Lääketieteellinen tiedekunta


Recent Submissions

  • Salminen, Aino (Helsingin yliopisto, 2016)
    Matrix metalloproteinases (MMPs) are enzymes that are responsible for the degradation of the extracellular matrix (ECM) during development, repair, and remodeling of tissues. MMP-8, also known as neutrophil collagenase, is released from neutrophils when they enter the tissues at the site of inflammation. An imbalance in MMP-8 activity leads to excess degradation of tissues and destructive inflammation, such as periodontitis. Elevated concentrations of MMP-8 in serum and plasma are also associated with cardiovascular diseases (CVDs). Doxycycline has inhibitory effects against MMPs in addition to its well-known property of being antimicrobial. This thesis investigated the genetics of MMP-8, the effect of MMP-8 and its inhibitors on lipid metabolism, and the potential of salivary MMP-8 in diagnostics of periodontitis. We performed a genome-wide association study (GWAS) in two independent populations with a total of 6049 individuals to identify genetic variants and molecular mechanisms that affect serum MMP-8 concentrations. In addition, we studied whether MMP-8-associated genetic variants are related to increased risk of CVDs in over 20 000 individuals. We discovered that genetic polymorphism in the gene of complement factor H (CFH) is strongly associated with the concentrations of MMP-8 in serum. By conducting functional experiments with isolated neutrophils, we found that genetic variation of CFH affected the release of MMP-8 from neutrophils in response to complement activation. In addition, genetic polymorphism in the locus containing the genes of S100 calcium binding proteins A8, A9, and A12 was associated with serum and plasma MMP-8 levels and also with the prevalence and incidence of CVDs. We studied the effect of MMP-8 and its inhibitors on lipid metabolism by conducting cell experiments, in an MMP-8-knockout mouse model, and in a placebo-controlled clinical trial of two years. We discovered that MMP-8 cleaved apolipoprotein A-I (apoA-I), the main protein component of HDL. MMP-8 significantly reduced the ability of HDL to promote cholesterol efflux from cholesterol-loaded macrophages. The cleavage of apoA-I by MMP-8 and the reduction in its cholesterol efflux capacity was inhibited by doxycycline at clinically attainable doses. MMP-8 deficient mice had significantly lower serum triglyceride levels and larger HDL particle size compared to wild type mice. In the clinical trial, the subjects treated with subantimicrobial-dose doxycycline (SDD) displayed a significant increase in cholesterol efflux from macrophages to the serum compared to the baseline, whereas the efflux levels did not change in the placebo group over the study period. We studied the association of three salivary biomarkers, MMP-8, interleukin-1β, and Porphyromonas gingivalis, with periodontal status in 463 subjects. The salivary concentrations of MMP-8, interleukin-1β, and P. gingivalis were associated with the number of deepened periodontal pockets and the extent of alveolar bone loss. The combination of the biomarkers was more strongly associated with moderate to severe periodontitis than any of the biomarkers alone. Our results indicate that activation of the complement system, especially the alternative pathway, contributes significantly to the concentrations of MMP-8 in serum. Genetic polymorphism in S100A8/A9/A12 locus affects circulating MMP-8 levels, and is associated with CVDs. These results emphasize the role of inflammation and the immune system in CVDs. Proteolysis of apoA-I by MMP-8 may disturb HDL metabolism and reverse cholesterol transport, which leads to accumulation of cholesterol in the vessel walls and accelerated atherosclerosis. Inhibition of MMP-8 by doxycycline may reduce the risk of CVDs, especially in vulnerable individuals such as periodontitis patients. Saliva MMP-8, particularly when combined with other biomarkers, has great potential in the diagnostics of periodontitis. MMP-8 in saliva reflects the health of the oral cavity, whereas circulating MMP-8 is associated with systemic diseases such as CVDs. Our results suggest that MMP-8 functions as a link between inflammatory disorders, such as periodontitis, and cardiovascular disorders.
  • Castrén, Eeva Helena (Helsingin yliopisto, 2016)
    Background and aims: Vascular anomalies constitute a challenging patient group. Their pathogenesis and risk factors are understood only in part. Their management has dramatically developed during the last 20 years. The objective of this study was to examine the mode of inheritance of and the risk factors for infantile haemangioma (IH), the most common vascular anomaly, and to analyse the treatment complications of sclerotherapy, the current first-line treatment for venous malformations (VM). Patients and methods: For the IH studies, we included all IH patients who had visited Helsinki University Hospital s vascular anomaly clinic in 2004-2007. We collected data from hospital records on IH characteristics, complications, and interventions, and the child s perinatal data when available. We sent these patients and their caregivers a questionnaire on perinatal data, child s diseases, family history of IH, and the current subjective long-term discomfort due to IH. Perinatal and gestational data was compared to the Finnish Medical Birth Register data from the same catchment area in 2004-2007. Families reporting positive family history of IH were interviewed by phone to elucidate the pedigrees and inheritance patterns. For studies on sclerotherapy for VMs, we included all VM patients who had received sclerotherapy in our unit between 2007-2013. We recorded retrospectively VM characteristics, procedural data, and sclerotherapy complications, and analysed factors predisposing to complications. We graded complications from I to V according to the Clavien-Dindo classification for complications. Results: In addition to known IH risk factors, this study showed that preterm birth promotes ulceration of IH. Maternal gestational diabetes mellitus rate was significantly higher in our IH cohort than in the catchment area of our hospital district. One-third of IH patients reported a positive family history of IH. The inheritance pattern followed the autosomal dominant pattern, but in ten families, the transmission may also have been maternal. IH characteristics were similar in familial and sporadic cases. Of the head and neck venous malformations, 17% suffered from sclerotherapy complications. These patients needed overall more sclerotherapies, but also more surgery after sclerotherapy, and longer follow-up than those without a complication. Complication rate per procedure was 10%. Three treatment complications were severe, grade IIIb-IV, and necessitated surgical management and intensive post-complication care. Of the trunk and extremity VMs undergoing sclerotherapy, 24% suffered from complications. Complication rate per procedure was 13%. The use of ethanol and subcutaneous location predisposed to local complications. Four severe, grade IV to V complications occurred, one of which was lethal. Severe complications were related to blood coagulopathy. Conclusions: Our results imply that the role of maternal gestational diabetes as a potential IH risk factor is unclear and deserves further studies. Preterm IH infants higher risk for ulceration is a relevant concern for physicians treating IH-children. In addition to autosomal dominant inheritance, a relevant proportion of the familial IHs may be maternally transmitted. Complications of sclerotherapy for VMs mostly recover with conservative treatment, but severe complications do occur. Associated blood coagulopathy constitutes a major risk for the treatment. Interdisciplinary assessment of the treatment strategy is therefore crucial.
  • Mehine, Miika (Helsingin yliopisto, 2016)
    Uterine leiomyomas, often called fibroids, are highly common tumors arising from smooth muscle cells of the myometrium. Whereas cancers have the ability to metastasize, leiomyomas are benign tumors that grow only locally. Nevertheless, leiomyomas frequently cause a variety of health complications, including abdominal pain, abnormal menstrual bleeding, and impaired fertility. Leiomyomas are the leading indication for hysterectomy worldwide, and pose a significant socio-economic impact. Despite their major public health impact, this disease attracts relatively little research. Epidemiological and molecular studies have indicated that, in the etiology of leiomyomas, genetic factors play a central role. Early cytogenetic studies revealed that approximately half of all leiomyomas display non-random chromosomal abnormalities such as high mobility group AT-hook 2 (HMGA2) gene translocations. Furthermore, family-based linkage studies revealed that germline mutations in the fumarate hydratase (FH) gene result in high penetrance susceptibility to uterine leiomyomas. Sporadic leiomyomas, however, rarely harbor FH mutations and the majority lack chromosomal abnormalities, suggesting that some driver genes remain undiscovered. Recent advances in sequencing technologies have made it possible to examine tumor genomes on a previously unprecedented scale. The aim of this thesis was to characterize the molecular underpinnings of uterine leiomyomas by the use of genome-wide methods such as massively parallel sequencing technology and gene expression microarrays. Using exome sequencing, we discovered that 71% of leiomyomas display localized mutations in the mediator complex subunit 12 (MED12) gene, making it their most commonly mutated gene. Furthermore, with whole-genome sequencing, we discovered that a subset of leiomyomas display highly complex chromosomal rearrangements, ones previously undetectable by conventional cytogenetic techniques. These rearrangements closely resembled chromothripsis, a phenomenon in which one or a few chromosomes are shattered into multiple pieces and randomly stitched together in a single event. We also found these events to have occurred multiple times, and some had resulted in genetic changes with a selective value, such as collagen type IV alpha 5 chain and collagen type IV alpha 6 chain (COL4A5-COL4A6) deletions. Patients affected by leiomyomas frequently harbor multiple distinct tumor nodules. Whereas the majority of studies have proposed that each leiomyoma arises independently, we found some leiomyomas to display identical chromosomal abnormalities, suggesting a common clonal origin. Whole-genome sequencing of clonally related leiomyomas revealed intratumor genetic heterogeneity suggestive of a branching model of tumor growth. Furthermore, we also discovered DEP domain containing 5 (DEPDC5) as a novel tumor suppressor gene, acting as a secondary driver gene in a subset of leiomyomas. Our integrative analyses demonstrated that specific genetic defects were the major determinants of expression changes in leiomyomas. Our observations indicate that at least four molecular subtypes exist: leiomyomas harboring a MED12 hotspot mutation, HMGA2 overexpression, FH inactivation, or COL4A5-COL4A6 deletion. We also detected subtype-specific expression differences in key tumorigenic pathways, including Wnt/β-catenin, Prolactin, IGF-1, and NRF2 signaling. Using genome-wide methods in this thesis work, we have discovered several novel molecular defects that underlie leiomyoma etiology. These studies emphasize the importance of stratification in leiomyoma research and offer a set of candidate biomarkers that may facilitate the molecular classification of uterine leiomyomas. Millions of women suffer from uterine leiomyomas, and the ability to classify each lesion should pave the way towards personalized treatments.
  • Poutiainen, Hannele (Helsingin yliopisto, 2016)
    The universal services and regular, extensive health check-ups at child health clinics and school health services provide an opportunity to reach the entire age cohort. This enables the assessment of risk factors affecting children, adolescents and families, the identification of potential need for support, and the targeted provision of assistance and support at an early stage. The purpose of the study was to chart the concerns that arise in public health nurses during health check-ups of children and adolescents, as well as the to identify connections between the concerns and the family's socioeconomic factors, health, and health habits. Since the aim was to obtain a comprehensive and extensive view of the phenomenon under study, i.e., the concerns of public health nurses, a combination of quantitative and qualitative approaches was chosen for the study. The quantitative data for the study consisted of the Child Health Monitoring Study (LATE) data (n=6,506) from children's health check-ups, collected between 2007 and 2009. Information regarding the well-being of children nurses and families was collected using questionnaires aimed at guardians as well as a separate questionnaire for students in grades 8 and 9. (n=9,58). The qualitative data were gathered in 2011 and in 2013 with open group interviews. The public health nurses (n=17) participating in the interviews had taken part in the Child Health Monitoring Study (LATE) pilot study from 2007 to 2008. The public health nurses were concerned about the child's and/or family's health and well-being during one in four child health check-ups (25 %). The nurses were especially concerned about the health and development of children from families with health, social or financial problems. Changes in family structure, such as divorce, single parenthood, and reconstituted families, as well as the effects of these situations on the health and psychosocial development of children and adolescents, especially boys in lower comprehensive school, were topics that were frequently raised by the public health nurses. The nurses' concerns were also connected to the possible effects of the father s low education level, the poor employment situation of the parents and insufficient income on children's health and well-being. The nurses voiced their concern over the increase in the number of families under child protection supervision. Obesity in children and parents, smoking in parents and adolescents, and children s excessive screen time were connected to public health nurses' concerns about the physical health and psycho¬social development of children and adolescents. Smoking in single mothers was particularly linked to public health nurses' concerns about the psychosocial development of girls. The public health nurses were concerned about the increasing number of school absences and the fact that parents accept the absences too easily as being due to illness. Online bullying among lower comprehensive school children was also a topic of concern for the public health nurses. Annual, regular health check-ups, as stipulated by the Decree on Child Health Services and School Health Care provide a good opportunity for public health nurses to recognise problems and the need for support related to the different life situations of children, adolescents, and families.
  • Aho, Vilma (Helsingin yliopisto, 2016)
    Sleep is an essential physiological function. It is conserved across the animal kingdom; no animal species studied has been shown not to sleep. The timing of sleep and wakefulness is regulated by two processes. The circadian process works like a clock and accounts for the timing of sleep and arousal, synchronised by the light dark rhythm. The homeostatic process acts like an hourglass balancing the amount of sleep vs wakefulness. In case waking is prolonged, the homeostatic sleep need drives more sleep to take place, i.e. the sleep rebound. The homeostatic aspect of sleep can be studied by restricting sleep in laboratory conditions and assessing the effects on e.g. cognitive functions and physiological processes. Sleep is not merely a function of the brain, occurring in the brain and for the brain. The brain acts in concert with other organs and tissues in physiological and pathophysiological processes. During the recent decades, epidemiological studies have suggested that there is a connection between short or insufficient sleep with higher mortality. Increased risk for cardiovascular diseases, atherosclerosis, type II diabetes, and obesity has been reported in individuals who sleep less than the average. Laboratory studies have partly supported these findings, suggesting a causative role of sleep loss in the development of metabolic diseases, particularly type II diabetes. Experimental sleep restriction has been shown to alter glucose metabolism towards insulin resistance. Studies on the effects of sleep loss on lipid metabolism have been more inconclusive. Sleep is tightly interconnected with the immune system. Experimental sleep restriction increases proinflammatory cytokines, which in turn promote sleep. Atherosclerosis is the pathophysiological process underlying ischaemic heat disease and stroke, the two leading causes of death worldwide. The immune system plays a major role in the development of this metabolic disease characterised by plaque-formation in the arterial walls. Altered cholesterol transport by low density and high density lipoproteins (LDL and HDL) triggers an immune response involving macrophages and other white blood cells. Chronic low-grade inflammation has been shown to in turn predict future cardiovascular diseases. Thus, the development of atherosclerosis is a complex process with both metabolic and immunological components. In the current thesis, I have investigated the effects of sleep loss on gene expression and metabolites in the blood, focusing on changes that may participate in the development of cardiovascular diseases, especially atherosclerosis. Short-term sleep loss was studied in carefully controlled laboratory conditions with an experimental protocol simulating a working week with restricted sleep (4 h sleep/night for 5 nights; N=21). Sleep loss occurring chronically in real-life conditions was assessed in two Finnish epidemiological cohorts. Subjective sleep insufficiency (SSI) was estimated using questionnaire information on sleep, and a prevalence of 16-18% was found in these samples (FINRISK2007/DILGOM; N=518; SSI 16%, and Young Finns Study, YFS; N=2221; SSI 18%). In both the experimental and epidemiological samples, whole-genome expression profiles were assessed with RNA microarrays and serum lipoprotein profiles with NMR metabolomics. Immune response-related gene pathways were enriched among transcripts with higher expression in experimental sleep loss. Pathways involved in reverse cholesterol transport (RCT) were down-regulated in both experimental and epidemiological sleep loss. Concentration of large high density lipoprotein (HDL) particles was lower in subjects with SSI, even though in experimental sleep loss the low density lipoproteins (LDL) decreased. Up-regulation of low-grade inflammation-related pathways, and down-regulation of RCT-related pathways with decreased serum large HDL in chronic sleep loss may participate in the development of cardiovascular diseases, such as atherosclerosis. Experimental and epidemiological sleep studies in human volunteers can complement each other, but still yield information mostly from blood samples. Other methods are needed to elucidate mechanisms involving e.g. the liver. As sleep is a complex phenomenon involving synchronised activity of neuronal networks and integration with other systems and organs, it is not feasible to be studied in vitro, i.e. in cultured cells. Thus, animal models are needed to further study the effects of sleep loss at the level of molecular mechanisms. Zebrafish is a small diurnal vertebrate whose genome has been sequenced. It has a short generation time, readily available genetic tools, and it is well suited for in vivo imaging studies thanks to its transparent larval stage. Sleep or sleep-like states have been reported in this species using behavioural criteria. According to these studies, adult and larval zebrafish exhibit behavioural quiescence periods with circadian timing and increased arousal threshold. However, the homeostatic sleep rebound after prolonged wakefulness has not been unquestionably proven in this species. To confirm sleep homeostasis and validate this model for further studies on the effects of sleep loss, I developed a method for naturally prolonging the waking activity of zebrafish larvae. After 6 hours of this water flow protocol applied during the night, the larvae showed less responses to sensory stimuli than control larvae. Thus, I suggest that zebrafish larvae do have sleep homeostasis and they can be a useful model to study the sleep loss-related mechanisms involved in disease development.
  • Viinamäki, Jenni (Helsingin yliopisto, 2016)
    In Finland, 800-900 deaths are caused by fatal poisonings due to drugs or alcohols annually. Of these, around ten are caused by toxic alcohols, mainly methanol and ethylene glycol, and 400-500 are caused by drugs, both medicinal and illicit. Despite the fact that the majority of fatal poisonings are caused by relatively few individual compounds, toxicological laboratories must be capable of identifying and quantifying a wide range of possible toxicants. Small sample volumes and post-mortem changes impose special demands on both the laboratory investigation and the interpretation of results. The two main objectives of the present thesis are, first, to develop new analytical laboratory methods for toxic alcohols and drugs and, second, to apply these methods to post-mortem toxicology cases in order to generate reference data on lethal concentrations of especially metabolites for interpretation purposes. Metabolites of toxic substances constitute an interpretive resource that is far from fully exploited in post-mortem toxicology. The therapeutic, toxic, and lethal concentrations of alcohols and drugs are usually expressed as the concentration of the parent compound in blood. Yet the toxicity of methanol and ethylene glycol, for example, is mainly caused by their toxic metabolites and many therapeutic drugs are transformed to metabolites that possess pharmacological activity similar to or greater than that of the parent drug. A method for the quantitative analysis of ethylene glycol, glycolic acid, and formic acid and for the screening of volatile and hydroxylic organic compounds was developed, using headspace in-tube extraction gas chromatography-mass spectrometry. In fatal poisonings by methanol or ethylene glycol, the concentration of the parent alcohol varied significantly, whereas the concentrations of formic acid and glycolic acid in blood were found to be more uniform. Fatal metabolite threshold concentrations were established for those poisoning cases that had not received hospital treatment. In cases involving putrefaction, significant post-mortem production of formic acid was detected, and consequently the concentrations of metabolites should always be interpreted together with those of the parent alcohols. A comprehensive method for the quantitative monitoring of over 150 basic drugs and metabolites in blood samples was developed utilizing ultra-high performance liquid chromatography coupled with two consecutive detectors: diode array detector and corona charged aerosol detector (UHPLC-DAD-CAD). Based on the universal response of CAD, the method was evaluated for the quantification of drug metabolites using a secondary calibration method with the corresponding parent drug as a calibration standard. This approach offers a straightforward way of quantifying especially N-demethylated metabolites. It was found that the metabolite to parent ratios of certain toxicologically relevant drugs in post-mortem blood samples were generally comparable to the standard ratios defined in clinical therapeutic drug monitoring. With the highest post-mortem parent drug concentrations, the ratios were below the clinical normal ranges, suggesting acute ingestion or poisoning. These findings encourage forensic toxicologists to actively use the metabolite to parent ratio in the interpretation of post-mortem toxicology results.
  • Evilä, Anni (Helsingin yliopisto, 2016)
    Muscular dystrophies are a clinically and genetically heterogeneous group of inherited disorders that cause progressive weakness and atrophy of muscles. They are divided into several subgroups based on the clinical and genetic findings. Mutations in dozens of different genes are known to cause the disorders, but unsolved cases exist. Accurate molecular diagnosis is important for genetic counseling and management of the diseases. The aims of this doctoral study were to identify still unknown genetic defects in muscular dystrophy patients using Sanger sequencing and next-generation sequencing (NGS) methods and to develop a targeted NGS gene panel for molecular diagnostics. In the course of this study more than 30 muscular dystrophy families or sporadic patients obtained a molecular diagnosis. A majority of the identified mutations were located in TTN. The gene encodes titin, which is a giant muscle protein having a central role in aspects of development, structure and function of the striated muscle. Due to the huge size of the gene it was not routinely sequenced before the NGS era. Muscle disorders caused by mutations in TTN are called titinopathies. In the first study, missense mutations in TTN exon 343 were identified in 12 families from seven different countries having hereditary myopathy with early respiratory failure (HMERF). The findings considerable expanded the mutational spectrum of HMERF and highlighted the geographically wide occurrence of the disease as well as the role of TTN exon 343 as a mutational hotspot region. In the second study, additional mutations in patients having previously reported tibial muscular dystrophy (TMD) mutation in TTN exon 362 or 363, but atypical phenotypes, were identified. The patients had a frameshift mutation in the other TTN allele causing a more severe TMD phenotype or limb-girdle muscular dystrophy (LGMD). In the last study, recessive distal myopathy patients were identified to have novel mutations in TTN exon 362 or 363 in either homozygous state or in compound heterozygosity with a nonsense or frameshift mutation. The patients had similar more severe TMD phenotype as the patients in the previous study. The findings expanded the list of distal myopathies with a new category: juvenile or early adult onset recessive distal titinopathy. In addition, a few patients having other novel recessive TTN mutations and novel phenotypes were identified in this doctoral study. The targeted NGS gene panel, MyoCap, was designed to capture and sequence 180 myopathy related genes simultaneously. Altogether 61 myopathy patients, negative for previous candidate gene approaches, were screened using the assay and 19 (31%) of them obtained a molecular diagnosis. MyoCap has high sensitivity and specificity and it is easily updated when new discoveries of myopathy causing genes are made. It provides fast and reliable detection of mutations in myopathy patients and it has been implemented in the molecular diagnostics of myopathy patients in Finland. It is now the first method of choice in diagnostics if no clear candidate gene can be determined by conventional clinical examinations.
  • 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.
  • 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.