Molecular Genetic Studies of Melanocortin Receptors in Morbid Obesity

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.

Ion channel gene variants predisposing to severe human ventricular arrhythmias

Congenital long QT syndrome (LQTS) with an estimated prevalence of 1:2000-1:10 000 manifests with prolonged QT interval on electrocardiogram and risk for ventricular arrhythmias and sudden death. Several ion channel genes and hundreds of mutations in these genes have been identified to underlie the disorder. In Finland, four LQTS founder mutations of potassium channel genes account for up to 40-70% of genetic spectrum of LQTS. Acquired LQTS has similar clinical manifestations, but often arises from usage of QT-prolonging medication or electrolyte disturbances. A prolonged QT interval is associated with increased morbidity and mortality not only in clinical LQTS but also in patients with ischemic heart disease and in the general population. The principal aim of this study was to estimate the actual prevalence of LQTS founder mutations in Finland and to calculate their effect on QT interval in the Finnish background population. Using a large population-based sample of over 6000 Finnish individuals from the Health 2000 Survey, we identified LQTS founder mutations KCNQ1 G589D (n=8), KCNQ1 IVS7-2A>G (n=1), KCNH2 L552S (n=2), and KCNH2 R176W (n=16) in 27 study participants. This resulted in a weighted prevalence estimate of 0.4% for LQTS in Finland. Using a linear regression model, the founder mutations resulted in a 22- to 50-ms prolongation of the age-, sex-, and heart rate-adjusted QT interval. Collectively, these data suggest that one of 250 individuals in Finland may be genetically predisposed to ventricular arrhythmias arising from the four LQTS founder mutations. A KCNE1 D85N minor allele with a frequency of 1.4% was associated with a 10-ms prolongation in adjusted QT interval and could thus identify individuals at increased risk of ventricular arrhythmias at the population level. In addition, the previously reported associations of KCNH2 K897T, KCNH2 rs3807375, and NOS1AP rs2880058 with QT interval duration were confirmed in the present study. In a separate study, LQTS founder mutations were identified in a subgroup of acquired LQTS, providing further evidence that congenital LQTS gene mutations may underlie acquired LQTS. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by exercise-induced ventricular arrhythmias in a structurally normal heart and results from defects in the cardiac Ca2+ signaling proteins, mainly ryanodine receptor type 2 (RyR2). In a patient population of typical CPVT, RyR2 mutations were identifiable in 25% (4/16) of patients, implying that noncoding variants or other genes are involved in CPVT pathogenesis. A 1.1 kb RyR2 exon 3 deletion was identified in two patients independently, suggesting that this region may provide a new target for RyR2-related molecular genetic studies. Two novel RyR2 mutations showing a gain-of-function defect in vitro were identified in three victims of sudden cardiac death. Extended pedigree analyses revealed some surviving mutation carriers with mild structural abnormalities of the heart and resting ventricular arrhythmias suggesting that not all RyR2 mutations lead to a typical CPVT phenotype, underscoring the relevance of tailored risk stratification of a RyR2 mutation carrier.

Pharmacogenetics of SLCO1B1: Population genetics and effect on statins

Kohonneiden kolesterolipitoisuuksien alentamisessa käytettävien statiinien hyödyt sydän- ja verisuonisairauksien estossa on vahvasti osoitettu ja niiden käyttö on niin Suomessa kuin muuallakin maailmassa kasvanut voimakkaasti – Suomessa statiininkäyttäjiä on noin 600 000. Statiinilääkitys on pitkäaikaisessakin käytössä melko hyvin siedetty, mutta yleisimpinä haittavaikutuksina voi ilmetä lihasheikkoutta, -kipua ja -kramppeja, jotka voivat edetä jopa henkeä uhkaavaksi lihasvaurioksi. Lihashaittariski suurenee suhteessa statiiniannokseen ja plasman statiinipitoisuuksiin. Statiinien plasmapitoisuuksissa, tehossa ja haittavaikutusten ilmenemisessä on suuria potilaskohtaisia eroja. SLCO1B1-geenin koodaama OATP1B1-kuljetusproteiini kuljettaa monia elimistön omia aineita ja lääkeaineita verenkierrosta solukalvon läpi maksasoluun, mm. statiineja, joiden kolesterolia alentava vaikutus ja poistuminen elimistöstä tapahtuvat pääosin maksassa. Erään SLCO1B1-geenin nukleotidimuutoksen (c.521T>C) tiedetään heikentävän OATP1B1:n kuljetustehoa. Tässä väitöskirjatyössä selvitettiin SLCO1B1-geenin perinnöllistä muuntelua suomalaisilla ja eri väestöissä maailmanlaajuisesti. Lisäksi selvitettiin SLCO1B1:n muunnosten vaikutusta eri statiinien pitoisuuksiin (farmakokinetiikka) ja vaikutuksiin (farmakodynamiikka) sekä kolesteroliaineenvaihduntaan. Näihin tutkimuksiin valittiin SLCO1B1-genotyypin perusteella terveitä vapaaehtoisia koehenkilöitä, joille annettiin eri päivinä kerta-annos kutakin tutkittavaa statiinia: fluvastatiinia, pravastatiinia, simvastatiinia, rosuvastatiinia ja atorvastatiinia. Verinäytteistä määritettiin plasman statiinien ja niiden aineenvaihduntatuotteiden sekä kolesterolin ja sen muodostumista ja imeytymistä kuvaavien merkkiaineiden pitoisuuksia. Toiminnallisesti merkittävien SLCO1B1-geenimuunnosten esiintyvyydessä todettiin suuria eroja eri väestöjen välillä. Suomalaisilla SLCO1B1 c.521TC-genotyypin (geenimuunnos toisessa vastinkromosomissa) esiintyvyys oli noin 32 % ja SLCO1B1 c.521CC-genotyypin (geenimuunnos molemmissa vastinkromosomeissa) esiintyvyys noin 4 %. Globaalisti geenimuunnosten esiintyvyys korreloi maapallon leveyspiirien kanssa siten, että matalaan transportteriaktiivisuuteen johtavat muunnokset olivat yleisimpiä pohjoisessa ja korkeaan aktiivisuuteen johtavat päiväntasaajan lähellä asuvilla väestöillä. SLCO1B1-genotyypillä oli merkittävä vaikutus statiinien plasmapitoisuksiin lukuun ottamatta fluvastatiinia. Simvastatiinihapon plasmapitoisuudet olivat keskimäärin 220 %, atorvastatiinin 140 %, pravastatiinin 90 % ja rosuvastatiinin 70 % suuremmat c.521CC-genotyypin omaavilla koehenkilöillä verrattuna normaalin c.521TT-genotyypin omaaviin. Genotyypillä ei ollut merkittävää vaikutusta minkään statiinin tehoon tässä kerta-annostutkimuksessa, mutta geenimuunnoksen kantajilla perustason kolesterolisynteesinopeus oli suurempi. Tulokset osoittavat, että SLCO1B1 c.521T>C geenimuunnos on varsin yleinen suomalaisilla ja muilla ei-afrikkalaisilla väestöillä. Tämä geenimuunnos voi altistaa erityisesti simvastatiinin, mutta myös atorvastatiinin, pravastatiinin ja rosuvastatiinin, aiheuttamille lihashaitoille suurentamalla niiden plasmapitoisuuksia. SLCO1B1:n geenimuunnoksen testaamista voidaan tulevaisuudessa käyttää apuna valittaessa sopivaa statiinilääkitystä ja -annosta potilaalle, ja näin parantaa sekä statiinihoidon turvallisuutta että tehoa.

Probiotics in the prevention of clinical manifestations of common infectious diseases in children and in the elderly

Infectious diseases put an enormous burden on both children and the elderly in the forms of respiratory, gastrointestinal and oral infections. There is evidence suggesting that specific probiotics may be antagonistic to pathogens and may enhance the immune system, but the clinical evidence is still too sparce to make general conclusions on the disease-preventive effects of probiotics. This thesis, consisting of four independent, double-blind, placebo-controlled clinical trials, investigated whether Lactobacillus GG (LGG) or a specific probiotic combination containing LGG would reduce the risk of common infections or the prevalence of pathogens in healthy and infection-prone children and in independent and institutionalised elderly people. In healthy day-care children, the 7-month consumption of probiotic milk containing Lactobacillus GG appeared to postpone the first acute respiratory infection (ARI) by one week (p=0.03, adjusted p=0.16), and to reduce complicated infections (39% vs. 47%, p<0.05, adjusted p=0.13), as well as the need for antibiotic treatment (44% vs. 54%, p=0.03, adjusted p=0.08) and day-care absences (4.9 vs. 5.8 days, p=0.03, adjusted p=0.09) compared to the placebo milk. In infection-prone children, the 6-month consumption of a combination of four probiotic bacteria (LGG, L. rhamnosus LC705, Propionibacterium freudenreichii JS, Bifidobacterium breve 99) taken in capsules appeared to reduce recurrent ARIs (72% vs. 82%, p<0.05; adjusted p=0.06), and the effect was particularly noticeable in a subgroup of children with allergic diseases (12% vs. 33%, p=0.03), although no effect on the presence of nasopharyngeal rhinovirus or enterovirus was seen. The 5-month consumption of the same probiotic combination did not show any beneficial effects on the respiratory infections in frail, institutionalised elderly subjects. In healthy children receiving Lactobacillus GG, the reduction in complications resulted in a marginal reduction in the occurrence of acute otitis media (AOM) (31% vs. 39%, p=0.08; adjusted p=0.19), and the postponement of the first AOM episode by 12 days (p=0.04; adjusted p=0.09). However, in otitis-prone children, a probiotic combination did not reduce the occurrence of AOM or the total prevalence of common AOM pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis), except in the case of children with allergic diseases, in whom probiotics reduced recurrent AOM episodes (0% vs. 14%, p=0.03). In addition, interaction between probiotics and bacterial carriage was seen: probiot-ics reduced AOM in children who did not carry any bacterial pathogens (63% vs. 83%), but the effect was the reverse in children carrying bacteria in the nasopharynx (74% vs 62%) (p<0.05). Long-term probiotic treatment, either LGG given in milk to healthy children for 7 months or a combination of probiotics given in capsules to institutionalised elderly subjects for 5 months, did not reduce the occurrence of acute diarrhoea. However, when the probiotic combination (LGG, L. rhamnosus LC705, Propionibacterium JS) was given in cheese to independent elderly subjects for 4 months, the oral carriage of high Candida counts was reduced in the probiotic group vs. the placebo group (21% vs. 34%, p=0.01, adjusted p=0.004). The risk of hyposalivation was also reduced in the probiotic group (p=0.05). In conclusion, probiotics appear to slightly alleviate the severity of infections by postponing their appearance, by reducing complications and the need for antimicrobial treatments. In addition, they appear to prevent recurrent infections in certain subgroups of children, such as in infection-prone children with allergic diseases. Alleviating ARI by probiotics may lead to a marginal reduction in the occurrence of AOM in healthy children but not in infection-prone children with disturbed nasopharyngeal microbiota. On the basis of these results it could be supposed that Lactobacillus GG or a specific combination containing LGG are effective against viral but not against bacterial otitis, and the mechanism is probably mediated through the stimulation of the immune system. A specific probiotic combination does not reduce respiratory infections in frail elderly subjects. Acute diarrhoea, either in children or in the elderly, is not prevented by the continuous, long-term consumption of probiotics, but the consumption of a specific probiotic combination in a food matrix is beneficial to the oral health of the elderly, through the reduction of the carriage of Candida.

Conformal Field Theory Methods for Variants of Schramm-Loewner Evolutions

This thesis consists of an introduction, four research articles and an appendix. The thesis studies relations between two different approaches to continuum limit of models of two dimensional statistical mechanics at criticality. The approach of conformal field theory (CFT) could be thought of as the algebraic classification of some basic objects in these models. It has been succesfully used by physicists since 1980's. The other approach, Schramm-Loewner evolutions (SLEs), is a recently introduced set of mathematical methods to study random curves or interfaces occurring in the continuum limit of the models. The first and second included articles argue on basis of statistical mechanics what would be a plausible relation between SLEs and conformal field theory. The first article studies multiple SLEs, several random curves simultaneously in a domain. The proposed definition is compatible with a natural commutation requirement suggested by Dubédat. The curves of multiple SLE may form different topological configurations, ``pure geometries''. We conjecture a relation between the topological configurations and CFT concepts of conformal blocks and operator product expansions. Example applications of multiple SLEs include crossing probabilities for percolation and Ising model. The second article studies SLE variants that represent models with boundary conditions implemented by primary fields. The most well known of these, SLE(kappa, rho), is shown to be simple in terms of the Coulomb gas formalism of CFT. In the third article the space of local martingales for variants of SLE is shown to carry a representation of Virasoro algebra. Finding this structure is guided by the relation of SLEs and CFTs in general, but the result is established in a straightforward fashion. This article, too, emphasizes multiple SLEs and proposes a possible way of treating pure geometries in terms of Coulomb gas. The fourth article states results of applications of the Virasoro structure to the open questions of SLE reversibility and duality. Proofs of the stated results are provided in the appendix. The objective is an indirect computation of certain polynomial expected values. Provided that these expected values exist, in generic cases they are shown to possess the desired properties, thus giving support for both reversibility and duality.

Sampled longest common prefix array

When augmented with the longest common prefix (LCP) array and some other structures, the suffix array can solve many string processing problems in optimal time and space. A compressed representation of the LCP array is also one of the main building blocks in many compressed suffix tree proposals. In this paper, we describe a new compressed LCP representation: the sampled LCP array. We show that when used with a compressed suffix array (CSA), the sampled LCP array often offers better time/space trade-offs than the existing alternatives. We also show how to construct the compressed representations of the LCP array directly from a CSA

Streptomyces scabies and S. turgidiscabies as pathogens causing potato common scab, and potential methods for their control

Tieteellinen tiivistelmä Common scab is one of the most important soil-borne diseases of potato (Solanum tuberosum L.) in many potato production areas. It is caused by a number of Streptomyces species, in Finland the causal agents are Streptomyces scabies (Thaxter) Lambert & Loria and S. turgidiscabies Takeuchi. The scab-causing Streptomyces spp. are well-adapted, successful plant pathogens that survive in soil also as saprophytes. Control of these pathogens has proved to be difficult. Most of the methods used to manage potato common scab are aimed at controlling S. scabies, the most common of the scab-causing pathogens. The studies in this thesis investigated S. scabies and S. turgidiscabies as causal organisms of common scab and explored new approaches for control of common scab that would be effective against both species. S. scabies and S. turgidiscabies are known to co-occur in the same fields and in the same tuber lesions in Finland. The present study showed that both these pathogens cause similar symptoms on potato tubers, and the types of symptoms varied depending on cultivar rather than the pathogen species. Pathogenic strains of S. turgidiscabies were antagonistic to S. scabies in vitro indicating that these two species may be competing for the same ecological niche. In addition, strains of S. turgidiscabies were highly virulent in potato and they tolerated lower pH than those of S. scabies. Taken together these results suggest that S. turgidiscabies has become a major problem in potato production in Finland. The bacterial phytotoxins, thaxtomins, are produced by the scab-causing Streptomyces spp. and are essential for the induction of scab symptoms. In this study, thaxtomins were produced in vitro and four thaxtomin compounds isolated and characterized. All four thaxtomins induced similar symptoms of reduced root and shoot growth, root swelling or necrosis on micro-propagated potato seedlings. The main phytotoxin, thaxtomin A, was used as a selective agent in a bioassay in vitro to screen F1 potato progeny from a single cross. Tolerance to thaxtomin A in vitro and scab resistance in the field were correlated indicating that the in vitro bioassay could be used in the early stages of a resistance breeding program to discard scab-susceptible genotypes and elevate the overall levels of common scab resistance in potato breeding populations. The potential for biological control of S. scabies and S. turgidiscabies using a non-pathogenic Streptomyces strain (346) isolated from a scab lesion and S. griseoviridis strain (K61) from a commercially available biocontrol product was studied. Both strains showed antagonistic activity against S. scabies and S. turgidiscabies in vitro and suppressed the development of common scab disease caused by S. turgidiscabies in the glasshouse. Furthermore, strain 346 reduced the incidence of S. turgidiscabies in scab lesions on potato tubers in the field. These results demonstrated for the first time the potential for biological control of S. turgidiscabies in the glasshouse and under field conditions and may be applied to enhance control of common scab in the future.

Content of zinc, iron and their absorption inhibitors in Nicaraguan common beans (Phaseolus vulgaris L.).

Protein-energy malnutrition and mineral deficiencies are two of the three forms of nutritional deficiencies that affect most developing countries due to inadequate access to food and diets based on a sole crop. Common bean (Phaseolus vulgaris L.) is the staple crop of Nicaragua and it has the potential to improve the nutritional status of the poorest group of the nation. Its high content of both protein and nonhaem iron provides many nutrients, but inhibitors also may prevent absorption of iron and zinc by the human consumer. A proper production chain must be followed to ensure the best grain quality for the consumer. To achieve food security, both production and high nutritional content must be maintained. Four nationally important accessions of common bean, with different harvesting dates, were selected to be submitted to two treatments: to evaluate the impact of storage conditions on the end quality of the grain. The duration of the study was six months with sampling every six weeks, and the two treatments were controlled one stored at 40°C and 75 RH %, and the other was stored in in-situ conditions. Proximate and mineral composition was evaluated as well as tannin, phytate and bioavailability. Significant differences among different accessions were found, being the most significant in protein, Fe and Zn content, tannins and phytate. Protein values ranged from 21-23%. Iron content was 61-81 mg/kg but only 3-4% was bioavailable. Zinc content was 21-25 mg/kg and 10-12% was bioavailable. The concentration of phytate ranged from 8.6-9.6 mg/g while tannin values ranged within 37.7-43.8 mg/g. Storage at high temperatures was demonstrated to have an impact on certain nutritional compounds and proved detrimental to final grain quality. Soluble sugar content and tannin content decreased after six months in both storage conditions, IDF decreased in the in-situ and SDF in the stress. The iron content and bioavailability in INTA Biofortificado were not as outstanding as expected, so experiments should be conducted to compare its iron uptake and delivery with other cultivars.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Integration or disintegration? Human resource implications of a common corporate language decision in a crossborder merger

The primary purpose of introducing a common corporate language in crossborder mergers is to integrate two previously separate organizations and facilitate communication. However, the present case study of a cross-border merger between two Nordic banks shows that the common corporate language decision may have disintegrating effects, particularly at organizational levels below top management. We identify such effects on performance appraisal, language training and management development, career paths, promotion and key personnel. Our findings show that top management needs to work through the consequences of the language decision upon those who are expected to make such a decision work.

Common past, divided memories: historical memory of the Polish minority members in Lithuania

The Master’s thesis examines historical memory of the Polish minority members in Lithuania with regard to how their interpretation of the common Polish-Lithuanian history reiterates or differs from the official Polish and Lithuanian narratives conveyed by the school textbooks. History teaching in high schools carries a crucial state-supported role of “identity building policies” – it maintains a national narrative of memory, which might be exclusive to minorities and their peculiar understanding of history. Lithuanians Poles, in this regard, represent a national minority, which is exposed to two conflicting national narratives of the common past – Polish and Lithuanian. As members of the Polish nation, their understanding of the common Polish-Lithuanian history is conditioned by the Polish historical narrative, acquired as part of the collective memory of the family and/or different minority organizations. On the other hand, they encounter Lithuanian historical narrative of the Polish-Lithuanian past throughout the secondary school history education, where the curriculum, even if taught in Polish, largely represents the Lithuanian point of view. The concept of collective memory is utilized to refer to collective representations of national memory (i.e. publicly articulated narratives and images of collective past in history textbooks) as well as to socially framed individual memories (i.e. historical memory of minority members, where individual remembering is framed by the social context of their identity). The thesis compares the official national historical narratives in Lithuania and Poland, as conveyed by the Polish and Lithuanian history textbooks. The consequent analysis of qualitative interviews with the Polish minority members in Lithuania offers insights into historical memory of Lithuanian Poles and its relation to the official Polish and Lithuanian national narratives of the common past. Qualitative content analysis is applied in both parts of the analysis. The narratives which emerge from the interview data could be broadly grouped into two segments. First, a more pronounced view on the past combines the following elements: i) emphasis on the value of multicultural and diverse past of Lithuania, ii) contestation of “Lithuanocentricity” of the Lithuanian narrative and iii) rejection of the term “occupation”, based on the cultural presuppositions – the dominant position of Polish culture and language in the Vilnius region, symbolic belonging and “Lithuanianness” of the local Poles. While the opposition to the term of “occupation” is in accord with the official Polish narrative conveyed by the textbooks, the former two elements do not neatly adhere to either Polish or Lithuanian textbook narratives. They should rather be considered as an expression of claims for inclusion of plural pasts into Lithuanian collective memory and hence as claims for symbolic enfranchisement into the Lithuanian “imagined community”. The second strand of views, on the other hand, does not exclude assertions about the historically dominant position of Polish culture in Lithuania, but at the same time places more emphasis on the political and historical continuity of the Lithuanian state and highlights a long-standing symbolic connectedness of Vilnius and Lithuania, thus, striking a middle way between the Polish and Lithuanian interpretations of the past.

Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)

Pituitary adenomas are common benign neoplasms. Although most of them are sporadic, a minority occurs in familial settings. Heterozygous germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were found to underlie familial pituitary adenomas, a condition designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of mostly growth hormone (GH) secreting adenomas in young patients, who often lack a family history of pituitary adenomas. This thesis work aimed to clarify the molecular and clinical characteristics of PAP. Applying the multiplex ligation-dependent probe amplification assay (MLPA), we found large genomic AIP deletions to account for a subset of PAP. Therefore, MLPA could be considered in PAP suspected patients with no AIP mutations found with conventional sequencing. We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. The heterozygous Aip mutation conferred complete penetrance of pituitary adenomas that were mostly GH-secreting, rendering the phenotype of the Aip mouse similar to that of PAP patients. We suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. To clarify molecular mechanisms of tumorigenesis, we elucidated the expression of AIP-related molecules in human and mouse pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) was reduced in mouse Aip-deficient adenomas, and similar ARNT reduction was also evident in human AIP mutation positive adenomas. This suggests that in addition to participating in the hypoxia pathway, estrogen receptor signaling and xenobiotic response pathways, ARNT may play a role in AIP-related tumorigenesis. We also studied the characteristics and the response to therapy of PAP patients and found them to have an aggressive disease phenotype with young age at onset. Therefore, improvement in treatment outcomes of PAP patients would require their efficient identification and earlier diagnosis of the pituitary adenomas. The possible role of the RET proto-oncogene in tumorigenesis of familial AIP mutation negative pituitary adenomas was evaluated, but none of the found RET germline variants were considered pathogenic. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIP mutation positive pituitary adenomas an observation that merits further investigation.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.