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  • Lizarazo Torres, Clara Isabel (2010)
    Drought is one of the most important abiotic stresses that causes significant reductions in crop yield, and thus hinders the food security of the growing world population. In consequence, it is urgent to select crops able to resist drought, maintain high yield and have a good nutritive content. The purpose of this project was to evaluate the responses of different accessions of Andean lupin to drought stress, and identify if there are significant differences in their physiological responses. To identify germplasm for further investigation, thirty accessions of Lupinus mutabilis Sweet. and one accession of L. albococcineus Hort. were screened in two sets, A of 15 and B of 16 accessions. From these sets, four lines were chosen on the basis of extreme values in the measurements, and this set of four was investigated in depth (set C). In all experiments, ten seeds of each chosen accession were sown in pots and grown in a glasshouse with 22°C, 18 h days and 18°C, 6 h nights, and were well watered until day 50 after planting. The pots were arranged in a randomized complete block design. The eight most uniform plants were chosen, and four were exposed to water stress while watering of the other four was continued. Water stress consisted of controlled water loss from the soil, 20% water holding capacity at 2% per day over 9 days, so the soil reached 2% moisture content and was held at this level for 2 days more. In all experiments, leaf temperature, stomatal conductance, relative water content, water potential, ion membrane leakage, and shoot dry weight were measured and transpiration efficiency was calculated. In set C, carbon isotope discrimination, root length, root dry matter, proline content and soluble sugar content were also determined. The analysis of set A and B revealed significant differences between treatments for all the parameters measured, except for relative water content, and there were also differences amongst accessions in certain parameters. PI 457972 and PI 457981 were selected for further investigation because of their low stomatal conductance under water stress conditions and low water use, and PI 510572 was selected as sensitive to drought stress due to its high water use, ion membrane leakage and water potential under water stress conditions. In addition AC 2792 (L. albococcineus) was selected due to its low stomatal conductance and water use, and high leaf temperature under water stress conditions. In the final experiment, PI 457981 and PI 457972 appeared to avoid drought through appropriate stomatal characteristics. PI 457981 showed low stomatal conductance, high leaf temperature and also high root length, similarly, accession PI 457972 showed low water potential, low stomatal conductance, low carbon isotope discrimination and accumulation of soluble sugars. Accession PI 510572 contrasted for these stomatal traits, but interestingly it showed low membrane ion leakage, high proline content and soluble sugars content, suggesting that it was capable of drought tolerance by osmotic adjustment. Finally, accession AC2792 showed low water use, low water potential and low carbon isotope discrimination. This survey thus identified accessions of Andean lupin that were able to avoid drought stress through stomatal traits and root traits, and other that were able to tolerate drought through the accumulation of osmotically active substances. Thus, there are good prospects for breeding of Andean lupin to improve its drought resistance.
  • Pietarinen, Paavo (2012)
    Most xenobiotics are biotransformed by phase I enzymes to a more hydrophilic form in order to get excreted out from the body. In most cases xenobiotics are in lipophilic form when entering body. The most important group in phase I enzymes is cytochrome P450 (CYP) superfamily. Of CYP enzymes probably the most studied is CYP2D6, which is responsible for metabolism of 20-25% of drugs currently on market. Many CYP2D6 substrates belong to therapeutically important drug groups, such as antiarrhytmics, antidepressants, beta-blockers, or neuroleptics. CYP2D6 gene, which encodes the enzyme, exhibits large interindividual variability, which has an effect on the metabolic activity of the enzyme. The frequencies of these genetic variances differ globally on wide scale between and inside populations. Through genotyping it is possible to predict the CYP2D6 metabolic rate, which can be divided into four classes: ultra-rapid metabolizers (UM), extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). The purpose of our study was to examine the frequencies of CYP2D6 genotypes in Finnish population in detail and compare the results to previous studies. Our study population consisted of 857 healthy volunteers whose DNA was extracted. From DNA sample we genotyped 10 different CYP2D6 genetic variants and the copy number of the gene using Applied Biosystems TaqMan genotyping and copy number assays. This study was the largest CYP2D6 genotype frequency study in Finnish population so far. The results supported the findings of a similar study in a Finnish population of smaller scale. Large majority of study subjects were EMs (87.3%) and the second largest group was Ums (7.2%). IMs and PMs were in clear minority (3.0% and 2.5%, respectively). The expected frequencies for UMs (1-2%) are much lower and for PMs higher (~8%) in other North European populations than in Finns. Accordingly, CYP2D6 genetic profile of Finnish population differs from its neighbours, which may be important for the dose requirements, efficacy, and safety for drugs metabolized by CYP2D6.
  • Lahtinen, Annukka (Helsingin yliopisto, 2012)
    Arrhythmogenic right ventricular cardiomyopathy (ARVC) and long QT syndrome (LQTS) are inherited cardiac arrhythmia disorders that predispose to sudden cardiac death (SCD). In ARVC, structural and electrical abnormalities of the heart occur together with progressive replacement of the right ventricular myocardium by adipose and fibrous tissue. Mutations in desmosomal cell adhesion genes may cause ARVC. LQTS manifests with a prolonged electrocardiographic QT interval in a structurally normal heart. LQTS is caused by mutations in cardiac ion channel genes, delaying the repolarization of the ventricular myocardium. The aims of this study were to identify genetic variants predisposing to ARVC, LQTS, and SCD and to assess their prevalence and clinical significance in the Finnish population. Six (18%) of 33 ARVC probands were identified to carry mutations in desmosomal genes. Desmosomal mutations occurred together with disorganization of the intercalated disk structure and demonstrated reduced disease penetrance. The carrier frequency of the desmosomal mutations identified in this study was 1:250 as estimated in a sample of 27 670 Finns. Therefore, an unexpectedly large number of individuals could be at risk of developing ARVC in Finland. However, another trigger is likely to be needed for disease expression. In a clinical sample of 712 LQTS founder mutation carriers, KCNE1 D85N was associated with a 26-ms prolongation of QT interval in males with KCNQ1 G589D, representing thus a potential sex-specific disease-modifying factor. In a population sample of 6808 Finns, KCNE1 D85N together with 13 other genetic variants explained 8.6% of the variation in QT interval, and a 10-ms prolongation of QT interval was associated with a 19% increased risk of SCD. This information could ultimately contribute to assessment of individual susceptibility to LQTS. When studied in a total of 28 323 individuals, two novel common variants, rs41312391 in SCN5A and rs2200733 in 4q25 near PITX2, were associated with risk of SCD. In addition, the associations for rs2383207 in 9p21 and for clinical risk factors for coronary heart disease were replicated. Rare mutations in desmosomal and ion channel genes had a combined carrier frequency of 1:130 in the Finnish population and were detected in individual SCD victims. These results provide novel information for SCD risk prediction and prevention.
  • Lappalainen, Maarit (Helsingin yliopisto, 2008)
    Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.
  • Saarinen, Anne (Helsingin yliopisto, 2011)
    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.
  • Mäkinen, Ulla-Maija (1984)
  • Jaakola, Minna (1994)
  • Gustafsson, K. R. (1913)
  • Badeau, Maija (Helsingin yliopisto, 2008)
    Soy-derived phytoestrogen genistein and 17β-estradiol (E2), the principal endogenous estrogen in women, are also potent antioxidants protecting LDL and HDL lipoproteins against oxidation. This protection is enhanced by esterification with fatty acids, resulting in lipophilic molecules that accumulate in lipoproteins or fatty tissues. The aims were to investigate, whether genistein becomes esterified with fatty acids in human plasma accumulating in lipoproteins, and to develop a method for their quantitation; to study the antioxidant activity of different natural and synthetic estrogens in LDL and HDL; and to determine the E2 esters in visceral and subcutaneous fat in late pregnancy and in pre- and postmenopause. Human plasma was incubated with [3H]genistein and its esters were analyzed from lipoprotein fractions. Time-resolved fluoroimmunoassay (TR-FIA) was used to quantitate genistein esters in monkey plasma after subcutaneous and oral administration. The E2 esters in women s serum and adipose tissue were also quantitated using TR-FIA. The antioxidant activity of estrogen derivatives (n=43) on LDL and HDL was assessed by monitoring the copper induced formation of conjugated dienes. Human plasma was shown to produce lipoprotein-bound genistein fatty acid esters, providing a possible explanation for the previously reported increased oxidation resistance of LDL particles during intake of soybean phytoestrogens. Genistein esters were introduced into blood by subcutaneous administration. The antioxidant effect of estrogens on lipoproteins is highly structure-dependent. LDL and HDL were protected against oxidation by many unesterified, yet lipophilic derivatives. The strongest antioxidants had an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups. E2 ester levels were high during late pregnancy. The median concentration of E2 esters in pregnancy serum was 0.42 nmol/l (n=13) and in pre- (n=8) and postmenopause (n=6) 0.07 and 0.06 nmol/l, respectively. In pregnancy visceral fat the concentration of E2 esters was 4.24 nmol/l and in pre- and postmenopause 0.82 and 0.74 nmol/l. The results from subcutaneous fat were similar. In serum and fat during pregnancy, E2 esters constituted about 0.5 and 10% of the free E2. In non-pregnant women most of the E2 in fat was esterified (the ester/free ratio 150 - 490%). In postmenopause, E2 levels in fat highly exceeded those in serum, the majority being esterified. The pathways for fatty acid esterification of steroid hormones are found in organisms ranging from invertebrates to vertebrates. The evolutionary preservation and relative abundance of E2 esters, especially in fat tissue, suggest a biological function, most likely in providing a readily available source of E2. The body s own estrogen reservoir could be used as a source of E2 by pharmacologically regulating the E2 esterification or hydrolysis.
  • Almusa, Henrikki (2013)
    The next-generation sequencing (NGS) platforms create a large amount of sequence in short amount of time, when compared to first generation sequencers. An overview of the NGS platforms is provided with more in-depth look into Illumina Genome Analyzer II as that is used to create the data for the thesis. There were two main aims in this thesis. First, to create a pipeline which can be used to analyse genomic sequencing. Second, to use the pipeline to compare whole human exome capture methods from two manufacturers, Roche Nimblegen and Agilent. The pipeline is describe in detail in material and methods. All the inputs for the pipeline are described and examples shown. In the pipeline the given sequences are first aligned against the reference genome. Then various separate analysis is performed to retrieve variants and coverage of the sequencing. Supplementary results include paired-end anomalies, larger insertion and deletion polymorphisms and assembly of non-aligned sequences. The two capture methods are also described and changes to the manufacturers' recommended protocols are listed. Finally, the section has the options and various inputs used in the pipeline runs of the exome data. The results of the pipeline is a basic level of analysis of the sequencing as well as various graphs showing the quality of the run. All the output files intended for user are described. By using the results of the pipeline, the user can do more in-depth analysis as required by the project. When comparing the two exome capture methods, the Nimblegen capture was shown to be more efficient in capturing the CCDS exome. While the Agilent capture kit provided better one fold coverage over the exome, higher fold coverage (over 10 fold), which is required for reliable variant calling in nextgeneration sequencing, was better reached using the Nimblegen capture kit. Also, significantly fewer false positive paired-end anomalies were observed in the library created by using the Nimblegen capture.
  • Wang, Hao (Helsingin yliopisto, 2014)
    Natural products are small molecules produced by a range of living organisms. They may be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and anti-fungal agents. Natural products are commonly synthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), such as microcystins. Ribosomal pathways in cyanobacteria are also known for the synthesis of bacteriocins, lantibiotics, cyanobactins and microviridins. Genes encoding biosynthetic enzymes of these systems are often found together and form gene clusters. The filamentous cyanobacterium Anabaena sp. strain 90, a hepatotoxin producer isolated from a bloom of a Finnish lake, was selected for genome sequencing, in order to explore its full capacity of bioactive compound production. The 5.3-Mb Anabaena sp. 90 genome displays a multi-chromosomal composition with five circular replicons: two chromosomes and three plasmids. A total of four non-ribosomal biosynthetic gene clusters, which are responsible for the production of anabaenopeptilides, anabaenopeptins, microcystins and the novel glycolipopeptides hassallidins, were identified in chromosome I. Genome annotation revealed that Anabaena sp. 90 genome also harbors an anacyclamide-encoding cyanobactin gene cluster and seven putative bacteriocin gene clusters, which belong to the ribosomal pathways. These biosynthetic gene clusters amount to a total of ~250 kb, and 5% of the genome. Analysis of the Anabaena sp. 90 genome suggested that cyanobacteria might produce bacteriocins. A thorough genome mining at the phylum level was conducted targeting the discovery of cyanobacterial bacteriocin biosynthetic pathways. The results demonstrated the common presence of bacteriocin gene clusters in cyanobacteria. A total of 145 bacteriocin gene clusters were discovered, the majority of them were previously unknown. Based on their gene organization and domain composition, these gene clusters were classified into seven groups. This classification is supported by the phylogenetic analysis, which also indicates independent evolutionary trajectories of the gene clusters in different groups. By scrutinizing the surrounding regions of these gene clusters, a total of 290 putative precursors were located. They showed diverse structures and very little sequence conservation of the core peptide. To explore the distribution of NRPSs and PKSs, a comprehensive genome-mining study was carried out and demonstrated their widespread occurrence across the three domains of life, with the discovery of 3,339 gene clusters from 991 organisms, by examining a total of 2,699 genomes. The majority of these gene clusters were found in bacteria, in which high correlation between bacterial genome size and the capacity of NRPS and PKS biosynthetic pathways was observed. Currently, PKSs are classified into three types. Type I PKSs and NRPSs are known to share a modular scheme with a multidomain structure. Surprisingly, a large number (8,906) of enzymes encoding a single NRPS or type I PKS functional domain were found. These monodomain enzymes have a similar genetic organization to type II PKSs, which are nonmodular enzymes. The finding of common occurrence of nonmodular NRPSs and type I PKSs substantially differs from the current knowledge. Furthermore, a total of 314 gene clusters comprised mostly of monodomain enzymes were found. In addition, sequence analysis suggested that the evolution of NRPS machineries was a combination of common descent and horizontal gene transfer.
  • Sahu, Biswajyoti (Helsingin yliopisto, 2012)
    The androgen receptor (AR) mediates the effects of the two physiological male sex hormones (androgens) testosterone and 5α-dihydrotestosterone (DHT) that are critical in the development and maintenance of the male sexual characteristics. Androgens and AR signaling play an important role in the growth of prostate gland and in the development and progression of prostate cancer (PCa). AR is a member of the steroid receptor family and is a ligand-inducible transcription factor. Upon binding to male sex steroids, AR acquires a new conformational state, translocates to the nucleus, homodimerizes and binds to regulatory regions called androgen-response elements on target cell chromatin, as well as interacts with coregulatory proteins, sequence-specific transcription factors, and the basal transcription machinery to regulate gene expression (transcription). AR regulates distinct transcriptional programs in androgen-dependent and castration-resistant (androgen-independent) prostate cancer (CRPC). Gene transcription is a well-orchestrated process of multiple events involving chromatin modifiers, coregulators and collaborating transcription factors, occurring in an ordered and sequential fashion. This strictly regulated process is tightly controlled at every stage to govern cell growth and homeostasis. Details of the ways by which AR regulates gene expression in a genome-wide fashion are still not clear. This warrants an in-depth study of the molecular basis of genome-wide AR function in response to cognate ligands under androgen-dependent and CRPC conditions, their recruitment to specific genomic sites and the role of other transcription factors, chromatin modifiers and coregulators in modulating AR function. This study aimed at investigating genome-wide AR binding sites in prostate cancer cells by using ChIP-sequencing technique to understand the androgen-regulated gene expression. Genome-wide AR binding sites (ARBs) in the presence of physiological androgens and partial agonist/antagonists were profiled and respective downstream target genes were characterized. Particular emphasis was paid to investigate the role of the collaborating transcription factor FoxA1 in mediating recruitment of AR to the chromatin and in androgen signaling, and prognostic value of FoxA1 expression in PCa. Furthermore, we explored the role of FoxA1 in defining cell type-specific recruitment of AR in two different PCa cell lines, and its influence on defining site-specific occupancy of two steroid receptors, namely AR and glucocorticoid receptor (GR), in PCa cells. Finally, the consequences of AR overexpression and its repercussions on genomic recruitment of AR to chromatin were investigated. Our work showed that nuclear receptors, such as AR and GR, employ distal modes of transcriptional regulation, as majority of the binding sites are located far from the transcription start sites. The physiological androgen DHT was more potent in recruitment of AR to the chromatin and mediating subsequent gene expression than partial androgen agonist/antagonists. This demonstrates that agonist-bound AR forms a more favorable conformation compared to partial agonist/antagonists-bound AR, and the resulting binding profiles as well as the transcription programs exhibit quantitative differences. The genome-wide DHT-bound ARBs were significantly over-represented for FoxA1 cis-element, suggesting a global role of FoxA1 in AR function. The identification of genome-wide FoxA1 binding sites correlating with AR binding sites led to a paradigm shift in understanding the role of FoxA1 in androgen signaling. FoxA1 has previously been considered as a pioneering factor with the ability to bind compact chromatin and prime it for nuclear receptor binding. Our findings reveal that FoxA1 has a dual role in regulating AR functions and we defined three distinct classes of ARBs in PCa cells that were commensurate with the androgen-regulated transcription programs. Importantly, FoxA1 levels significantly correlate with prostate cancer-specific disease survival as low FoxA1 levels predict good clinical outcome in primary PCa patients. Further analysis of the ARBs in two different PCa cell lines revealed that FoxA1 defines unique receptor binding sites in each cell line. Moreover, AR and GR binding specificity to chromatin was determined by FoxA1, when analyzed in a single PCa cell line in response to their respective ligands. Furthermore, the results also highlight the role of glucocorticoid as an anti-androgen, which attenuates AR-dependent transcription programs. In AR overexpressing PCa cells, we demonstrated sensitization to low levels of androgens due to enhancement in receptor binding. The association between AR levels and chromatin occupancy was also validated in two PCa xenografts, one with high and another with low AR expression levels.
  • Borze, Ioana (Helsingin yliopisto, 2011)
    Myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders whose etiology and molecular pathogenesis are poorly understood. During the past decade, enormous developments in microarray technology and bioinformatics methods have made it possible to mine novel molecular alterations in a large number of malignancies, including MPN and MDS, which has facilitated the detection of new prognostic, predictive and therapeutic biomarkers for disease stratification. By applying novel microarray techniques, we profiled copy number alterations and microRNA (miRNA) expression changes in bone marrow aspirate and blood samples. In addition, we set up and validated an miRNA expression test for bone marrow core biopsies in order to utilize the large archive material available in many laboratories. We also tested JAK2 mutation status and compare it with the in vitro growth pattern of hematologic progenitors cells. In the study focusing on 100 MPN cases, we detected a Janus kinase 2 (JAK2) mutation in 71 cases. We observed spontaneous erythroid colony growth in all mutation-positive cases in addition to nine mutation negative cases. Interestingly, seven JAK2V167F negative ET cases showed spontaneous megakaryocyte colony formation, one case of which also harbored a myeloproliferative leukemia virus oncogene (MPL) mutation. We studied copy number alterations in 35 MPN and 37 MDS cases by using oligonucleotide-based array comparative hybridization (array CGH). Only one essential thrombocythemia (ET) case presented copy number alterations in chromosomes 1q and 13q. In contrast, MDS cases were characterized by numerous novel cryptic chromosomal aberrations with the most common copy number losses at 5q21.3q33.1 and 7q22.1q33, while the most common copy number gain was trisomy 8. As for the study of the bone marrow core biopsy samples, we showed that even though these samples were embedded in paraffin and underwent decalcification, they were reliable sources of miRNA and suitable for array expression analysis. Further, when studying the miRNA expression profiles of the 19 MDS cases, we found that, compared to controls, two miRNAs (one human Epstein-Barr virus (miR-BART13) miRNA and one human (has-miR-671-5p) miRNA) were downregulated, whereas two other miRNAs (hsa-miR-720 and hsa-miR-21) were upregulated. However, we could find no correlation between copy number alterations and microRNA expression when integrating these two data. This thesis brings to light new information about genomic changes implicated in the development of MPN and MDS, and also underlines the power of applying genome-wide array screening techniques in neoplasias. Rapid advances in molecular techniques and the integration of different genomic data will enable the discovery of the biological contexts of many complex disorders, including myeloid neoplasias.
  • Lakspere, Triin (2014)
    Influenza viruses are a group of pathogens in the family Orthomyoviridae, which are classified into 6 genera (A,B,C, Thogotovirus, Isavirus and new unnamed genus). Type A influenza viruses are categorized based on their surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). So far 17 HA and 9 NA subtypes have been identified. Influenza genome comprises of eight single-stranded negative-sense RNA segments that encode ten to twelve proteins (HA, NA, NP M1/M2, NS1/NS2, PA, PB1 and PB2). Influenza replication cycle depends on the surface proteins binding to host cell receptors, pH mediated fusion and cell-mediated transcription and replication of the viral genome. Virus particles leave the host cell via budding. Influenza viruses cause global epidemic infections each year, the peak is from December to March. These pathogens have also contributed to six global pandemics identified so far. The latest pandemic outbreak was announced by WHO in 2009 which caused over 5000 hospitalizations in Finland. Factors contributing to the severity of clinical outcome can be either genetic, environmental or caused by human host features. This study aims to identify the susceptibility factors for severe influenza A infections and describe the phylodynamics of the latest pandemic A(H1N1)pdm09 from Finnish patient nasopharyngeal aspirates collected between 2009-2013. One-step reverse-transcription PCR was used to amplify all of the 8 segments equally. Fast and precise next-generation sequencing with Illumina 2000 sequencer was used to generate the sequences. Results were bioinformatically analysed using Bayesian modelling with Markov Chain Monte Carlo algorithms of probability distributions. Models analysed showed highest mutation rate in hemagglutinin protein. Phylodynamic analysis revealed higher mutation rate of HA and NA compared to other proteins. Subgroup specific polymorphisms (either in severe or mild cases) were not identified. In total 4657 amino acid substitutions were located in 135 pandemic A(H1N1)pdm09 patient isolates and 238 in 10 seasonal patient samples. Viral HA, NA and PB2 were more frequently mutated than other proteins. Interestingly this study identified double-resistant markers (E119K and S31N) to two antiviral drugs (amantadine and oseltamivir) in one patient isolate (A/Helsinki/598/2013). Previously reported D222 polymorphism (without the signalling peptide)causing more severe clinical outcome was not identified in any of the patient isolates in this study.
  • Myllykangas, Samuel (Helsingin yliopisto, 2008)
    Helicobacter pylori infection is a risk factor for gastric cancer, which is a major health issue worldwide. Gastric cancer has a poor prognosis due to the unnoticeable progression of the disease and surgery is the only available treatment in gastric cancer. Therefore, gastric cancer patients would greatly benefit from identifying biomarker genes that would improve diagnostic and prognostic prediction and provide targets for molecular therapies. DNA copy number amplifications are the hallmarks of cancers in various anatomical locations. Mechanisms of amplification predict that DNA double-strand breaks occur at the margins of the amplified region. The first objective of this thesis was to identify the genes that were differentially expressed in H. pylori infection as well as the transcription factors and signal transduction pathways that were associated with the gene expression changes. The second objective was to identify putative biomarker genes in gastric cancer with correlated expression and copy number, and the last objective was to characterize cancers based on DNA copy number amplifications. DNA microarrays, an in vitro model and real-time polymerase chain reaction were used to measure gene expression changes in H. pylori infected AGS cells. In order to identify the transcription factors and signal transduction pathways that were activated after H. pylori infection, gene expression profiling data from the H. pylori experiments and a bioinformatics approach accompanied by experimental validation were used. Genome-wide expression and copy number microarray analysis of clinical gastric cancer samples and immunohistochemistry on tissue microarray were used to identify putative gastric cancer genes. Data mining and machine learning techniques were applied to study amplifications in a cross-section of cancers. FOS and various stress response genes were regulated by H. pylori infection. H. pylori regulated genes were enriched in the chromosomal regions that are frequently changed in gastric cancer, suggesting that molecular pathways of gastric cancer and premalignant H. pylori infection that induces gastritis are interconnected. 16 transcription factors were identified as being associated with H. pylori infection induced changes in gene expression. NF-κB transcription factor and p50 and p65 subunits were verified using elecrophoretic mobility shift assays. ERBB2 and other genes located in 17q12- q21 were found to be up-regulated in association with copy number amplification in gastric cancer. Cancers with similar cell type and origin clustered together based on the genomic localization of the amplifications. Cancer genes and large genes were co-localized with amplified regions and fragile sites, telomeres, centromeres and light chromosome bands were enriched at the amplification boundaries. H. pylori activated transcription factors and signal transduction pathways function in cellular mechanisms that might be capable of promoting carcinogenesis of the stomach. Intestinal and diffuse type gastric cancers showed distinct molecular genetic profiles. Integration of gene expression and copy number microarray data allowed the identification of genes that might be involved in gastric carcinogenesis and have clinical relevance. Gene amplifications were demonstrated to be non-random genomic instabilities. Cell lineage, properties of precursor stem cells, tissue microenvironment and genomic map localization of specific oncogenes define the site specificity of DNA amplifications, whereas labile genomic features define the structures of amplicons. These conclusions suggest that the definition of genomic changes in cancer is based on the interplay between the cancer cell and the tumor microenvironment.