Genetic Mechanisms of Tumourigenesis in von Hippel-Lindau-Associated Tumours with the Emphasis on Capillary Hemangioblastoma

The von Hippel-lindau (VHL) disease is a dominantly inherited neoplastic disorder which predisposes patients to multiple tumours including capillary haemangioblastomas (CHBs), pheochromocytomas (PCCs), renal cell carcinomas (RCCs). CHBs are the most common manifestations of VHL disease, occurring sporadically or as a manifestation of VHL disease. Inactivation of the VHL gene at 3p25-26 is believed to cause both familial and sporadic VHL-associated tumours and germ-line mutation of the VHL gene have been detected in 100% of the CHBs studied. However, a limited number of sporadic CHBs, PCCs display VHL inactivation. Other molecular alterations involved in tumourigenesis of sporadic CHBs, PCCs remain largely unknown. The purpose of the present work was to search for genetic alterations, or other mechanisms of inactivation, in addition to the VHL gene, that may be important in the development of VHL-associated tumours. Though less satisfactory than cure, prevention and early detection are the most promising and feasible means reducing cancer morbidity and mortality. This work is based on the view that increasing knowledge about the molecular events underlying tumour development will eventually aid in early detection and lead to improved treatment. We evaluated a large set of VHL-associated patients, searched for a clinical and radiologic signs of the disease. We succesfully performed a germ-line mutation analysis and characterised three patient groups, VHL, suspect VHL and sporadic, a germ-line mutation analysis revealed a 50% mutation rate only in the VHL groups, no sporadic or suspect cases displayed any mutation. We also utilized comparative genomic hybridization (CGH) to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed (27%) DNA copy number losses. The most common finding was loss of chromosomal arm 6q, seen in (23%) cases, No differences were noted between VHL-associated and sporadic tumours. Furthermore a loss of heterozygosity (LOH) study on chromosome 3p and 6q was done with the purpose to determine allele losses not observable by CGH, and to uncover the location of putative tumour suppressor genes important in CHB and PCC tumourigenesis. We identified loss of chromosome 6q and a minimal deleted area at 6q23-24 in CHBs. We also showed LOH at 6q23-24 in PCCs and identified the ZAC1 (6q24-25) as a candidate gene, ZAC1 is a maternally imprinted tumour suppressor gene with anti proliferative properties. To study further the role of ZAC inactivation in CHBs, we investigated LOH, promoter hypermethylation and expression status of the ZAC1 gene in mainly sporadic CHBs. Our LOH analysis revealed that the majority of the tumours with allele loss. The gene promoter methylation analysis similarly detected predominance of the methylated ZAC sequence in almost all tumours. Immunohistochemistry exhibited a strongly reduced expression of ZAC in stromal cells of all CHBs studied. Our current results indicate that the absence of the unmethylated, ZAC1 promoter sequence was highly concurrent with LOH for the ZAC1 region or 6q loss. This observation together with lack of ZAC expression, points to preferential loss of the non imprinted, expressed ZAC allele in CHB, in summary, our series of studies reveal a new chromosomal region 6q, emphasizes the importance of ZAC1 gene in the development of CHB and PCC, particularly in non-VHL associated cases.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Novel Insights into the Molecular Genetic Background of Colorectal Tumors

Many of the genes predisposing to highly penetrant colorectal cancer (CRC) syndromes, including hereditary non-polyposis colorectal cancer (MLH1, MSH2, MSH6, PMS2), familial adenomatous polyposis (APC), Peutz-Jeghers syndrome (LKB1), juvenile polyposis (SMAD4, BMPR1A), MYH-associated polyposis (MYH), and Cowden syndrome (PTEN) have already been discovered. Identification of these genes has allowed a more precise classification of the hereditary CRC syndromes and provided a means for predictive genetic testing and surveillance. Some of the genes are also involved in sporadic cancer forms, and therefore the investigation of the rare CRC syndromes has been a breakthrough for general cancer research. Despite the accumulating knowledge on hereditary cancer syndromes, a significant number of familial CRCs remain molecularly unexplained after genetic testing, reflecting the possibility of other predisposing genes or existence of novel syndromes. Moreover, genetic variants conferring low-penetrance risk are still largely unknown. In this study, we examined the role of some new high- and low-penetrance alleles on CRC predisposition. We identified disease causing MYH mutations in a subset (9%) of patients with APC and AXIN2 mutation negative adenomatous polyposis. Due to differences in the pattern of inheritance and clinical manifestation, screening for mutations in MYH is beneficial in view of genetic counselling and surveillance. A novel functionally deficient MYH founder mutation A459D was identified in the Finnish population, and this finding had immediate clinical implications for genetic counselling of at risk families. Many patients with hamartomatous polyposis remain without molecular diagnosis due to atypical phenotypes. We therefore sought to classify 49 patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analyses of PTEN, LKB1, BMPR1A, SMAD4, ENG, BRAF, MYH, and BHD along with revision of polyp histology. Mutations were identified in 11/49 (22%) of the patients. In 6 cases the molecular diagnosis was re-classified guiding surveillance and decisions for prophylactic surgery. Re-evaluation of polyp histology with subsequent more accurate selection of candidate gene analyses is beneficial and can be recommended for patients with unexplained polyposis. Furthermore, germline mutations in ENG underlying juvenile polyposis were described for the first time, characterizing a possible novel genetically defined form of hereditary CRC. Association analyses on two putative low-penetrance alleles, NOD2 3020insC and MDM2 SNP309 were performed in a population-based series of 1042 Finnish CRC patients and in cancer-free controls. In contrast to previous results, NOD2 3020insC did not associate with CRC or age at disease onset in the Finnish population. These data suggest that NOD2 3020insC alone might not be sufficient for CRC predisposition. MDM2 SNP309 was as common in the CRC cohort as in the healthy controls. Interesting trends, however, were observed, which after correction for multiple testing did not reach statistical significance. SNP309 was more common in female CRC patients and a trend towards an earlier age at disease onset was observed in women with SNP309. Subsequent studies have supported this observation and SNP309 could affect gender- or hormone-related tumorigenesis. Finally, a large-scale unbiased effort was designed to characterize the complete mutatome of CRC with microsatellite instability (MSI). Using an approach combining expression microarray and genome database searches, we were able to identify putative MSI target genes. Further characterization of one of the genes suggested that it might play a role also in microsatellite stable CRC and Peutz-Jeghers syndrome pathogenesis.

Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate

Positional cloning has enabled hypothesis-free, genome-wide scans for genetic factors contributing to disorders or traits. Traditionally linkage analysis has been used to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and finally sequencing of regions of interest. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization. Here, we have utilized modern molecular genetic methods combined with the advantages provided by the special population structure in Finland to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as previously identified linkage to 3p14-p24 was replicated in an independent family set of families with Asperger syndrome, but no other ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between autism and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). We also used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure of Finns. We observed significant population substructure which correlates with the known history of multiple consecutive bottle-necks experienced by the Finnish population. We used this information to ascertain a genetically homogenous subset of autism families to identify possible rare, enriched risk variants using genome-wide SNP data. No rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with others in the field. The underlying genetic architecture for this group of disorders appears highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors.

Amyloid diseases at old age : a pathological, epidemiological, and genetic study

Along with the increased life span of individuals, the burden of old age-associated diseases has inevitably increased. Alzheimer s disease (AD), probably the most well known geriatric disease, belongs to the old age-associated amyloid diseases. The purpose of this study was to investigate the frequency, genetic and health-associated risk factors, mutual association, and amyloid proteins in two old age-associated amyloid disorders senile systemic amyloidosis (SSA) and cerebral amyloid angiopathy (CAA) as part of the prospective population-based Vantaa 85+ autopsy study on a Finnish population aged 85 years or more (Studies I-III), completed with a case report on a patient with advanced AGel amyloidosis (Study IV). The numbers of patients investigated in the studies (I-III) were 256, 74, and 63, respectively. The diagnosis and grading of amyloid were based upon histological examination of tissue samples obtained post mortem and stained with Congo red. The amyloid fibril and associated proteins were characterized by immunohistochemical staining methods. The genotype frequencies of 20 polymorphisms in 9 genes and information on health-associated risk factors in subjects with and without SSA and CAA were compared. In a Finnish population ≥ 95 years of age, SSA and CAA occurred in 36% and 49% of the subjects, respectively. In total, two-thirds of these very elderly individuals had SSA, CAA, or both. However, in only 14% of the population these two conditions co-occurred. In subjects 85 years or older, the prevalence of SSA was 25%. In this population, SSA was associated with age at the time of death (p=0.002), myocardial infarctions (MIs; p=0.004), the G/G (Val/Val) genotype of the exon 24 polymorphism in the alpha2-macroglobulin (α2M) gene (p=0.042) and with the H2 haplotype of the tau gene (p=0.016). In contrast, the presence of CAA was strongly associated with APOE e4 (p=0.0003), with histopathological AD (p=0.0005), and with clinical dementia (p=0.01) in both e4+ (p=0.02) and e4- (p=0.06) individuals. Apart from demonstrating the amyloid fibril proteins, complement proteins 3d (C3d) and 9 (C9) were detected in the amyloid deposits of CAA and AGel amyloidosis, and α2M protein was found in fibrous scar tissue close to SSA. In conclusion, this first population based study on SSA shows that both SSA and CAA are common in very elderly individuals. Old age, MIs, the exon 24 polymorphism of the α2M gene, and H1/H2 polymorphism of the tau gene associate with SSA while clinical dementia and APOE ε4 genotype associate with CAA. The high prevalence of CAA, combined with its association with clinical dementia independent of APOE genotype, neuropathological AD, or SSA, also highlights its clinical significance in the very aged, among which the serious end stage complications of CAA, namely multiple infarctions and hemorrhages, are rare. The report on a patient having advanced AGel amyloidosis added knowledge on the disease and showed that this generally benign condition occasionally may lead to death. Further studies are warranted to confirm the findings in other populations. Also, the role of α2M and tau in the pathogenesis of SSA and the involvement of complement in the process of amyloid beta (Aβ) protein elimination from the brain remain to be clarified. Finally, the high prevalence of SSA in the elderly raises the need for prospective clinical studies to define its clinical significance.

Genetic diversity of Atlantic salmon (Salmo salar) and European whitefish (Coregonus lavaretus) in the Baltic Sea

We described the patterns and extent of microsatellite DNA variation in historical and present-day Atlantic salmon (Salmo salar L.) stocks in the Baltic Sea and neighbouring areas, and in European whitefish (Coregonus lavaretus) ecotypes, populations and run-timing types in Finland. Moreover, the amount and pattern of genetic diversity in historical salmon populations before human impact were described, and the proportion of diversity maintained in the present hatchery stocks evaluated. Salmon populations in the Baltic Sea were, on average, significantly less variable than eastern Atlantic populations, and the diversity of landlocked populations (Lakes Vänern, Saimaa, Onega and Ladoga) was in turn significantly lower than that of anadromous salmon populations in the Baltic Sea populations. Within the Baltic Sea, the anadromous populations of Atlantic salmon formed three clear groups, corresponding to the northern (Gulf of Bothnia), eastern (Gulf of Finland and eastern Baltic Main Basin) and southern (western Baltic Main Basin) regions. Based on microsatellite data, three salmon population groups in the Baltic Sea were considered potentially different colonization lineages. In short- and long-term breeding programmes of Atlantic salmon, the average observed rate of loss of alleles was 4.9% and 2.0% per generation and the average rate of loss of heterozygosity was 1.4% and 1% per generation, respectively. When comparing the genetic parameters of stocks before and after hatchery breeding of several successive generations (Rivers Iijoki and Oulujoki), statistically significant changes in allele frequencies were common, while large wild stock in the Teno River has remained temporally very stable over 56 years. Despite the observed losses of genetic diversity in broodstock breeding, a large proportion of the genetic resources of the extirpated stocks are still conserved in the broodstocks. Genetic differentiation among European whitefish ecotypes was generally low, thus giving support to the hypothesis of one native European whitefish species in Fennoscandia. Among the ecotypes, the northern, large sparsely rakered, bottom-dwelling whitefish was the most unique. The known genetic differences in quantitative traits have thus either developed independently of potential phylogenetic lineages, or the lineages have mixed and the quantitative traits of the ecotypes, like gill-raker number, have later changed according to environment and selection pressures. Overall, genetic distances between the anadromous whitefish populations along the Finnish coast, especially in the Bothnian Bay area, were small. Wild whitefish populations studied had slightly higher allelic diversity than hatchery-reared populations in corresponding rivers.

Genetic Diversity and Adaptation of Date Palm (Phoenix dactylifera L.)

Acquiring sufficient information on the genetic variation, genetic differentiation, and the ecological and genetic relationships among individuals and populations are essential for establishing guidelines on conservation and utilization of the genetic resources of a species, and more particularly when biotic and abiotic stresses are considered. The aim of this study was to assess the extent and pattern of genetic variation in date palm (Phoenix dacttylifera L) cultivars; the genetic diversity and structure in its populations occurring over geographical ranges; the variation in economically and botanically important traits of it and the variation in its drought adaptive traits, in conservation and utilization context. In this study, the genetic diversity and relationships among selected cultivars from Sudan and Morocco were assessed using microsatellite markers. Microsatellite markers were also used to investigate the genetic diversity within and among populations collected from different geographic locations in Sudan. In a separate investigation, fruits of cultivars selected from Sudan, involved morphological and chemical characterization, and morphological and DNA polymorphism of the mother trees were also investigated. Morphological and photosynthetic adjustments to water stress were studied in the five most important date palm cultivars in Sudan, namely, Gondaila, Barakawi, Bitamoda, Khateeb and Laggai; and the mechanism enhancing photosynthetic gas exchange in date palm under water stress was also investigated. Results showed a significant (p < 0.001, t-test) differentiation between Sudan and Morocco groups of cultivars. However, the major feature of all tested cultivars was the complete lack of clustering and the absence of cultivars representing specific clones. The results indicated high genetic as well as compositional and morphological diversity among cultivars; while, compositional and morphological traits were found to be characteristic features that strongly differentiate cultivars as well as phenotypes. High genetic diversity was observed also in different populations. Slight but significant (p < 0.01, AMOVA) divergence was observed for soft and dry types; however, the genetic divergence among populations was relatively weak. The results showed a complex genetic relationships between some of the tested populations especially when isolation by distance was considered. The results of the study also revealed that date palm cultivars and phenotypes possess specific direct or interaction effects due to water availability on a range of morphological and physiological traits. Soft and dry phenotypes responded differently to different levels of water stress, while the dry phenotype was more sensitive and conservative. The results indicated that date palm has high fixation capacity to photosynthetic CO2 supply with interaction effect to water availability, which can be considered as advantageous when coping with stresses that may arise with climate change. In conclusion, although a large amount of diversity exists among date palm germplasm, the findings in this study show that the role of biological nature of the tree, isolation by distance and environmental effects on structuring date palm genome was highly influenced by human impacts. Identity of date palm cultivars as developed and manipulated by date palm growers, in the absence of scientific breeding programmes, may continue to mainly depend on tree morphology and fruit characters. The pattern of genetic differentiation may cover specific morphological and physiological traits that contribute to adaptive mechanisms in each phenotype. These traits can be considered for further studies related to drought adaptation in date palm.

Genetic and environmental influences on human responses to odors

Olfaction, the sense of smell, has many important functions in humans. Human responses to odors show substantial individual variation. Olfactory receptor genes have been identified and other genes may also influence olfaction. However, the proportion of phenotypic variation in odor response due to genetic variation remains largely unknown. Little is also known about which genes modify specific responses to odors. This study aimed to elucidate genetic and environmental influences on human responses to odors. Individuals from Finnish families (n=146) and Australian (n=413), British (n=163), Danish (n=336), and Finnish (n=399) twins rated intensity and pleasantness of a set of 12 (families) or 6 (twins) odors and tried to identify the odors. In addition, the participants rated their own sense of smell and annoyance experienced with different environmental odors. The odor stimuli of a commercial smell test (The Brief Smell Identification Test; banana, chocolate, cinnamon, gasoline, lemon, onion, paint thinner, pineapple, rose, smoke, soap, and turpentine) were presented in the family study. Based on the results of the family study and a literature survey, a new set of odor stimuli (androstenone, chocolate, cinnamon, isovaleric acid, lemon, and turpentine) was designed for the twin studies. In the family sample, heritabilities of the traits were estimated and underlying genomic regions were searched using a genome-wide linkage scan. In the pooled twin sample, variation in the measured traits was decomposed into genetic and environmental components using quantitative genetic modeling. In addition, associations between nongenetic factors (e.g., sex, age, and smoking) and olfactory-related traits were explored. Suggestive evidence for a genetic linkage for pleasantness of cinnamon at a locus on chromosome 4q32.3 emerged from the family sample. High heritability for the pleasantness of cinnamon was found in the family but not the twin study. Heritability of perceived intensity of androstenone odor was determined to be ~30% in the twin sample. A strong genetic correlation between perceived intensity and pleasantness of androstenone, in the absence of any environmental correlation, indicated that only the genetic correlation explained the phenotypic correlation between the traits (r=-0.27) and that the traits were influenced by an overlapping set of genes. Self-rated olfactory function appeared to reflect the odor annoyance experienced rather than actual olfactory acuity or genetic involvement. Results from nongenetic analyses supported the speculated superiority of females' olfactory abilities, the age-related diminishing of olfactory acuity, and the influences of experience-dependent factors on odor responses. This was the first study to estimate heritabilities and perform linkage screens for individual odors. A genetic effect was detected for only a few responses to specific odors, suggesting the predominance of environmental effects in odor perceptions.

A matter of taste : genetic and environmental influences on responses to sweetness

Diet is a major player in the maintenance of health and onset of many diseases of public health importance. The food choice is known to be largely influenced by sensory preferences. However, in many cases it is unclear whether these preferences and dietary behaviors are innate or acquired. The aim of this thesis work was to study the extent to which the individual differences in dietary responses, especially in liking for sweet taste, are influenced by genetic factors. Several traits measuring the responses to sweetness and other dietary variables were applied in four studies: in British (TwinsUK) and Finnish (FinnTwin12 and FinnTwin16) twin studies and in a Finnish migraine family study. All the subjects were adults and they participated in chemosensory measurements (taste and smell tests) and filled in food behavior questionnaires. Further, it was studied, whether the correlations among the variables are mediated by genetic or environmental factors and where in the genome the genes influencing the heritable traits are located. A study of young adult Finnish twins (FinnTwin16, n=4388) revealed that around 40% of the food use is attributable to genetic factors and that the common, childhood environment does not affect the food use even shortly after moving from the parents home. Both the family study (n=146) and the twin studies (British twins, n=663) showed that around half of the variation in the liking for sweetness is inherited. The same result was obtained both by the chemosensory measurements (heritability 41-49%) and the questionnaire variables (heritability 31-54%). By contrast, the intensity perception of sweetness or the responses to saltiness were not influenced by genetic factors. Further, a locus influencing the use-frequency of sweet foods was identified on chromosome 16p. A closer examination of the relationships among the variables based on 663 British twins revealed that several genetic and environmental correlations exist among the different measures of liking for sweetness. However, these correlations were not very strong (range 0.06-0.55) implying that the instruments used measure slightly different aspects of the phenomenon. In addition, the assessment of the associations among responses to fatty foods, dieting behaviors, and body mass index in twin populations (TwinsUK n=1027 and FinnTwin12 n=299) showed that the dieting behaviors (cognitive restraint, uncontrolled eating, and emotional eating) mediate the relationship between obesity and diet. In conclusion, the work increased the understanding of the background variables of human eating behavior. Genetic effects were shown to underlie the variation of many dietary traits, such as liking for sweet taste, use of sweet foods, and dieting behaviors. However, the responses to salty taste were shown to be mainly determined by environmental factors and thus should more easily be modifiable by dietary education, exposure, and learning than sweet taste preferences. Although additional studies are needed to characterize the genetic element located on chromosome 16 that influences the use-frequency of sweet foods, the results underline the importance of inherited factors on human eating behavior.

Genetic diversity and microcystin production by Anabaena in the Gulf of Finland, Baltic Sea

Syanobakteerit (sinilevät) ovat olleet Itämeressä koko nykymuotoisen Itämeren ajan, sillä paleolimnologiset todisteet niiden olemassaolosta Itämeren alueella ovat noin 7000 vuoden takaa. Syanobakteerien massaesiintymät eli kukinnat ovat kuitenkin sekä levinneet laajemmille alueille että tulleet voimakkaimmiksi viimeisten vuosikymmenien aikana. Tähän on osasyynä ihmisten aiheuttama kuormitus, joka rehevöittää Itämerta. Suomenlahti, jota tämä tutkimus käsittelee, on kärsinyt tästä rehevöitymiskehityksestä muita Itämeren altaita enemmän. Syanobakteerit muodostavat jokakesäisiä kukintoja Suomenlahdella - niin sen avomerialueilla kuin rannoillakin. Yleisimmät kukintoja muodostavat syanobakteerisuvut ovat Nodularia, Anabaena ja Aphanizomenon. Kukinnat aiheuttavat paitsi esteettistä haittaa myös terveydellisen riskitekijän. Niiden myrkyllisyys liitetään usein Nodularia-suvun tuottamaan nodulariini-maksamyrkkyyn. Itämeren Aphanizomenon-suvun on todettu olevan myrkytön. Vaikka Itämeren kukintoja aiheuttavista Nodularia- ja Aphanizomenon-syanobakteereista tiedetään varsin paljon, on molekyylimenetelmiin pohjautuva syanobakteeritutkimus ohittanut Itämeren Anabaena-suvun monelta osin. Tämän työn tarkoituksena oli syventää käsitystämme Itämeren Anabaena-syanobakteerista, sen mahdollisesta myrkyllisyydestä, geneettisestä monimuotoisuudesta ja fylogeneettisista sukulaisuussuhteista. Tässä työssä eristettiin 49 planktista Anabaena-kantaa, joista viisi tuottivat mikrokystiinejä. Tämä oli ensimmäinen yksiselitteinen todiste, että Itämeren Anabaena tuottaa maksamyrkyllisiä mikrokystiini-yhdisteitä. Jokainen eristetty myrkyllinen Anabaena-kanta tuotti useita mikrokystiini-variantteja. Lisäksi mikrokystiinejä löydettiin kukintanäytteistä, joissa oli myrkkyä syntetisoivia geenejä sisältäneitä Anabaena-syanobakteereita. Myrkkyjä löydettiin molempina tutkimusvuosina 2003 ja 2004. Myrkkyjen esiintyminen ei siten ollut vain yksittäinen ilmiö. Tässä työssä saimme viitteitä siitä, että maksamyrkyllinen Anabaena-syanobakteeri esiintyisi vähäsuolaisissa vesissä. Tämä riippuvuussuhde jää kuitenkin tulevien tutkimuksien selvitettäväksi. Tässä työssä havaittiin mikrokystiinisyntetaasi-geenien inaktivoituminen Itämeren Anabaena-kannassa ja kukintanäytteissä. Kuvasimme Anabaena-kannan mikrokystiinisyntetaasigeenien sisältä insertioita, jotka hyvin todennäköisesti inaktivoivat myrkyntuoton. Insertion sisältäneeltä kannalta löysimme kuitenkin kaikki mikrokystiinisyntetaasigeenit osoittaen, että geenien olemassaolo ei välttämättä varmista kannan mikrokystiinintuottoa. Mielenkiintoista oli se, että inaktivaation aiheuttavia insertioita löytyi kukintanäytteistä molemmilta tutkimusvuosilta. Vastaavia insertioita ei kuitenkaan löydetty makean veden Anabaena-kannoista tai järvinäytteistä. On yleistä, että syanobakteerikukinnoista löytyy usean syanobakteerisuvun edustajia. Myrkyllisiä sukuja tai lajeja ei voida kuitenkaan erottaa mikroskooppisesti myrkyttömistä. Käsillä olevassa tutkimuksessa kehitettiin molekyylimenetelmä, jolla on mahdollista määrittää kukinnan mahdollisesti maksamyrkylliset syanobakteerisuvut. Tätä menetelmää sovellettiin Itämeren kukintojen tutkimiseen. Itämeren pintavesistä ja ranta-alueiden pohjasta eristetyt Anabaena-kannat osoittautuivat geneettisesti monimuotoisiksi. Tämä Anabaena-syanobakteerien geneettinen monimuotoisuus vahvistettiin monistamalla geenejä suoraan kukintanäytteistä ilman kantojen eristystä. Makeiden vesien ja Itämeren Anabaena-kannat ovat geneettisesti hyvin samankaltaisia. Geneettisissä vertailuissa kävi kuitenkin ilmi, että pohjassa elävien Anabaena-kantojen geneettinen monimuotoisuus oli suurempaa kuin pintavesistä eristettyjen kantojen. Itämeren Anabaena-kantojen sekvenssit muodostivat omia ryhmiä sukupuun sisällä, jolloin on mahdollista, että nämä edustavat Itämeren omia Anabaena-ekotyyppejä. Tämä tutkimus oli ensimmäinen, jossa uusin molekyylimenetelmin systemaattisesti selvitettiin Itämeren Anabaena-syanobakteerin geneettistä populaatiorakennetta, fylogeniaa ja myrkyntuottoa. Tulevaisuudessa monitorointitutkimuksissa on otettava huomioon myös Itämeren Anabaena-syanobakteerin mahdollinen maksamyrkyntuotto – erityisesti vähäsuolaisemmilla rannikkovesillä.