter, T wave morphology was also analyzed. The effect of cetirizine was studied in LQTS carriers and also with supra- therapeutic dose in healthy volunteers. At rest, LQTS mutation carriers had a slightly longer heart rate adjusted QTc interval than healthy subjects (427 ± 31 ms and 379 ± 26 ms; p<0.001), but significant overlapping existed. LQT2 mutation carriers had a conspicuously long Tpe-interval (113 ± 24 ms; compared to 79 ± 11 ms in LQT1, 81 ± 17 ms in LQT3 and 78 ± 10 ms in controls; p<0.001). In exercise stress tests, LQT1 mutation carriers exhibit a long QT interval at high heart rates and during recovery, whereas LQT2 mutation carriers have a long Tpe interval at the beginning of exercise and at the end of recovery at low heart rates. LQT3 mutation carriers exhibit prominent shortening of both QT and Tpe intervals during exercise. A small epinephrine bolus revealed disturbed repolarization, especially in LQT2 mutation carriers, who developed prolonged Tpe intervals. A higher epinephrine bolus caused abnormal T waves with a different T wave profile in LQTS mutation carriers compared to healthy controls. These effects were seen in LQT3 as well, a group that may easily escape other provocative tests. In the cetirizine test, the QT and Tpe intervals were not prolonged in LQTS mutation carriers or in healthy controls. Subtype-specific findings in exercise test and epinephrine bolus test help to diagnose silent LQTS mutation carriers and to guide subtype-specific treatments. The Tpe interval, which signifies the repolarization process, seems to be a sensitive marker of disturbed repolarization along with the QT interval, which signifies the end of repolarization. This method may be used in studying compounds that are suspected to affect repolarization. Cetirizine did not adversely alter ventricular repolarization and would not be pro-arrhythmic in common LQT1 and LQT2 subtypes when used at its recommended doses.

Formale Aspekte dreier finnischsprachiger Untertitelungsversionen des Films Das Leben der Anderen und bei den Untertitelungen angewandte Übersetzungsstrategien

Työssä tutkitaan yhtäältä saksankielisen elokuvan Muiden elämä ( Das Leben der Anderen ) kolmen suomenkielisen tekstitysversion tekstityksen muotoseikkoja, kuten välimerkkien käyttöä, repliikki- ja rivijakoa, ja toisaalta tekstityksen sisältöseikkoja, joita tarkastellaan tekstityksessä käytettävien käännösstrategioiden avulla. Tutkielmassa tarkastellaan, ovatko tekstityksissä käytetyt muotoseikat konventioiden mukaisia ja minkälaisia käännösstrategioita tekstityksissä on käytetty sekä johtaako tietyn käännösstrategian käyttö merkityksen muutoksiin ja kaventumisiin tekstityksessä. Tutkielman aineistona on elokuva Muiden elämä ja sen kolme suomenkielistä tekstitystä: elokuvateattereissa esitetty versio, DVD-tekstitys ja televisiossa esitetty versio. Hypoteesina oli, että kaikkien versioiden muotoseikat ovat konventioiden mukaisia ja että versioilla on eroavuuksia ruututekstien määrässä ja repliikkijaossa. Lisäksi oletettiin, että tietyn käännösstrategian käytön ja merkityksen muutoksien välillä on yhteys, niin että merkityksen muutoksia esiintyy eniten silloin, kun kaikkea sanottua ei voida kääntää. Tutkielman teoriataustassa esitellään aluksi lyhyesti av-kääntämisen muita lajeja, kuten ns. dubbausta eli jälkiäänitystä, selostusta ja voice-overia, jonka jälkeen syvennytään tekstityksen teoriaan, tekstityksen muotoseikkoihin ja konventioihin sekä tekstityksen käännösstrategioihin. Tekstityksen sisältöseikkojen analyysin viitekehyksenä ovat Henrik Gottliebin (1994, 1997) kymmenen käännösstrategiaa: lisäys (engl. expansion), para-fraasi (paraphrase), suora käännös (transfer), imitaatio (imitation), transkriptio (transcription), siirtäminen (dislocation), tiivistäminen (condensation), lyhentäminen (decimation), poisjättäminen (deletion) ja resignaatio (resignation). Tutkielmassa analysoitiin kaikkien tekstitysversioiden muotoseikat. Tekstityksen sisältöseikkojen tutkimuksessa jokainen dialogin lause tai sivulause analysoitiin omana verbaalisena segmenttinään. Yhteensä analysoitiin 1311 verbaalista segmenttiä ja tutkittiin, mitä käännösstrategiaa missäkin tekstitysversiossa oli missäkin osakäännöksessä käytetty, sekä tarkasteltiin, muuttuuko repliikissä merkitys alkutekstiin verrattuna. Strategiat kuvataan ja niiden käyttöä valaistaan esimerkkien avulla. Tulokset ovat hypoteesia tukevia: Tutkielmassa havaittiin, että kaikkien tekstitysversioiden muotoseikat ovat suurimmalta osin konventioiden mukaisia. Eroja oli havaittavissa tavuviivan, pilkun ja kursiivin käytössä. Versiot eroavat myös ruututekstien määrässä: elokuvaversiossa on eniten ruututekstejä (1091), vaikka siinä on vähiten sanoja (3968); DVD-tekstityksessä on 876 ruututekstiä ja 4278 sanaa ja TV-tekstityksessä on 983 ruututekstiä ja 5293 sanaa. Eroavaisuudet selittyvät sillä, että elokuvatekstityksen ruututekstit ovat lyhyempiä ja nopeammin ilmestyviä kuin DVD- tai TV-tekstitysten. Tutkittu DVD-tekstitys on elokuvatekstityksen muokkaus, mistä johtuu sen vähäinen sana- ja ruututekstimäärä. Tutkielmassa havaittiin myös, että kaikissa versioissa yleisimmin käytetty käännösstrategia oli suora käännös, mutta TV-tekstityksessä sitä oli käytetty yli 50 %:ssa kaikista osakäännöksistä ja DVD- ja elokuvaversiossa yli 35 %:ssa, eli TV-tekstityksessä sitä oli käytetty huomattavasti enemmän kuin muissa versioissa. Lyhentämistä ja poisjättämistä oli DVD- ja elokuvatekstityksessä käytetty enemmän kuin TV-tekstityksessä. Nämäkin erot selit-tynevät elokuvatekstityksen pienemmällä tilalla ja tämän tekstityksen muokkaamisesta DVD-tekstitykseksi. Merkityksen muutoksia ja kaventumisia esiintyi eniten strategioiden poisjättäminen ja lyhentäminen yhteydessä, muiden strategioiden yhteydessä merkityksen muutoksia ja kaventumisia esiintyi vain marginaalisesti. Tulokset tukevat hypoteesia siitä, että merkitys muuttuu ja kaventuu eniten silloin, kun kaikkea sanottua ei voida kääntää.

Die mündliche Sprachkompetenz finnischer Deutschlernender aus Sicht der grammatikalischen Korrektheit : Eine Fehleranalyse im Rahmen des HY-TALK-Projekts

Tutkielman päätavoitteena on analysoida suomalaisten, saksaa opiskelevien lukiolaisten suullisen testin suorituksia kielen oikeellisuuden näkökulmasta virheanalyysin avulla. Aineistona on osa Helsingin yliopiston HY-TALK-hankkeen saksan kielen tutkimusaineistosta: videoinnit suullisen kielitaidon testeistä sekä asiantuntijaraadin arvioinnit suorituksista. Koska aineisto on suullinen, tutkielmassa tarkastellaan aluksi suullisen kielen piirteitä. Virheanalyysin pohjana ovat muun muassa Corderin, Nickelin, Kleppinin ja Jamesin esittämät teoriat. Tutkielmassa analysoidaan kolmentoista, selkeästi joko A1.3- tai A2.2-taitotasolle arvioidun opiskelijan suoritukset. Virheanalyysin tavoitteena on saada selville kielioppivirheiden määrät, kullekin taitotasolle tyypilliset kielioppivirheet sekä selvittää tasojen välisiä eroja. Virheanalyysissä ei huomioida leksikaalisia eikä ääntämisvirheitä, vaan keskitytään morfologisiin ja syntaktisiin virheisiin. Virheet jaotellaan eri virheluokkiin (nominaalilausekkeiden deklinaatio, pronomini, taipumattomat sanaluokat, kysymyssanat, kieltoilmaukset, verbit ja lauserakenne) ja ne tilastoidaan taulukoihin. Lisäksi pohditaan virheiden syitä sekä virheiden vaikutusta kielen ymmärrettävyyteen. Jälkimmäistä selvitetään saksaa äidinkielenään puhuvan, kolmihenkisen raadin avulla. Raadin sekä numeeriset että verbaaliset arvioinnit ovat myös osa tutkielman aineistoa. Toinen tutkielman päätavoite on opetussuunnitelman taitotasoasteikon validointi. Tarkoituksena on selvittää oikeellisuuden taitotasokuvausten toimivuutta arvioinnissa A1.3- ja A2.2-tasojen osalta. Tätä tavoitetta varten selvitetään myös kielitaidon arvioinnin taustalla olevia teorioita. HY-TALK-asiantuntijaraadin arviointeja verrataan virheanalyysin tuloksiin korrelaatiokertoimen ja regressiosuoran avulla. Virheanalyysin perusteella voidaan todeta, että molemmilla tasoilla olevat opiskelijat tekevät eniten virheitä nominaalilausekkeiden deklinaatiossa (A2.2-tasolla 60,0 prosenttia kaikista virheistä ja A1.3-tasolla 42,2 prosenttia). A1.3-tasolle arvioidut opiskelijat tekevät paljon virheitä myös verbeissä ja lauserakenteissa, kun taas A2.2-tason opiskelijat hallitsevat nämä rakenteet paremmin. A1.3-tasolle arvioidut opiskelijat tekevät enemmän virheitä (22,10 virhettä sataa sanaa kohden) kuin A2.2-tasolle arvioidut (9,80 virhettä sataa sanaa kohden). Virheiden syitä ovat muun muassa testitilanne, puhekumppanin vaikutus ja interferenssi. Kielen ymmärrettävyyteen vaikuttavat saksalaisen raadin mukaan kielioppivirheiden lisäksi muun muassa puheen sujuvuus ja leksikaaliset ongelmat. Vakavimpina virheinä pidetään virheitä lauserakenteessa, sillä ne haittaavat usein ymmärrettävyyttä. Deklinaatiovirheitä puolestaan ei yleensä pidetä vakavina virheinä. Taitotasokuvaukset toimivat A1.3- ja A2.2-tasojen osalta hyvin, sillä virheanalyysin virhemäärät ja HY-TALK-raadin arvioinnit korreloivat keskenään, kun otetaan huomioon molemmat tasot A1.3 ja A2.2. Kielioppivirheiden määrä siis vaikuttaa kielen oikeellisuuden arviointiin. Taitotasokuvauksia olisi kuitenkin joiltain osin syytä tarkentaa ja yhtenäistää esimerkiksi konkreettisten esimerkkien avulla (esim. mikä on peruskielioppivirhe?) ja kuvaamalla myös kielen ymmärrettävyyttä joka tasolla.

HDL subspecies : association with low HDL-C, obesity, and metabolic syndrome

AIMS An independent, powerful coronary heart disease (CHD) predictor is a low level of high-density lipoprotein cholesterol (HDL-C). Discoidal preβ-HDL particles and large HDL2 particles are the primary cholesterol acceptors in reverse cholesterol transport, a key anti-atherogenic HDL mechanism. The quality of HDL subspecies may provide better markers of HDL functionality than does HDL-C alone. We aimed I) to study whether alterations in the HDL subspecies profile exist in low-HDL-C subjects II) to explore the relationship of any changes in HDL subspecies profile in relation to atherosclerosis and metabolic syndrome; III) to elucidate the impact of genetics and acquired obesity on HDL subspecies distribution. SUBJECTS The study consisted of 3 cohorts: A) Finnish families with low HDL-C and premature CHD (Study I: 67 subjects with familial low HDL-C and 64 controls; Study II: 83 subjects with familial low HDL-C, 65 family members with normal HDL-C, and 133 controls); B) a cohort of 113 low- and 133 high-HDL-C subjects from the Health 2000 Health Examination Survey carried out in Finland (Study III); and C) a Finnish cohort of healthy young adult twins (52 monozygotic and 89 dizygotic pairs) (Study IV). RESULTS AND CONCLUSIONS The subjects with familial low HDL-C had a lower preβ-HDL concentration than did controls, and the low-HDL-C subjects displayed a dramatic reduction (50-70%) in the proportion of large HDL2b particles. The subjects with familial low HDL-C had increased carotid atherosclerosis measured as intima-media-thickness (IMT), and HDL2b particles correlated negatively with IMT. The reduction in both key cholesterol acceptors, preβ-HDL and HDL2 particles, supports the concept of impaired reverse cholesterol transport contributing to the higher CHD risk in low-HDL-C subjects. The family members with normal HDL-C and the young adult twins with acquired obesity showed a reduction in large HDL2 particles and an increase in small HDL3 particles, which may be the first changes leading to the lowering of HDL-C. The low-HDL-C subjects had a higher serum apolipoprotein E (apoE) concentration, which correlated positively with the metabolic syndrome components (waist circumference, TG, and glucose), highlighting the need for a better understanding of apoE metabolism in human atherosclerosis. In the twin study, the increase in small HDL3b particles was associated with obesity independent of genetic effects. The heritability estimate, of 73% for HDL-C and 46 to 63% for HDL subspecies, however, demonstrated a strong genetic influence. These results suggest that the relationship between obesity and lipoproteins depends on different elements in each subject. Finally, instead of merely elevating HDL-C, large HDL2 particles and discoidal preβ-HDL particles may provide beneficial targets for HDL-targeted therapy.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.

Early Growth and Later Health, Focus on Metabolic Syndrome, Obesity and Physical Activity

The Developmental Origins of Health and Disease Hypothesis proposes that adverse health outcomes in adult life are in part programmed during fetal life and infancy. This means that e.g. restricted nutrition during pregnancy programmes the offspring to store fat more effectively, to develop faster and to reach puberty earlier. These adaptations are beneficial in terms of short term survival. However, in developed countries these adaptations often lead to an increased risk of obesity and metabolic disturbances in later life, due to a mismatch between the prenatal and postnatal environment. This thesis aimed to study the role of early growth in people who are obese as adults, but metabolically healthy as well as in those who are normal in weight but metabolically obese. Other study aims were to assess whether physical activity and cardiorespiratory fitness are programmed early in life. The role of socioeconomic status in the development of obesity from a life course setting was also studied. These studies included 2003 men and women born in Helsinki between 1934 and 1944 with detailed information of their prenatal and childhood growth as well as living conditions. They participated in the detailed clinical examination during the years 2001-2004. A sub-group of the subjects participated in the UKK Institute 2-kilometre walk test. Metabolic syndrome was defined according to the 2005 criteria of the International Diabetes Federation. Among the obese men and women 20 % were metabolically healthy. Those with metabolic syndrome did not differ in birth size compared to the healthy ones, but by two years of age, they were lighter and thinner, and remained so up to 11 years. The period when changes in BMIs were predictive of the metabolic syndrome was from birth to 7 years. Of the normal weight individuals 17 % were metabolically obese. Again, there were no differences in birth size. However, by the age 7 years, those men who later developed metabolic syndrome were thinner. Gains in BMI during the first two years of life were protective of the syndrome. Children who were heavier, and especially taller, were more physically active, exercised with higher intensity and had higher cardiorespiratory fitness in their adult life than those who were shorter and thinner as children. Lower educational attainment and lower adult social class were associated with obesity in both men and women. Childhood social class was inversely associated with body mass index only in men while lower household income was associated with higher BMI in women. These results support the role of early life factors in the development of metabolic syndrome and adult life style. Early detection of risk factors predisposing to these conditions is highly relevant from a public health point of view.