Lääketieteellinen tiedekunta

 

Recent Submissions

  • Riihijärvi, Sari (Helsingin yliopisto, 2016)
    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in the western world. It is an aggressive disease and rapidly fatal if left untreated. With immunochemotherapy, a combination of chemotherapy and CD20 antibody rituximab, 70% of the patients can be cured. DLBCL comprises several distinct molecular subgroups, which differ in the expression of specific gene signatures and in the oncogenic pathways involved, corresponding to discrete prognostic categories. Research on biological prognostic factors and targeted therapies is active and it is likely that in the near future the patients will be treated according to risk-adapted and biomarker driven therapies. The aim of this study was to identify novel prognostic factors in the immunochemotherapy era. The study population comprised tumor tissue, blood samples and clinical data of DLBCL patients treated with immunochemotherapy. Methods included immunohistochemistry, ELISA and gene expression microarrays. PKCβII is an enzyme involved in cellular proliferation and it is overexpressed in cancer, including DLBCL. We observed that the patients with high tumor PKCβII expression had poorer survival than the patients with low expression. Consistently, high PKCβII gene expression correlated with inferior survival in an independent patient set. PKCβII is also known for its ability to stimulate tumor angiogenesis. We found no association between tumor microvessel density and survival data. Vascular endothelial growth factor (VEGF) is a signalling protein which stimulates angiogenesis. It is overexpressed in cancer and essential in tumor growth and development. We observed that high pretreatment serum VEGF levels are associated with inferior survival after therapy. However, VEGF mRNA levels correlated neither with the survival nor the serum VEGF levels. Thus, it is possible that the serum VEGF is not derived from the tumor itself but may be a host response instead. Tumor associated macrophages (TAMs) reprogrammed by malignant cells are a major source of angiogenic factors. Also, mechanism of rituximab is partly mediated by macrophages. The function of TAMs is known to be controversial: they have both anti-tumor and pro-tumor features. In our study, tumor macrophage counts correlated with favorable outcome. The findings were validated in independent cohorts of immunochemotherapy-treated patients. In contrast, in patients treated with chemotherapy, high macrophage count was associated with inferior survival. We also measured serum levels of CCL18 and CD163, proteins commonly expressed in M2 type macrophages. A significant association with inferior survival and low CCL18 serum levels was observed. To summarize, high PKCβII expression, low tumor TAM content, high serum VEGF and low serum CCL18 levels serve as prognostic factors for inferior outcome in immunochemotherapy-treated DLBCL. Their use as prognostic biomarkers in the clinical setting, however, calls for further study. The results underline the complex biology of DLBCL and the interplay between the tumor microenvironment, host responses and the malignant cells.
  • Mäki-Nevala, Satu (Helsingin yliopisto, 2016)
    Lung cancer is a common cancer with a poor prognosis. Prognostic and predictive molecular markers for lung cancer have become available and along those treatment options have multiplied. Treatments can be targeted to aberrant molecules driving the tumorigenesis. Thus, clarification of molecular characteristics of a tumor is important for the optimized therapy. Further development in this field would be aided by the identification of (novel) significant markers and understanding how their incidence is associated with clinical characteristics. The aim of this thesis work was to examine known and novel molecular markers, more specifically mutations in selected genes which could be potential molecular markers of cancer, and to link findings with clinical data in Finnish lung cancer patients. Mutations were studied in genes encoding ephrin receptors (Ephs), EGFR, and in 22 other lung cancer related genes. In addition, coding regions of the genome, i.e. exons, were studied for mutations potentially associated with asbestos-exposure. The study material consisted of more than 600 patients, their tumor specimens and clinical data. The majority of the specimens were formalin-fixed, paraffin-embedded non-small cell lung cancer (NSCLC) and malignant mesothelioma (MM) samples. Most of the specimens were subjected to next generation sequencing (NGS); the suitability of this technology in cancer diagnostics was also assessed. Mutations in Ephs were common; 18 % of the patients carried one or more novel mutation. In MM, in particular EPHB1 was found to be mutated. The mutations did not associate with any particular clinical characteristic and they were found often concurrently with known pathogenic driver mutations, which points to a probable passenger mutation nature for these mutations. However, when considering their diverse role in cellular function, as well as their oncogenic and tumor suppressive properties, therapeutically they represent a very intriguing group of molecules. Clinically significant EGFR mutations were found in 11 % of tumors from NSCLC patients. The mutations were associated with adenocarcinoma histology, female gender and never-smoking status, as has been reported in previous studies. The incidence of EGFR mutations resembled that described in previous studies conducted on other Western patients. Similarly, screening of mutational hot spot regions in 22 genes revealed the mutation profile to be rather similar to that described in Western NSCLC patients with some exceptions, such as the higher BRAF mutation and lower STK11 mutation frequency. TP53 and KRAS as the most frequently mutated genes, being mutated in 46 % and 26 % of the NSCLC patients, respectively. In particular, TP53 mutations were found to co-occur recurrently with other mutations, also with pathogenic EGFR and KRAS mutations. Of the 425 patients, 77 % carried one or more mutations. Statistically significant associations were found between the following mutated genes and clinical characteristics: TP53 and PIK3CA and squamous cell carcinoma, KRAS and adenocarcinoma, and CTNNB1 and light ex-smoking status. In the study comparing asbestos-exposed and non-exposed lung cancer, eight candidate genes (BAP1, COPG1, INPP4A, MBD1, SDK1, SEMA5B, TTLL6 and XAB2) were found to be recurrently mutated exclusively in the asbestos-exposed patients. Mutations in BAP1 and COPG1 were found exclusively in MM. However, the well-established lung cancer-related, pathogenic clinically relevant mutations, such as EGFR and KRAS, do not associate with asbestos-exposure. Finally, the application of NGS technology proved to be very suitable for cancer diagnostics. One major advantage of this technology is the possibility to test for different alterations in multiple genes simultaneously as well as the ability to detect and characterize both known and novel alterations.
  • Lehto, Elviira (Helsingin yliopisto, 2016)
    Most children consume less fruit and vegetables (FV) than recommended for a healthy diet. FV intake is especially low among boys and children from lower socioeconomic backgrounds. The aims of the study were to clarify which factors could explain socioeconomic and gender differences in children s FV intake and liking for FV. Furthermore, it was of interest to find out how the implementation level of an intervention affects children s FV intake and which factors mediate this association. The study sample consisted of 11-year-old children, who participated in the PRO GREENS project in 2009 and 2010. The cross-sectional data used in Study I was derived from ten European countries (n=8159). Finnish longitudinal data from children in control schools (n=424) was used in Studies II and III and from children in intervention and control schools (n=727) in Study IV and. Children reported their FV intake frequency and several environmental and psychosocial factors. Parents stated their highest level of education and teachers documented the implementation level of the intervention. The results indicate that I) in most studied countries, children with higher socioeconomic background were more likely to eat FV daily. The most important mediators were higher availability of FV at home and greater knowledge of the FV recommendation among children from higher socioeconomic backgrounds. II) Descriptive norms about FV intake among friends were as important as parental descriptive norms in predicting children s FV intake among both girls and boys. III) Girls higher liking for vegetables and greater variety in preferences was partly explained by girls higher previous vegetable intake and fewer perceived barriers. IV) Higher implementation level of the intervention increased children s fruit intake via increasing children s knowledge and liking for fruits. Lower implementation levels were less effective, but increased children s fruit intake via increasing the frequency children brought fruits to school for snacking. In conclusion, schoolchildren s FV intake is intertwined with liking for FV and affected by both parents and friends. Interventions should be implemented at the higher level and target all above-mentioned factors. Plentiful availability of FV and their consumption in a supportive environment should be ascertained, especially for boys and children from lower socioeconomic backgrounds.
  • Kuuskeri, Jaana (Helsingin yliopisto, 2016)
    The wood-decaying white-rot fungi have the profound ability to completely degrade lignocelluloses and all wood components. These fungi and their enzymes have evolved to modify the various lignocellulose feedstocks in nature, and thereby, they are important organisms for bioconversions as well as in fundamental research on fungal biology. The enzymes have many potential applications in biotechnology and industrial purposes including bioenergy production. Evolutionary background of the fungal species and their organelles thus requires deeper understanding to aid in elucidating the relationship of the species to their lifestyles. This PhD study concentrated on the white-rot fungal species Phlebia radiata, Finnish isolate number 79 (FBCC0043). The phylogenetic studies confirmed positioning of P. radiata species in the systematic class Agaricomycetes of Basidiomycota, and in the phlebioid clade of the order Polyporales. The sequenced and annotated mitochondrial genome of P. radiata was discovered to have features that indicate evolutionary pressure and structural diversity in fungal mitogenomes, not being as stable and compact entities than was previously believed. In this study, P. radiata together with species like Phlebia acerina and Phlebia brevispora was demonstrated to form a Phlebia sensu stricto group which consists of efficient producers of lignin-modifying enzymes. The results pinpointed that there is a species-level connection of fungal molecular systematics to the efficiency in the production of wood-decaying enzymes and activities. Norway spruce (Picea abies) is a common tree species in the boreal forests providing an important source of biomass for forest-based industry. Therefore, P. radiata was cultivated on Norway spruce wood under conditions mimicking natural solid-wood colonization, up to six weeks of growth, and the dynamics of fungal enzyme production and gene expression was studied. The lignin-modifying class-II peroxidases (LiPs and various MnPs) were produced, especially in the beginning of fungal growth and colonization of wood, thus indicating the essence of class-II peroxidase as the primary enzymes to function against coniferous wood lignin. Moreover, these extracellular oxidoreductases enhance the accessibility of lignocellulose carbohydrates and thereby, they promote fungal growth in wood. Simultaneously, lytic polysaccharide monooxygenases and several CAZyme glycoside hydrolases attacking cellulose, hemicellulose and pectin were produced, which demonstrates ongoing depolymerization of the polysaccharides to monomers and oligomers. Electron microscopic examination of fungal-colonized wood after six weeks of growth indicated that the decay of wood cell walls was initiated at the tracheid lumen side apparently proceeding towards the middle lamellae. Furthermore, degradation of spruce wood lignin was detected by pyrolysis-GC/MS as decrease in the amount of phenylpropane units with concomitant increase in the number of smaller fragmented products from these lignin units. Thus, the previously observed unique and strong ability of P. radiata to degrade wood lignin and lignin-like aromatic compounds was confirmed. According to the results of this PhD study, P. radiata produces the white-rot type of decay of wood components when growing on Norway spruce. This is due to the efficient ability of the fungus to express and produce a versatile enzyme repertoire for degradation of wood lignocellulose, and in consequence, to generate diverse reactions and bioconversions important for carbon cycling in the forest ecosystems.
  • Viitasalo, Katriina (Helsingin yliopisto, 2016)
    Epidemiologic evidence supports an association between shift work and increased risk of cardiovascular disease. Circadian misalignment, shortened and disturbed sleep, and alterations of lifestyle aspects are the main factors related to shift workers health problems. Shift work is carried out by 17% of workers across the EU. In a globally operating airline, irregular working hours and time-zone flights are challenging to workers coping strategies and their health, especially as they age. The main purpose of this study was to evaluate the feasibility and effectiveness of screening and prevention of cardiovascular risk factors and type 2 diabetes among the workers of a Finnish airline. Two kinds of interventions were tested; changes in shift systems and lifestyle counselling. We found that metabolic syndrome, a clustering of risk factors for cardiovascular disease, was more prevalent among former male shift workers than workers who had never worked shifts. In shift rotation, a faster speed together with a change from the backward to forward direction alleviated daytime sleepiness and older workers had less sleep complaints in a quickly forward rotating shift system than their younger colleagues. In addition, lower systolic blood pressure and a declining trend for heart rate in a flexible shift system indicated a decrease in psychophysiological stress. Health check-ups effectively identified employees with increased risk of type 2 diabetes and cardiovascular diseases. The FINDRISC questionnaire proved to be a practicable first-step screening method. Low intensity lifestyle intervention was feasible in an occupational health care setting. However, only modest health benefits were observed among the men with increased risk. The study revealed that a number of factors are associated with participation in the lifestyle interventions. Employees with higher baseline FINDRISC score and clinical and lifestyle risk factors were eager to take part in lifestyle counselling by a dietician or a diabetes nurse. Also problems in sleep and mood increased attendance in the interventions. From research to practice: former shift workers increased risk for metabolic syndrome should be recognized in occupational health care and taken into account in preventive work. In the design of new shift systems, a faster speed, together with the forward rotation of the shift system enhances older workers well-being and coping strategies for shift work. In addition, combining individual elements with company-based working-time flexibility may have favourable effects on shift workers cardiovascular health. Low intensity lifestyle interventions targeted at risk individuals may be profitable. It is important to identify the work and individual related characteristics affecting intervention project attendance to develop more effective methods for the preventive work of occupational health care.
  • Koskenvuo, Laura (Helsingin yliopisto, 2016)
    Familial adenomatous polyposis (FAP) is an autosomal dominant inherited syndrome, which is characterized by the development of hundreds or thousands of polyps in the colon and rectum. The first representative of the family (proband) is usually found because he/she presents with the symptoms that usually arise from multiple polyps or from cancer in the large intestine. After this diagnosis family members of that proband are called for screening. The prevention of otherwise inevitable colorectal cancer by prophylactic surgery should preferably be performed in early adulthood. The main surgical options are colectomy with ileorectal anastomosis and proctocolectomy with an ileal pouch-anal anastomosis or ileostomy. The screening of FAP has been shown to be effective in terms of diminishing the number of deaths from colorectal cancer, but the reduction in overall mortality remains unclear. Patients with FAP also carry an elevated risk of desmoid tumours, which are histologically benign proliferations of myofibroblasts, but are often difficult to treat. Desmoid tumours of FAP patients may also act more aggressively than their sporadic counterparts. The aims of this PhD study were to analyse the short-term and long-term outcomes of the two different surgical procedures: colectomy with ileorectal anastomosis (IRA) and proctocolectomy with ileal pouch-anal anastomosis (IPAA). Further analysis was done on the need and the results of secondary proctectomies after IRA. The authors aimed to determine, whether familial screening reduces the overall mortality. The causes of death among Finnish FAP patients were studied. The risk of FAP among desmoid tumour patients was also studied. The disease outcome of patients with FAP-related tumours was compared with that of sporadic desmoid tumours in the Finnish population. Patient files of all 421 Finnish FAP patients archived since the year 1963 were studied. There were a total of 228 patients who had undergone IRA or IPAA between years 1963-2012. During the same period, 39 secondary proctectomies were performed for IRA patients. All the Finnish FAP patients until April 30th 2015 were included in the study for which the effect of screening was evaluated. Patients with a diagnosis of sporadic desmoid tumours between years 2000-2012 in Helsinki University Hospital district were invited to the FAP screening. They were offered both endoscopic screening and gene mutation testing. All 221 desmoid tumour patients from the year 1980 were included into the comparison of treatment between FAP associated and sporadic desmoid tumour. There were no significant differences in short term complications between IRA and IPAA. In the long run, however, more patients in the IRA group ended up with ileostomy than in the IPAA group. The total cumulative survival was better after IPAA than IRA, but if the analysis only took into account IRA performed after the IPAA era (from the year 1992 onwards) there were no significant difference between the groups. Secondary proctectomy was performed on 28% of IRA patients. The cumulative risk for secondary proctectomy at 30 years was 53%. The majority of operations were performed for cancer or suspicion of cancer. The risk of rectal cancer after IRA was 13% and the risk of rectal cancer death was 7%. The crude mortality ratio of probands was 34.9 per 1000 person years and 8.3 among call-ups. The relative survival of probands was significantly lower than for their call-up counterparts, and 20 year relative survival for the call-ups was as high as 94%. Over two-thirds of all deaths were FAP related. Among sporadic desmoid tumour patients the prevalence of FAP was 4.8%. FAP diagnosis of these patients was evident by endoscopy. No cases of AFAP, which could sometimes be detectable only by gene mutation testing, were found. There were more intra-abdominal desmoids in the FAP desmoid tumour group, and the desmoid tumours were bigger and more often multiple than those in the sporadic desmoid tumour group. Majority of sporadic desmoid tumour patients were women, whereas among the FAP-related desmoid tumour population the gender distribution was equal and the FAP related desmoid tumour patients were younger. The treatment of FAP-related desmoids was more difficult, intralesional resections were more common and there are desmoid-related deaths (14% of all deaths) among FAP patients in contrast to sporadic desmoids. Patients who underwent IPAA did not have more postoperative complications than patients with IRA. Substantial risk of rectal cancer remains after colectomy and IRA, so the IPAA procedure should be favored for the FAP patients with intermediate or severe polyposis. The risk of permanent stoma is also higher when proctectomy was performed in the second phase. The survival of probands is significantly lower than that of the general population whereas that of call-ups was comparable to the general population for up to 20 years after diagnosis. This is why the screening effort for the family members of the proband must be done. Desmoid tumour patients carry an elevated risk of FAP and therefore screening is usually indicated. Only asymptomatic patients with desmoid tumours situated in the extra truncal region may not need to be routinely screened. Desmoid tumours among FAP patients carry a more complex course of disease compared to patients with a sporadic desmoids, and thus the treatment of FAP-related desmoids is also more complex. If R0 resection is not achieved, the wait-and-see strategy might be a better choice than resection with involved margins.
  • Hafrén, Lena (Helsingin yliopisto, 2016)
    Introduction: Otitis media (OM), inflammation or infection of the middle ear, is a very common childhood disease with a significant burden for the child, the family and society. Most children have sporadic acute otitis media (AOM), but some develop recurrent AOM (RAOM). In chronic otitis media with effusion (COME) the middle ear effusion is prolonged without signs of acute infection, creating hearing impairment. Both genetic and environmental factors affect the risk for OM. Inherited genetic factors have been shown in several twin- and family studies to be strong risk factors for OM. Earlier genetic studies on OM have suggested variants in several candidate genes as risk factors for OM, but the results have not always been replicated in other populations. Hence, very little is known about the underlying genes that contribute to the genetic risk of this common childhood disease. Having better understanding of the genetic of OM would greatly contribute to our knowledge of the disease pathogenesis, leading to improvements in diagnosis, prevention, and treatment. Study Subjects: Patients were recruited to the study at the Helsinki University Hospital’s (HUH) Department of Otorhinolaryngology, Head & Neck Surgery. Patients’ siblings and parents were also included as study subjects. Patient data was collected by questionnaires and DNA was extracted from blood samples. A previous OM cohort with 205 Finnish study subjects was used as a replication cohort and was also included in the genome wide association (GWA) study. Two OM cohorts from the US (Pennsylvania (403 families) and Oregon (N = 204)) and one from the UK (1269 trios) served as international replication cohorts. Methods: Heritability was estimated from the HUH cohort with the Solar software package. A candidate gene study was performed 53 markers from 35 different genes on the Sequenom platform. The loci around the initial significant association (pcorr < 0.05) was followed up by a tagging gene approach for 20 markers. The downstream cytokine Tumor necrosis factor alpha (TNFα) of Toll-like receptor 4 (TLR4) was used for functional studies. A genome wide association (GWA) study was performed on Finnish study patients and, serving as controls randomly selected subjects from the Finnish Health 2000 study. Verification studies and replication studies were done on other platforms. Results: Heritability in the Finnish cohort was estimated to 39% for RAOM, 22% for COME, and 48% for any OM. One marker (rs5030717) in the TLR4 gene was significantly associated to OM (OR 1.33, p = 0.003) in the initial candidate gene study on 624 patients and 778 controls. In the tagging gene approach three markers, in strong linkage disequilibrium, were associated with OM. The association was stronger in children with more severe phenotypes, a first AOM episode before the age of 6 months or those requiring multiple insertions of tympanostomy tubes. The association was verified in the Finnish replication cohort for the three variants, but not in the international cohorts. TNFα expression in myeloid dendritic cells was lower in the study subjects with the risk genotype compared to those with the alternative genotype, whereas TNFα mRNA expression was higher in those with the risk genotype compared to those with the alternative genotype. Assessing more than 300,000 genetic markers in 803 cases and 2073 controls revealed a locus on chromosome 19 associated with OM and one marker, rs16974263, proved to be genome-wide significant (p = 2.92 x 10-8) in 509 study subjects with COME. When genotyping the most significant variants in the UK cohort of 4860 study subjects, an association was revealed for the opposite allele. Conclusions: Genetic predisposition strongly influences the risk of childhood OM in the Finnish population. Analysis of candidate genes disclosed that genetic variants of the TLR4 gene might influence this predisposition to OM. Children with the previously unrecognized risk haplotype in the TLR4 locus have a higher risk for RAOM, especially early onset RAOM. GWA study analysis revealed a novel risk locus on chromosome 19q, which is highly associated with COME in the Finnish population, but due to lack of replication of the same allele in a UK population, it is unclear what, if any, function the risk locus has on the pathogenesis of OM. Further genetic work on OM in different populations must be carried out to investigate the complex pathogenesis of the different phenotypes of OM and eventually improve diagnostics and treatment.
  • Hepojoki, Satu (Helsingin yliopisto, 2016)
    Novel molecular tools for infectious disease diagnostics are constantly under development to reduce the time between onset of symptoms and diagnosis. Not only is it important to receive appropriate treatment, but also to avoid unnecessary use of antibiotics. The availability of rapid diagnostics is also important when epidemics or pandemics emerge. The purpose of this project was to examine the applicability of Förster resonance energy transfer (FRET) in homogeneous immunoassays, and to develop new diagnostic approaches. FRET has widely been applied in proximity-based assays, such as those measuring antigen-antibody binding. In FRET, energy is transferred between two chromophores, the donor and the acceptor, when in close proximity. Utilizing FRET as detection method for immunoassays enables the development of wash-free (homogeneous) simple workflow assays. In this thesis, of the three FRET-based rapid immunoassays that were set up, two served in clinical diagnosis. Study I (I) examined the possibility of detecting antibodies by FRET-pair forming fluorophore-labeled antigens, which upon binding to an antibody would induce a FRET signal. This homogeneous immunoassay, designated FRET-bridge, was successfully optimized for streptavidin (SA). By combining donor-labeled and acceptor-labeled SAs with anti-SA antibodies, FRET signals were recorded with high signal-to-noise ratios. When molecular determinants behind the FRET signals were examined, most of the FRET activity originated from fairly large immunocomplexes rather than from one IgG and two antigens. At the moment, SA represents the only antigen fully functioning in the FRET-bridge assay. This is most likely due to the multivalent nature of the antigen, which seems beneficial in FRET signal formation. Next, we introduced another homogeneous immunoassay (II), the LFRET assay. Here, a FRET pair forming fluorophore-labeled antigen and fluorophore-labeled protein L induce a signal if bound to the Fab (fragment antigen-binding) region of an immunoglobulin. To demonstrate the usefulness of the assay, SA served as test antigen. The assay was next optimized for virus diagnostics by use of Puumala virus (PUUV) nucleocapsid protein as antigen (III). In all, 211 serum samples underwent examination by the LFRET assay, representing acute (n=61) or past PUUV infection (n=27), and seronegative (n=123) individuals. With a simple workflow and an assay time of 30 minutes, the LFRET assay, compared to the reference tests, identified acute PUUV infection at 100% specificity and 95% sensitivity. The fourth study (IV) involved a competitive homogeneous immunoassay for the detection of PUUV antibodies from clinical samples. This assay, CFRET, is based on competition between fluorophore-labeled monoclonal antibodies (MAbs) and serum antibodies. Here, a donor-labeled antigen and an acceptor-labeled MAb form the FRET pair. If the clinical sample contains antibodies against the labeled antigen, they compete with the MAb for antigen binding, resulting in FRET signal decrease. Analysis of assay performance included a panel of 329 samples representing acute (n=101) or past (n=42) infection, and negative samples (n=186). The one-step CFRET assay performed at 99% specificity and 100% sensitivity in diagnosis of hantavirus disease compared to the reference tests, and with a rapid assay time of 30 minutes. The three rapid diagnostic approaches introduced herein represent simplicity, and show that diagnostics need not be time-consuming. Although the assays were optimized for accurate diagnosis of acute infection, both assays also recognized life-long immunity, albeit with lower sensitivity. By optimization, the assays could be developed towards more sensitive detection of past infection as well. The LFRET and CFRET assays thus represent innovative tools for rapid antibody detection, and their potential in serodiagnosis of diverse microbial infections and possibly even in detection of auto- and anti-allergen antibodies calls for further exploration.  
  • Raissadati, Alireza (Helsingin yliopisto, 2016)
    Pediatric heart surgery aims to correct or palliate cardiac malformations, and to maximize the quality of life of the patients. This thesis investigates the late outcome after surgery for congenital cardiac defects to assess the quality and progress of treatment during the last decades. It also studies outcomes after the arterial switch operation (ASO) for treatment of transposition of the great arteries (TGA) and pediatric heart transplantation. Data was obtained from a national database containing all pediatric heart operations performed since 1953 in one of five university- and one regional hospital in Finland. Causes of death were obtained from Statistics Finland and categorized into congenital heart defect (CHD)- related and non-CHD-related causes of death. CHD-related deaths were further divided into heart failure-, post-reoperative early-, cardiovascular-, and sudden deaths. Between 1953 and 2009, 13,786 cardiac operations were performed on 10,964 pediatric patients in Finland. Follow-up coverage was 98%. Early operative mortality was 5.6% for all operations. The 60-year survival for the entire study was 70% versus 86% for the general population. The number and proportion of severe cardiac defects increased in the 2000s, whereas surgery for simple defects decreased. Operative age and early operative mortality decreased significantly among all defect groups. Long-term survival of all patients improved significantly during recent decades. The most common cause of death was heart failure, which decreased among the majority of defect groups. Sudden death was reduced to zero among patients with simple defects and TGA, but remained a challenge among patients with univentricular heart configurations. ASO resulted in markedly improved early operative mortality and late outcome after correction of TGA when compared to the Mustard and Senning operations, with zero sudden deaths. Early mortality after pediatric heart transplantation was 10%, with a late outcome of 68% at 10 years after the transplantation. In conclusion, both early and long-term outcome after surgery for CHD s has improved. The reason is multifaceted, including advances in diagnostic tools, perioperative care, intensive care, and surgical techniques, allowing earlier treatment of increasingly severe defects. Sudden deaths and heart failure have markedly diminished, but remain a risk factor among patients with severe defects. These results underscore the importance of long-term follow-up after surgery for severe defects.
  • Stegajev, Vasili (Helsingin yliopisto, 2016)
    Sjögren's syndrome (SS) is a chronic autoimmune disease with yet unknown etiology and partly unclear pathogenesis that affects exocrine glands. Cardinal symptoms of the disease are dry mouth and eyes. Fas-apoptosis is considered as a key event in breach of autoimmune tolerance in SS. Increased levels of histamine in serum and tissue fluids characterize many autoimmune diseases, including SS. However, trials have showed ineffectiveness of H1 and H2 histamine receptor antagonists in the treatment of autoimmune diseases. More recently, it has been reported that many cells throughout the human body synthesize histamine in small nanomolar quantities in a non-professional manner. Unlike professional histamine-synthesizing, -storing and stimulus-mediated burst-like -releasing cells, such as mast cells, basophils and ECL-cells, many other cells, including dendritic- and T cells can synthesize histamine at 100-1000-fold lower rate and release it continuously into the extracellular milieu. In the beginning this discovery drew little attention until a new H4 receptor (H4R) was discovered in immune cells in 1994. H4R has about 10000-fold higher affinity to histamine than conventional H1R and H2R. This discovery shortly became a hot topic and started a new era for studies of immunomodulatory histamine/H4R-mediated effects. Today clinical trials of H4R antagonist as treatment for inflammatory diseases are ongoing. It was hypothesized that H4R is involved in SS pathogenesis. To test this hypothesis the following objectives were studied: (1) expression of H4R in salivary glands of healthy and SS individuals; (2) H4R internalization and functionality upon specific agonist stimulation; (3) transport of histamine in HSG cells and expression of histamine metabolizing enzymes in salivary glands of healthy and SS individuals; (4) mechanisms of anti-apoptotic activity of H4R; (5) mRNA levels of moH4R in orchidectomized and intact male mice. Briefly, this study was performed on snap-frozen and/or paraffin-embedded biopsies of minor salivary glands from SS patients, which were routinely taken as a part of the diagnostic procedure. Samples were considered as healthy if diagnostic criteria were not fulfilled. In vitro experiments were performed on two cell lines: human salivary gland (HSG) line, which is phenotypically a ductal epithelial line, and normal salivary Simian virus 40-immortalized acinar cell (NS-SV-AC) line. Additionally, we were gifted by snap-frozen murine salivary glands from BALB/c, NOD, orhydectomized and intact HDC+/HDC+ lines. Protein expression levels of histamine receptors, transporters and metabolizing enzymes in tissue samples and cell lines were tested by immunohistochemistry and immunofluorescence staining, mRNA were quantified by qRT-PCR technique (including TaqMan technology). Two specific H4R agonists ST-1006 and HST-10 were used for in vitro functional (HSG), internalization and apoptosis (NS-SV-AC) cell line experiments. Levels of IL-8 and VEGF produced by HSG cells ± ST-1006 were analyzed by xMAP and ELISA assays. [3H]histamine transport in HSG cells ± OCT3 inhibitor MMP was analyzed by liquid scintillation technique. hrTNFα/IMD0354-induced apoptosis of NS-SV-AC cells ± HST-10 was analyzed with following techniques: Western blot to study late apoptosis marker cPARP, anti- and pro-apoptotic proteins BAX and Bcl-XL and phosphorylation of JNK and ERK MAPKs; flow cytometry to study Annexin-V and PI labeling of apoptotic and necrotic cells; phase-contrast microscopy to study morphological changes of apoptotic cells; additionally, BAX and Bcl-XL mRNA levels were studied by qRT-PCR. H4R was found in the acinar and ductal epithelium in healthy salivary glands on both mRNA and protein levels. Immunohistochemical staining showed relatively low H4R expression in samples from SS patients as compared to those from healthy controls. Healthy ductal salivary epithelium is fully equipped with a histamine synthesizing, transporting and intracellular degrading machinery. SS salivary glands were characterized by drastically diminished expression of major histamine transporter OCT3 at both protein and mRNA levels. In vitro experiments showed time dependent up-regulated production of IL-8 and VEGF by HSG cell upon high-dose stimulation of H4R with ST-1006 agonist. NS-SV-AC cells express H4R, which internalization was delayed by clathrin inhibitor methyl-β-dextrin. qRT-PCR and [3H]histamine transport experiments proposed OCT3 as the major histamine transporter in HSG cells. hrTNFα/IMD0354-induced apoptosis of NS-SV-AC cells was successfully inhibited by HST-10-induced activation of H4R in a dose-dependent manner via inhibition of JNK MAPK pathways and up-regulation of Bcl-XL anti-apoptotic protein. mRNA levels of salivary moH4R relatively low in orhydectomized HDC+/HDC+ as compared to intact control. Salivary ductal epithelial cells are equipped with a HDC/OCT3/HNMT-machinery therefore considering them as non-professional histamine-producing cells. H4R-mediated locally produced low nanomolar levels of histamine maintain homeostasis of the salivary epithelium. H4R activation favors cell survival. Altered histamine transport together with decreased expression of H4R in salivary epithelium can contribute to SS pathogenesis by predisposing epithelial cells to an apoptotic pre-condition. Mast-cell-derived high concentrations of histamine in SS salivary glands may excessively stimulate H4R, leading to an up-regulated production of pro-inflammatory IL-8 and VEGF, followed by a down-regulation of H4R. Diminished expression of H4R in salivary epithelium in SS patients may also be aggravated by local/systemic androgen levels. In future, local use of low molecular weight H4R agonists may become an alternative for costly biologicals in the treatment of autoimmune diseases, including Sjögren´s syndrome.
  • Viiliäinen, Johanna (Helsingin yliopisto, 2016)
    Kaposi's sarcoma herpesvirus (KSHV) is a human gamma2-herpesvirus and the causative agent of three human cancers: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV establishes persistent life-long infection and manipulates host cell proliferation and apoptosis, which leads to the development of cancer in immunocompromised individuals. KSHV has two phases of life cycle, the latency phase and lytic replication phase. Latency is a silent mode of infection, while in the lytic replication phase new virus progeny are produced. Although the latent phase is the default mode of infection, the lytic cycle has an instrumental role in the emergence and progression of KS. KSHV encodes several microRNAs (miRNAs) that have a potential role in viral tumorigenesis. However, the targets of these miRNAs are still poorly known. In the first study we show that KSHV-encoded miRNAs inhibit apoptosis by silencing the expression of caspase 3, an apoptotic effector, in latent KSHV-infected cells. In the second study the aim is to discover new specific anti-viral drugs by targeting host cell anti-apoptotic mechanisms utilized by KHSV. To this end, Bcl-2 homology 3 domain (BH3) mimetic drugs are tested which bind and inhibit the anti-apoptotic function of the B-cell lymphoma 2 (Bcl-2) family of proteins in KSHV-infected cells. This study shows that the myeloid cell leukemia-1 (Mcl-1) inhibitor, Obatoclax, induces cell death in KSHV-infected cells undergoing lytic phase. The cellular mechanisms that regulate the triggering of the lytic cycle (reactivation) are not completely understood. To discover new regulators of KSHV reactivation and lytic cycle activation, a targeted RNAi screen was performed using a library of siRNA oligos targeting human epigenetic factors and their regulators. The screen identifies Mouse double minute-2 (MDM2), the main regulator of p53 protein stability, as a negative regulator of KSHV reactivation. Further studies demonstrate that efficient KSHV reactivation is favored by a p53 stress response and by the induction of p21Cip-1-mediated G2/M cell cycle arrest. KSHV encoded cyclin D homolog viral cyclin (v-cyclin) is able to overcome G1 cell cycle arrest and dysregulate mitosis of KSHV infected cells. V-cyclin operates by interacting with cellular proteins and consequently to study the role of v-cyclin in KSHV induced cancers, comprehensive interactome of v-cyclin and cellular proteins was studied in tetracycline inducible isogenic v-cyclin expressing human cell line. In summary, research in this thesis identifies a new cellular target of KSHV-encoded miRNAs and uncovers a cellular anti-apoptotic mechanism utilized by this virus to protect cells undergoing lytic replication from premature death. Inhibition of this mechanism enabled the identification of a potential new drug candidate against KSHV-associated cancers. Additionally, this study provides new insights into how KSHV can cope with and utilize cellular stress response to ensure efficient lytic replication and revealed 8 novel v-cyclin interacting cellular proteins.
  • Kinnunen-Amoroso, Maritta (Helsingin yliopisto, 2016)
    Work-related stress is the main risk factor to employees health. Therefore, organisations, with collaboration from the occupational health service (OHS), must strive to prevent or reduce stress factors in the workplace. Several studies have been conducted about work-related stress factors and their influence on employees psychological and physical well-being at work. Research has also indicated how to deal with work-related stress on both the individual and the organisational level, but information about actions in practice for handling work-related stress between the workplace and OHS is lacking. The employers have the responsibility to assess and to draw up measures to the work-related stress. They can use external resources as OHS to this if needed. The aim of this study was to clarify the collaboration between workplaces and OHS related to work-related stress and the methods to assess and manage stress. Preliminary study questions were generated through semi-structured interviews of 10 volunteer occupational physicians and 8 volunteer occupational nurses in the metropolitan area of Finland in May-June 2009. The interviews were analysed by qualitative methods. Based on this information, a questionnaire was developed for the cross-sectional study. The survey study was realised by email among Finnish occupational nurses and physicians, with 207 physicians and 335 nurses responding. Another self-administered email questionnaire based on the previous study was sent to a sample of enterprises (n=40) in the Finnish metropolitan area in May 2010. The data from these two questionnaires were analysed quantitatively using the SPSS 17.0 statistical programme. Work-related stress was well known to all participants. The OH specialists experienced that work stress was difficult to handle. Neither the OHS nor their client enterprises had standardised tools for assessing or handling work-related stress. Assessments of work-related stress were mostly made at the individual level with open interviews by OH. The management methods mentioned were often random at both the individual and organisational level, although actions remained mainly on the individual level to support the individual and were rarely allocated to the organisation. Collaboration between the workplace and OHS varied by mode of organising these services. All respondents mentioned the lack of administrative support for interventions for work-related stress. Standardised agreed-upon methods for assessing and handling stress at both the individual and organisational level should be developed. The procedures should be consistent across all occupational health service teams and companies to ensure the adoption of appropriate protocols.
  • Putkuri, Niina (Helsingin yliopisto, 2016)
    Inkoo virus (INKV) is a mosquito-borne virus belonging to the California serogroup (genus Orthobunyavirus, family Bunyaviridae) which includes many important human pathogens described especially in the USA. The association of INKV infection with clinical disease has not been confirmed, but occasional cases of meningitis and encephalitis have been diagnosed. However, the true incidence of acute infections is not known because ongoing research and laboratory diagnostics of INKV infections have been neglected for decades in the countries where the virus circulates. We established a serological test (IFA) to detect INKV antibodies, and studied protein-specific antibody responses. Antibody prevalence in humans in Finland and Sweden showed that 40-50% of the population had been infected with INKV or a related California serogroup virus. The seroprevalence was higher in older age groups, and in Finland, the prevalence increased northwards. We found that acute-phase sera had a distinct granular fluorescence pattern in IgG IFA, whereas those with pre-existing immunity showed a diffuse pattern. Using recombinant INKV proteins as antigens, the antibody response was showed predominantly to be against the N protein in early infection and also towards the Gc protein in the later stages of infection. We discovered a new California encephalitis virus isolate of the genus Orthobunyavirus in Finland from mosquitoes collected in 2007 and 2008 from the Ilomantsi and Sotkamo municipalities in Eastern Finland. The new isolates were named the Möhkö isolates of Chatanga virus (CHATV) since the genetic and serological findings suggested that the new virus isolates were most closely related to clusters of Russian orthobunyavirus CHATV isolates (99% N protein identity). Using samples from patients with febrile illness or neurological symptoms collected in the summertime between 2001 and 2013, we studied the frequency of acute INKV infections and the clinical picture of the patients. We found the frequency to be under 1%, but interestingly, not only INKV but also CHATV were confirmed to cause human infections. Most patients were not hospitalized, and they had visited a doctor most often once. The patients suffered from fever, headache, vomiting, disorientation, and seizures. INKV infections were more severe in children under 16 years, and CHATV infections were more severe in adults. In conclusion, we found a new mosquito-borne virus from Finland and showed for the first time that this virus was associated with a clinical disease. In addition, we described the clinical picture of INKV infection and showed that the infection is more severe in children if neurologic symptoms appear. These viruses are common in Finland, and their association with clinical disease in the summertime should not be forgotten.
  • Thomson, Annika (Helsingin yliopisto, 2016)
    Firesetters constitute a heterogeneous group, but some features have been found to be common, including frequent psychiatric (e.g. schizophrenia spectrum disorders) and substance use disorder comorbidities, and impulsivity. This study aimed to explore mortality rates and patterns among firesetters. Psychopathy is common in criminal and forensic settings and is associated with crimes and violence. The study investigated whether firesetters engaging in different types of crime (versatile firesetters) or repeating their fire-setting behavior show high rates of psychopathy. The study also analyzed whether fire-setting among young persons would predict onset of schizophrenia spectrum disorders. The firesetters were a consecutive sample of 441 pretrial men, who underwent a forensic psychiatric examination during 1973-98 in Helsinki University Hospital. The control group consisted of date and place of birth- and gender-matched persons. The follow-up began when the examination was completed and ended when the person died, moved abroad, or at the latest on 31.12.2012. Dates and causes of deaths and information on treatment were obtained from national registers. In one part of the study concerning psychopathy, a subgroup of firesetters comprising 135 men was assessed with PCL-R. In another part of the study, a subgroup consisting of 137 firesetters aged 15-25 years was used. The study assessed how many had onset of schizophrenia spectrum disorder during follow-up. Nearly half of firesetters and a fifth of control subjects had died at the end of follow-up. The firesetters died significantly younger, at an average age of 53.2 years, than the controls, whose average age at death was 61.6 years. Alcohol-related deaths were more frequent among firesetters and they were 8 times more likely to end their lives by suicide compared to control subjects. Suicide attempts leading to hospital treatment were nearly 13 times more common among firesetters than among controls. The versatile firesetters scored significantly higher on the PCL-R total and factor scores than the exclusive firesetters. A schizophrenia spectrum disorder was diagnosed significantly more often among firesetters than control subjects. Fire-setting behavior was associated with a high mortality rate. Alcohol use contributed substantially to suicide attempts, completed suicides, and deaths overall. Antisocial pathways may be the motivator for versatile firesetters, showing significant traits of psychopathy. Fire-setting among youths should be taken seriously, as these individuals are prone to schizophrenia spectrum disorders. Firesetters constitute a high-risk suicidal behavior group, which must be addressed when planning treatment and release.
  • Tervaniemi, Mari (2016)
    Psoriasis is a common skin disorder that is characterized by thickening of the most superficial layer of the skin, the epidermis, and accumulation of white blood cells, inflammation. The exact mechanism of how psoriasis develops is still unknown. Several gene expression studies have been conducted on psoriatic skin. Most of them, however, have focused on the expression in both the epidermis and dermis or were analyzed by microarrays. Here we used a novel approach to decipher the gene expression profile of the psoriatic skin, by utilizing a more specific and sensitive detection of transcripts by RNA sequencing (RNA-seq), implemented with an improved normalization method, and combined with samples that contain mainly the skin layer of interest: the epidermis. RNA-seq revealed more accurate expression profiles in different sample types that had varying amount of total mRNA per cell. Comparison with previous transcriptomics studies on psoriasis revealed that our approach provided more information about the transcriptional dysregulation in the epidermis. The expression profiling of epidermis highlighted the involvement of innate immunity and provided, for example, deeper understanding about the components of NOD-like receptor signaling pathway and inflammasome activation in keratinocytes. Some of the components have been associated with psoriasis in previous studies, yet the exact composition and activation mechanisms of inflammasomes have remained unclear. Our RNA-seq findings thus strengthen the role of keratinocytes as modulators of inflammation in the psoriatic lesions. The improved methods and focused analysis might help to pinpoint the most important pathways and functions, including broader knowledge in the involved components, in the psoriatic lesions. This, in turn, might improve the production of more specific treatments for psoriasis. The psoriasis candidate gene CCHCR1 is located in the major psoriasis predisposition locus PSORS1, contains a psoriasis-associated risk allele *WWCC, and its gene product is expressed by the basal keratinocytes of the epidermis and has been shown to have an effect on cell proliferation and differentiation. The gene has two different transcription start sites and is able to encode for a peptide with a longer N-terminus from the transcript 1, which, however, depends upon a SNP that encodes for either tryptophan or stop codon, therefore either enabling or disabling the production of the longer protein. Here we presented association of the stop codon-encoding SNP (named as *Iso3) with psoriasis in family trios. We detected that CCHCR1 localizes at the centrosome and P-bodies, and reported isoform-specific effects on the localization of the P-bodies. Our experiments exhibited haplotype-specific effects of CCHCR1 also on cytoskeletal organization and cell proliferation; functions relevant to the pathogenesis of psoriasis. Furthermore, our results suggest that CCHCR1 might function in EGFR-STAT3 signaling and innate immunity, which strengthens the role of innate immunity in psoriasis even further. In addition, RNA-seq revealed isoform- and haplotype-specific effects on the expression profiles of different CCHCR1 cell lines. Interestingly, the most dramatic changes in gene expressions were observed in the isoform 3 -overexpressing cells but also the Non-risk and Risk haplotypes had antagonistic effects. The observation that CCHCR1 influences multiple cell signaling pathways may result from its possible role as a centrosomal P-body protein, which suggests a role in post-transcriptional regulation as well as a role in the regulation of cell cycle. Its exact function in these cellular compartments and effect in psoriatic lesions remains to be studied further.