The genetics of behaviour and other adaptive traits in nine-spined sticklebacks (Pungitius pungitius)

One of the main goals in current evolutionary biology research is to identify genes behind adaptive phenotypic variations. The advances in genomic technologies have made it possible to identify genetic loci behind these variations, also concerning non-model species. This thesis investigates the genetics of the behaviour and other adaptive traits of the nine-spined stickleback (Pungitius pungitius) through the application of different genetic approaches. Fennoscandian nine-spined stickleback populations express large phenotypical differences especially in behaviour, life –history traits and morphology. However the underlying genetic bases for these phenotypical differences have not been studied in detail. The results of the project will lay the foundation for further genetics studies and provide valuable information for our understanding of the genetics of the adaptive divergence of the nine-spined stickleback. A candidate gene approach was used to develop microsatellite markers situating close to candidate genes for behaviour in the nine-spined stickleback. Altogether 13 markers were developed and these markers were used in the subsequent studies with the anonymous random markers and physiologically important gene markers which are already currently available for nine-spined sticklebacks. It was shown that heterozygosity correlated with behaviour in one of the marine nine-spined stickleback populations but with contrasting effects: correlations with behaviour were negative when using physiological gene markers and positive with random markers. No correlation was found between behavioural markers and behaviour. From the physiological gene markers, a strong correlation was found between osmoregulation-related gene markers and behaviour. These results indicate that both local (physiological) and general (random) effects are important in the shaping of behaviour and that heterozygosity– behaviour correlations are population dependent. In this thesis a second linkage map for nine-spined sticklebacks was constructed. Compared to the earlier nine-spined stickleback linkage map, genomic rearrangements were observed between autosomal (LG7) and sex-determing (LG12) linkage groups. This newly constructed map was used in QTL mapping studies in order to locate genomic regions associated with pelvic structures, behaviour and body size/growth. One major QTL was found for pelvic structures and Pitx1 gene was related to these traits as was predicted from three-spined stickleback studies, but this was in contrast to earlier nine-spined stickleback study. The QTL studies also revealed that behaviour and body size/growth were genetically more complex by having more QTL than pelvic traits. However, in many cases, pelvic structure, body size/growth and behaviour were linked to similar map locations indicating possible pleiotropic effects of genes locating in these QTL regions. Many of the gene related markers resided in the QTL area. In the future, studying these possible candidate genes in depth might reveal the underlying mechanism behind the measured traits.

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.

Early detection of severe sepsis in the emergency room in adults - Clinical utility of prognostic markers

Vaikean sepsiksen varhainen tunnistaminen päivystyspoliklinikalla – merkkiaineiden käyttökelpoisuus aikuispotilaiden arvioinnissa Päivystyspotilaan vakavan yleisinfektion eli sepsiksen varhainen tunnistaminen ja taudin vaikeusasteen arviointi on päivystävälle lääkärille tärkeä haaste. Arvioimme prospektiivisessa kohorttitutkimuksessa eri merkkiaineiden hyödyllisyyttä sepsiksen varhaisessa tunnistamisessa ja vaikeusasteen arvioinnissa. Työssä I ja III oli 539 päivystyspotilasta, joilta kliinikko päätti ottaa veriviljelyn sepsistä epäillen. Tutkimuksessa II oli 525 potilasta ja tutkimuksessa IV 537 potilasta. Tutkimuksessa I plasman C-reaktiivisen proteiinin (CRP) pitoisuuksia verrattiin plasman prokalsitoniinin (PCT) ja interleukiinin (IL-6) pitoisuuksiin. Tutkimuksessa II verrattiin plasman baktersidisen/ permeabiliteettia lisäävän proteiinin (BPI), ryhmän IIA fosfolipaasi A2:n (PLA2GIIA) ja CRP:n pitoisuuksia sekä valkosolujen määriä toisiinsa. Tutkimuksessa III arvioitiin liukoisen urokinaasi-tyyppisen plasminogeenin aktivaattorireseptorin (suPAR) ja tutkimuksessa IV pentraksiini 3:n (PTX3) määrityksen käyttökelpoisuutta. Tutkimuksessa I todettiin päivystystilanteessa mitattujen korkeiden PCT - ja IL-6 - pitoisuuksien ennustavan vaikean sepsiksen kehittymistä paremmin kuin korkean CRP:n. Tutkimuksessa II plasman PLA2GIIA vaikutti hiukan paremmalta vaikean sepsiksen ennustajalta kuin CRP tai veren valkosolutaso, mutta BPI ei ollut hyödyllinen. Tutkimuksessa III korkea plasman suPAR- pitoisuus osoittautui itsenäiseksi kuolleisuuden riskitekijäksi ja se liittyi myös vaikean sepsiksen kehittymiseen. Tutkimuksessa IV korkea PTX3 - pitoisuus toimi samaan tapaan kuin suPAR. Kokonaisuutena PCT osoittautui parhaaksi merkkiaineeksi ennustamaan elinhäiriön kehittymistä ja suPAR kuolleisuutta. PTX3 ei tarjonnut merkittävää lisäetua PCT:iin ja suPAR:iin verrattuna. CRP osoitti suhteellisen hyvin bakteeri-infektion esiintymistä, mutta ennusteellista arvoa sillä ei ollut. suPAR on kiinnostava kuolleisuuden ja elinhäiriön kehittymisen merkkiaine.

Quantitative Analysis of Novel Prostate Cancer Markers in Tissue

Prostate cancer is a heterogeneous disease affecting an increasing number of men all over the world, but particularly in the countries with the Western lifestyle. The best biomarker assay currently available for the diagnosis of the disease, the measurement of prostate specific antigen (PSA) levels from blood, lacks specificity, and even when combined with invasive tests such as digital rectal exam and prostate tissue biopsies, these methods can both miss cancers, and lead to overdiagnosis and subsequent overtreatment of cancers. Moreover, they cannot provide an accurate prognosis for the disease. Due to the high prevalence of indolent prostate cancers, the majority of men affected by prostate cancer would be able to live without any medical intervention. Their latent prostate tumors would not cause any clinical symptoms during their lifetime, but few are willing to take the risk, as currently there are no methods or biomarkers to reliably differentiate the indolent cancers from the aggressive, lethal cases that really are in need of immediate medical treatment. This doctoral work concentrated on validating 12 novel candidate genes for use as biomarkers for prostate cancer by measuring their mRNA expression levels in prostate tissue and peripheral blood of men with cancer as well as unaffected individuals. The panel of genes included the most prominent markers in the current literature: PCA3 and the fusion gene TMPRSS2-ERG, in addition to BMP-6, FGF-8b, MSMB, PSCA, SPINK1, and TRPM8; and the kallikrein-related peptidase genes 2, 3, 4, and 15. Truly quantitative reverse-transcription PCR assays were developed for each of the genes for the purpose, time-resolved fluorometry was applied in the real-time detection of the amplification products, and the gene expression data were normalized by using artificial internal RNA standards. Cancer-related, statistically significant differences in gene transcript levels were found for TMPRSS2-ERG, PCA3, and in a more modest scale, for KLK15, PSCA, and SPINK1. PCA3 RNA was found in the blood of men with metastatic prostate cancer, but not in localized cases of cancer, suggesting limitations for using this method for early cancer detection in blood. TMPRSS2-ERG mRNA transcripts were found more frequently in cancerous than in benign prostate tissues, but they were present also in 51% of the histologically benign prostate tissues of men with prostate cancer, while being absent in specimens from men without any signs of prostate cancer. PCA3 was shown to be 5.8 times overexpressed in cancerous tissue, but similarly to the fusion gene mRNA, its levels were upregulated also in the histologically benign regions of the tissue if the corresponding prostate was harboring carcinoma. These results indicate a possibility to utilize these molecular assays to assist in prostate cancer risk evaluation especially in men with initially histologically negative biopsies.

Genetic diversity and phenotypic variability of phytoplankton populations in the Baltic Sea

En av naturens mest grundläggande aspekter är den enorma mängd av variation som existerar mellan arter. Denna variation har lett oss till att klassificera olika organismer på basis av morfologiska skillnader och på senare tid till att jämföra genetiska skillnader på individens nivå. Den marina kiselalgen Skeletonema marinoi är en av de vanligaste växtplanktonarter i Östersjön under vårblomningen och anses viktig för den årliga produktionen. En av mina främsta målsättningar var att beskriva den intra-specifika diversiteten hos denna art längs med miljögradienter i Östersjön. Ett annat mål var att klargöra de faktorer som eventuellt är involverade i konfigurationen av genetisk diversitet och differentiering. Med hjälp av genetiska markörer visade jag att den genetiska diversiteten hos S. marinoi populationer i Östersjön är lägre jämfört med populationer i östra delen av Nordsjön. Arten är genetiskt uppdelad så att en utpräglad population förekommer i Östersjön och en annan, genetiskt åtskild population förekommer norr om de Danska sunden. Resultaten visar att de genetiskt åtskilda populationerna är anpassade till lokala salinitetsförhållanden. Genflödet mellan populationerna korrelerade kraftigt med havströmmar i området. Mina studier avslöjade även omfattande variation av fenotypiska, ekologiskt vikitga särdrag hos olika kloner. Djurplankton som äter kiselalger kunde modifiera den klonala mångfalden av fenotypiskt variabla S. marinoi populationer. En ökad klonal mångfald ledde till högre prestationsförmåga i fråga om primär produktion och stabiliserade ekofysiologiska funktioner. Som visas i denna avhandling består en art allt som oftast av åtskilliga genetiska varianter med fenotypiska skillnader. Kunskap om sådana intra-specifika skillnader är en förutsättning för att vi skall kunna förstå var och varför arter förekommer. Denna kunskap utgör även en grund för prognoser som siktar på att förutspå huruvida arter kan anpassa sig till framtida miljöförhållanden. ------------------------------------------------------ Suunnaton määrä variaatioita eliölajien välillä on perustavanlaatuinen ominaisuus luonnossa. Perinteisesti tätä monimuotoisuutta on käytetty organismien luokittelemiseen eri lajeihin niiden morfologisten eroavaisuuksien perusteella. Hiljattain myös geneettisten erojen huomioimista yksilötasolla on hyödynnetty lajien luokittelemisessa. Merialueilla esiintyvä piilevä, Skeletonema marinoi on yksi Itämeren tavallisimmista kasviplanktonlajeista kevätkukinnan aikana. Tavoitteenani oli selventää geneettistä ja fenotyyppistä monimuotoisuutta pitkin Itämeren ympäristögradienttejä. Geneettisen monimuotoisuuteen ja erkaantumiseen vaikuttavien tekijöiden selvittäminen oli tärkeä aspekti väitöstutkimuksessani. Geneettisiä markkereita käyttämällä pystyin toteamaan, että S. marinoi levän geneettinen monimuotoisuus on Itämeressä merkittävästi alhaisempi kuin läheisessä Pohjanmeren itäosassa. Tutkittu laji jakautuu geneettisesti yhteen erilliseen populaatioon Itämeressä ja toiseen selvästi erottuvaan populaatioon Tanskan salmien pohjoispuolella. Kokeellisten tulosten perusteella nämä geneettisesti erilaistuneet populaatiot ovat kumpikin sopeutuneet paikalliseen veden suolapitoisuuteen. Populaatioiden välisen geenivirran ja merivirtojen luoman yhteyden välillä havaittiin vahva korrelaatio. Tutkimukseni paljastivat myös laajaa vaihtelua Skeletonema-kloonien ekologisesti tärkeissä ominaisuuksissa. Kokeellisten tutkimusteni perusteella laiduntajat pystyivät muuttamaan geneettisten kloonien lukumäärää monimuotoisissa S. marinoi populaatioissa. Lisääntynyt kloonien lukumäärä paransi perustuotantokykyä ja vakautti ekofysiologisia toimintoja. Kuten tässä väitöstutkimuksessa osoitetaan, lajit koostuvat useimmiten lukuisista geneettisistä muunnelmista, jotka eroavat usein fenotyypeiltään. Ymmärtääksemme missä tietyt lajit esiintyvät ja miksi, tarvitsemme tietoa lajien sisäisistä vaihteluista. Tämä tieto on tarpeellista, jotta voimme ennustaa lajien sopeutumista tuleviin ympäristönmuutoksiin.

Clinical and genetic markers in disease progression – a study among subjects with obstructive lung diseases

Asthma, COPD, and asthma and COPD overlap syndrome (ACOS) are chronic pulmonary diseases with an obstructive component. In COPD, the obstruction is irreversible and the disease is progressive. The aim of the study was to define and analyze factors that affected disease progression and patients’ well-being, prognosis and mortality in Chronic Airway Disease (CAD) cohort. The main focus was on COPD and ACOS patients. Retrospective data from medical records was combined with genetic and prospective follow-up data. Smoking is the biggest risk factor for COPD and even after the diagnosis of the disease, smoking plays an important role in disease development and patient’s prognosis. Sixty percent of the COPD patients had succeeded in smoking cessation. Patients who had managed to quit smoking had lower mortality rates and less psychiatric diseases and alcohol abuse although they were older and had more cardiovascular diseases than patients who continued smoking. Genetic polymorphism rs1051730 in the nicotinic acethylcholine receptor gene (CHRNA3/5) associated with heavy smoking, cancer prevalence and mortality in two Finnish independent cohorts consisting of COPD patients and male smokers. Challenges in smoking cessation and higher mortality rates may be partly due to individual patient’s genetic composition. Approximately 50% of COPD patients are physically inactive and the proportion was higher among current smokers. Physically active and inactive patients didn’t differ from each other in regard to age, gender or comorbidities. Bronchial obstruction explained inactivity only in severe disease. Subjective sensation of dyspnea, however, had very strong association to inactivity and was also associated to low health related quality of life (HRQoL). ACOS patients had a significantly lower HRQoL than either the patients with asthma or with COPD even though they were younger than COPD patients, had better lung functions and smaller tobacco exposure.