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Astrosyytit ovat hermoston tukisoluja, joiden toiminnalliset ja morfologiset ominaisuudet vaihtelevat eri aivoalueilla. Astrosyyttien ominaisuuksien vaihtelun on todettu olevan erityisen suurta ihmisen aivoissa. Ihmisen pluripotentit kantasolut (hPS-solut) mahdollistavat astroglian monimuotoisuutta säätelevien mekanismien tutkimisen. Olemme luoneet menetelmän, joka tuottaa hPS-soluista ihmisen etuaivojen astrosyyttejä, ja kuvanneet tuotettujen astrosyyttien erityispiirteitä. Määritimme hPS-soluista erilaistettujen solujen geenien ilmentymisprofiilin päivänä 0 (D0), neuronaalisen induktion jälkeen D12 sekä solujen kasvutekijöillä monistamisen jälkeen D30 ja D60. Astrosyyttien lopullinen määräytyminen toteutettiin siliaarisella neurotrofisella tekijällä (ciliary neurotrophic factor; CNTF) ja D95-ikäisien astrosyyttien osoitettiin ilmentävän lähes 100 prosenttisesti yleisesti käytössä olevia astrosyyttimarkkereita. Erilaistamisen aikana tehty geeniprofilointi vahvisti solujen etuaivojen identiteetin. Kuvasimme solunsisäisen kalsiumkuvantamisen avulla, että erilaistamamme astrosyytit olivat elinkykyisiä ja antoivat toiminnallisia vasteita ATP:lle. Lisäksi määritimme astrosyyttien perustehtävää eli kykyä säädellä immuunivasteita aivoissa tutkimalla niistä erittyvien sytokiinien määriä. Totesimme D95-astrosyyttien viljelynesteessä merkittäviä pitoisuuksia MCP-1- ja TIMP-2-proteiinia yhteneväisesti näitä proteiineja ilmentävien geenien kohonneisiin mRNA-määriin. Astrosyyttien erilaistamismenetelmä oli toistettavissa usealla hPSC-linjalla, ja tutkimuksemme osoitti, että erilaistamamme etuaivojen astrosyytit tarjoavat uudenlaisen keinon sekä astrosyyttien soluspesifisten ominaisuuksien että yhteisviljelmissä muiden hermoston solujen kanssa hermoston solujen yhteisvaikutusten tutkimiseen. Potilaskohtaisista hPS-soluista erilaistettujen astrosyyttien avulla voidaan selvittää ihmisen astrosyyttien toimintaa myös sairaustiloissa.

Virusinduced gene silencing (VIGS) vectors based on tobacco rattle virus (TRV) are now widely used for characterizing the function of plant genes. However, previous TRV vectors using RNA2 to carry the targeted gene sequence had difficulties to induce gene silencing on some plant species (Gerbera hybrida etc.) due to the obstacle of RNA2 movement. To achieve efficient gene silencing in those species, it is necessary to develop new TRV vectors, in which the targeted gene will be included in TRV RNA1 and the 16K gene will be replaced. Based on TRV RNA1, two new VIGS vectors M1 and M2 were developed through deletion part of 16K gene. Another mutant 16Kstop was also constructed to carry an early terminator in the 4th codon of 16K gene. The infectivity and gene silencing efficiency of the new constructs were assessed through a series of infection experiments. It was found that the infectivity of M1 and M2 was lower than wild TRV RNA1. M1 and M2 could induce PDS gene silencing on Nicotiana benthamiana, but their gene silencing efficiency was limited as compared with previous TRV VIGS vectors in which the PDS gene fragment was contained in RNA2. We also found that the 16K gene sequence, rather than the 16K protein, was required for efficient virus movement and accumulation.

Objectives. Motivational contexts exert a profound influence on behavior biasing actions in sometimes detrimental ways. In Pavlovian bias, reward-predicting conditioned cues elicit approach behavior while aversively associated cues elicit withdrawal, with capacity to impact instrumental goal-driven behavior. Similar bias has been suggested to be produced by instrumental learning. Motivational biases have been linked to dopaminergic system but the precise role of dopamine in their modulation is unclear. The present study investigated genetically driven variation in Pavlovian and instrumental learning biases by comparing task performance in subjects carrying different variants of two dopaminergic SNPs, COMT Val108/158Met and DRD2/ANKK1Taq1A. Associations with BMI, diet, age and gender were studied. All subjects were expected to show motivational bias while no direct hypotheses were made concerning genotypic or lifestyle-mediated effects due to exploratory nature of the study. Methods. 160 subjects completed a probabilistic Go/NoGo learning task in an experimental within-subject design. Generalized mixed-model logistic regressions were used to predict differences by genotype in Go responding with and without covariants. Differences by genotype in computationally modelled latent bias estimates were studied with linear regression. Results and Conclusions. Confirming expectations, an overall effect of motivational bias and a general bias towards active responding were found. Relative to Val/Met and A1+, carriers of COMT Val/Val and Taq1A A1- variants showed superior learning of correct Go responses, indicating enhanced instrumental bias. BMI was inversely associated with learning rate while diet, age and gender did not explain variance. Results partly contradict previous findings and highlight the mixed nature of research regarding associations between dopaminergic SNPs and motivational biases.

Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.

Uterine leiomyomas are common benign smooth muscle tumors. They are a major gynecological problem affecting women’s health and contribute to a significant burden on national health expenditure. They can be classified based on their histopathology into conventional, and histopathological variants. Most of the conventional tumors exclusively harbor one of the three drivers (MED12 mutations, HMGA2 overexpression, and biallelic FH inactivation). Based on the genetic background, histopathological subtypes differ from each other and from conventional leiomyomas. Although histopathological variants are considered benign, they share some resemblance to malignant uterine leiomyosarcomas. The overall aim of the thesis was to characterize the mutational landscape of histopathological leiomyoma variants using exome sequencing. The specific aims were, to identify new causative mutations in the histopathological variants and within subtypes, and to analyze pathogenic cancer census gene mutations within the variants. Exome sequencing was performed on 35 tumors representing variant histopathology (14 highly cellular, 12 bizarre nuclei, and 9 mitotically active tumors). The sequences were analyzed using BasePlayer software. Mutations were filtered through the designed pipeline using gnomAD, and COSMIC controls. Interesting findings were validated using Sanger sequencing. Exome data analysis of the highly cellular and bizarre nuclei tumors separately resulted in 10 and 17 different mutations in each subtype, respectively. They were found to be pathogenic by in silico predictions. Analysis of all histopathological variants including mitotically active tumors did not reveal any frequently mutated candidate genes. The tumors harbored somatic mutations in 98 genes related to cancer. A mutation in TP53 was found in one bizarre nuclei sample. Specific tumors harbored multiple cancer-related mutations indicating their malignant potential. The highly cellular tumors had the least frequent amount of causative mutations, indicating that the tumorigenesis mechanisms are probably other than missense mutations or small indels in exomes. Tumors with bizarre nuclei displayed a noticeably larger amount of possible pathogenic mutations, in both cancer census and exome analysis, suggesting possible cancerous tendency. This was also supported by the TP53 finding, a gene associated with uterine leiomyosarcomas. The histopathological subtypes pathogenesis is conceivably caused by larger genomic alterations, epigenetic variations or intronic mutations that remain to be found. Tumors with frequent cancer census mutations might harbor malignant potential. Understanding the etiology of these tumors is needed for better diagnostics and possible targeted treatments

Introduction: Parkinson’s disease is the second most common neurodegenerative disease in the Western countries with a prevalence of about 0.3% in the population. Approximately 5 to 10% of patients are estimated to have a hereditary form of the disease. In recent years, 23 gene loci have been found, in which mutations cause hereditary Parkinson’s disease. In Finland, however, only a few disease linked gene variants have been found so far. Aims and methods: To find out if there are gene variants previously found in Parkinson’s disease patients in the Finnish population, we searched variants found in literature search in a novel genetic database, SISu, which contains genetic data of over 10 000 Finns. In addition, to confirm population findings and search for new gene variants, we analyzed 47 patient cohort with a designed gene panel and also another cohort, containing 147 patients, by minisequencing one variant found in the population data. Results: We found 16 variants in five different genes in the population data. Three of them were considered pathogenic and four likely benign after our analysis. In addition, we found nine potentially disease linked variants in eight different patients. Four of the variants were novel. Discussion: Finns seem to carry only few previously described gene variants in genes linked to Parkinson’s disease. It is likely that Finns carry their own unique variants, some of which we also found in our study. Our analysis brings valuable information about the still scarce knowledge of the genetics of this disease in the Finnish population. In addition, we were able to evaluate the disease risk of many variants further by studying their occurrence in Finns. The study of novel gene variants may bring valuable new information about the pathogenic processes related to the disease; especially the location of a novel variant in PARK2 gene found in our study turned out to be crucial for one of the previously suggested disease mechanisms.

Genetic composition of Theobroma cacao L., including 60 Nicaraguan farmers accessions, was investigated using nine microsatellite (SSR) markers. Fourteen breeders accessions from Experimental Center “El Recreo”, INTA, Nicaragua, two Criollos accession from CATIE, Costa Rica, and two accessions from Ecuador were included as reference material. The average PIC value (0.78) indicated a high power of discrimination for the nine loci used. A total of 155 alleles were detected at the nine loci. The number of alleles per marker ranged from 10 to 22 with a means of 17.22 alleles per locus. A heterozygosity deficiency (HExp < HObs) was registered for all microsatellite loci. The average expected heterozygosity was=0.68 among Nicaraguan farmers accessions. The Analysis of molecular variance (AMOVA) showed a low level of differentiation among populations. The genetic distances determined for the groups of farmers accessions RAAS and Pacifico Sur are closely similar, while a great genetic distance was observed between RAAN and RAAS groups. The cluster analysis presented a strong genetic relation between the Criollo 13 from CATIE and farmers accession MAT0404. The principal component analysis showed that 7 farmers accession from Nicaragua are genetically related with the accessions Criollo 13 and Yucatan from the international accessions. The present study suggested a good possibility to select farmers accessions to be included in breeding programs, especially those accessions related to know Criollo accessions.

Skeletal dysplasias are a group of rare monogenic bone disorders affecting joints and the skeleton. An increasing number of gene defects have been associated with skeletal dysplasias, but many cases remain without a known cause or a clear diagnosis. Exome sequencing data of the family with two siblings affected with an undiagnosed type of bone dysplasia was examined in this study with the aim of determining the genetic cause behind the phenotype. The causal variant was assumed to be in a novel disease-causing gene, since a previously performed gene panel of skeletal disease-causing genes had not revealed any positive results. The search for potential rare pathogenic variants in genes linked to the skeleton was done with VarAFT filtering software. The search revealed a short list of candidate variants confirmed first with Broad Institute’s Integrative Genomics Viewer (IGV) and then with targeted Sanger sequencing. Conservation analysis on the affected amino acids, in silico functional analysis on the variants and a comprehensive literature review on all candidate genes were performed to evaluate the likelihood of them being the variant behind the phenotype. A shortlist of three genes were obtained with the analyses, with one of them seeming to be the most likely candidate. However, to assuredly identify the disease-causing variant, further testing should be performed. Functional analyses should be done to test the functions of the proteins encoded by the candidate genes and the consequences of the pathogenic variants.

Arapaima gigas is one of the world’s largest freshwater fishes and it is native to the Amazon region. The species is over-exploited and sustainable long-term conservation strategies are needed to maintain the genetic diversity of the species. The aim of this study was to analyze the genetic diversity of Peruvian Arapaima gigas populations. The microsatellite data was collected as a part of a three-year project by the Regional Government of San Martín (GORESAM) and Finnish Game and Fisheries Research Institute (FGFRI). The data consisted of 15 microsatellite loci and 324 samples from three populations, Iquitos, Paiche, and Pucallpa. The samples for Iquitos and Pucallpa were collected from populations in the Amazon basin. Samples of Paiche were collected from a captive population in a fish farming research center. The average numbers of alleles and genotypes ranged between 1.9-3.3 and 2.5-4.6, respectively. Population Pucallpa showed the highest average level of heterozygosity (0.41), whereas the lowest level was observed in population Iquitos (0.25). There were altogether 13 loci which showed a statistically significant excess of heterozygosity, and nine loci with significant deficiency of heterozygosity across the three populations. The FIS-values were in accordance with most of the significant deviations indicating the excess or deficiency of heterozygosity. The average FIT-value (0.226) indicated a slight increase of homozygotes. Populations Iquitos and Paiche were on a state of Hardy-Weinberg equilibrium, but population Pucallpa showed a statistically significant deviation from the state of equilibrium. The pairwise FST-values ranged between 0.169-0.373, and they indicate that the three studied populations are genetically different. In addition, the values of Nei’s genetic distance (D) and full-pedigree likelihood analysis indicate a genetic differentiation between the populations. The number of migrants (Nm) between the three populations was estimated based on the mean frequency of private alleles (p(1) = 0.085) and the mean sample size (108 individuals). The number of migrants was 0.273 after the correction for sample size. The genetic diversity within and between the Peruvian populations resembles the results obtained in other studies of Arapaima gigas in the Amazon basin. Sustainable fish farming could offer a solution in maintaining the genetic diversity of the species.

Five wild sea buckthorn populations in Finland were studied using 8 SSR markers to reveal genetic diversity within and among populations. Population samples were collected from four different locations on mainland and one from the Åland Islands. Allelic data obtained from SSR markers were subjected to statistical analysis and AMOVA, and rarefaction was used to correct effects of uneven population sample sizes on some population diversity indices. Results showed moderate to high genetic diversity levels, as mean HE of all populations was 0.55 and HO 0.50. Most populations showed larger observed heterozygosity levels than expected, one showed equal amounts and one population showed lower heterozygosity levels than expected. Most genetic variation of populations was found within individuals across populations (84%) and only some among populations (16%). A weighted neighbor-joining phylogenetic tree showed only some clustering based on populations. The results of this study show, that there is mentionable diversity in Finnish wild sea buckthorn populations, and that populations are only moderately differentiated. This information can be beneficial particularly in sea buckthorn breeding efforts, but also from a possible genetic resources’ and conservation standpoint.