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  • Markkula, Ritva (Helsingin yliopisto, 2014)
    Fibromyalgia: Background factors and impact on mortality and ability to work The aim was to update the prevalence of fibromyalgia (FM) symptoms, their predictors, and their impact on mortality and ability to work, in an epidemiologically representative cohort. For this we used the older Finnish Twin Cohort, which enabled evaluation of genetic backgrounds, as well. This cohort (originally 17 357 twins born before 1958) participated in three surveys with extensive health questionnaires in 1975, 1981, and 1990; response rates were 89%, 84%, and 77%. Applying their responses and clinical FM patients´ responses to an FM question-set from 1990, we could classify this cohort into three latent symptom classes: LC1 with no or few FM-associated symptoms, LC2 with some symptoms, and LC3 with a high number of symptoms, resembling FM patients and thus serving as a proxy for FM. Of all responders, almost equally from each gender 13% were classified into LC3. The statistical analyses estimated heritability behind this symptom classification as 51% (95% CI 45-56%). Of the suggested risk factors (assessed by the surveys in 1975 and 1981), what emerged as independent predictors of FM-associated symptoms (LC3) were headache and back and neck pain. Obesity or high body mass index and sleep problems also predicted FM symptoms, but confounding by familial factors seemed probable. For analysis of disability retirement, 8 448 individuals at risk remained after exclusions, and for analysis of mortality, 9 759. During the 14-year follow-up, the cumulative incidence for disability retirement in general was 6.8% in LC1, 10.6% in LC2, and 25.7% in LC3. The multi-adjusted hazard ratio for general disability retirement in LC3 was three-fold and for musculoskeletal disability five-fold that in LC1. As for mortality, the follow-up lasted 19 years and revealed the age- and gender-adjusted hazard ratio of death to be 1.4 (95% CI 1.2-1.8) in LC3 and 1.2 (95% CI 1.0-1.4) in LC2. This excess risk disappeared with further adjustment for lifestyle factors: smoking, alcohol intake, and body mass index. FM symptoms and associated lifestyle factors and co-morbidities require active management in health-care systems, not only to alleviate individual suffering, but also to prevent disability and increased mortality. Further research is essential to clarify whether early active management of confirmed risk factors would prevent FM-symptom development.
  • Salmenperä, Pertteli (Helsingin yliopisto, 2012)
    Cancer is a complex disease. It is a multistep process where genetic changes lead to cellular transformation and uncontrolled proliferation. However, cancer is not only a disease of these transformed cells, since tumor stroma and microenvironment synchronously evolve and become activated together with these genetic changes. The interactions between different cell types in tumor microenvironment are mediated by soluble factors, such as cytokines, chemokines, growth factors and proteases. They modulate cell proliferation, activation and differentiation, as well as the composition of the extracellular matrix in tumor and its microenvironment. Nemosis is an in vitro model of fibroblast activation, which is initiated by forcing fibroblast to cluster together instead of providing solid support for attachment. This results in a multicellular spheroid that upregulates soluble paracrine molecules known to be important mediators of tumor microenvironment. Furthermore, nemotic fibroblasts affect cancer cell proliferation, invasion and differentiation through these soluble factors. In addition to direct effects on cancer cells they stimulate angiogenesis and the chemotaxis of leukocytes. This thesis study shows that fibroblast spheroid formation depends on the interaction between fibronectin (FN) with its integrin receptors, more accurately α5 and β1 integrin subunits, whereas fibroblasts activation in spheroids was mediated by the interaction of FN with α5, αV and β1 integrin subunits. The activation was mediated by the binding of integrins to the RGD -motif in FN molecules and the synergy site that is known to stimulate RGD-motif binding to integrins enhanced it. Unexpectedly, FN-matrix assembly was not essential for the activation of fibroblasts in spheroids although it had an effect on spheroid formation. FN deposit to matrix is an acknowledged consequence of integrin binding to fibronectin. Nemosis was accompanied by a dramatic change in gene expression. The change could be roughly categorized in three classes; the upregulation of secreted molecules and downregulation of cell cycle and cytoskeleton. Nemosis was associated with a quiescent withdrawal from the cell cycle, as the cells downregulated cyclin D and upregulated p27, the driver and the inhibitor of the cell cycle, respectively. Furthermore, nemotic fibroblasts resumed to the cell cycle when taken out from the spheroid, indicating reversible cell cycle arrest, a known characteristic of quiescence. Fibroblast activation by spheroid formation was accompanied by stress-related changes in the cellular ultrastructure, such as dilated endoplasmic reticulum, increased lipofuscin and degenerated organelles. Hence, nemosis is a cellular stress response. This observation was in agreement with the induction of autophagy in fibroblasts spheroids. Autophagy is a well-known stress response that helps cell survival under stress conditions. Furthermore, nemosis resembled another cellular stress condition, the cellular senescence. They both had a similar secretory phenotype, expressed senescence-associated β-galactosidase and lipofuscin, and there was a cell cycle arrest in both. However, there were also features to distinguish nemosis from senescence, such as nemosis being a reversible phenotype, and cell cycle inhibitors that regulate senescence being downregulated in nemosis. Nemosis attenuated tumor growth in vivo in a mouse xenograft model. The attenuation was associated with the expression of senescence-associated β-galactosidase and the expression of the p14ARF cell-cycle inhibitor in human RT3 malignant keratinocytes. This suggests that nemosis causes cellular senescence in the RT3 keratinocytes in vivo. In addition to the senescence response, nemosis was found to increase the cytokeratin-7 (CK7) mediated differentiation of RT3 cells in xenografts. It is becoming obvious that cancer is not just a disease of uncontrolled proliferation of cancer cells, but a disease where normal stromal fibroblasts actively participate in its progression. The current work reveals new mechanistic insights of fibroblast activation and concludes that nemosis can be a useful model to study the activation of fibroblasts and interactions between fibroblasts and cancer cells.
  • Erra, Elina (Helsingin yliopisto, 2013)
    The mosquito-borne flavivirus infections Japanese encephalitis (JE) and dengue pose a considerable disease burden in the tropical and subtropical regions of the world. Sometimes these infections also affect international travelers visiting areas endemic for the diseases. This thesis addresses the prevention of Japanese encephalitis and diagnostics of dengue, with a focus on traveler s health. The main aim was to find solutions to some practical clinical questions, and thus provide clinically important new data on travel-associated flavivirus infections. The first three studies assessed the immunogenicity of two inactivated JE vaccines, the new Vero cell derived (JE-VC) and the traditional mouse brain derived (JE-MB) preparation, in 120 Finnish and Swedish adult travelers, by determining the pre- and post-vaccination titers of JE virus neutralizing antibodies with plaque-reduction neutralization test (PRNT). A PRNT50 titer ≥10 was considered protective. Study I addressed the boosting capacity of the SA14-14-2 based JE-VC vaccine in subjects previously primed with the Nakayama strain based JE-MB preparation. The response rates were 91% after a homologous (JE-MB) and 95%-98% after a heterologous booster dose (JE-VC). Among those with no seroprotection at baseline, a higher proportion of JE-MB primed (100%) than non-primed (40%) subjects seroconverted after a single JE-VC dose. The data suggest that a single JE-VC dose suffices for boosting JE-MB immunity, and call for re-evaluation of guidelines recommending two JE-VC doses for JE-MB primed subjects. Study II demonstrated that both JE-VC and JE-MB elicit neutralizing antibodies, not only against the vaccine genotype, but also against heterologous JE virus genotypes. The seroprotection rates against the heterologous strains were 93%-97% after JE-VC, and 83%-92% after JE-MB primary series. The data imply that the two vaccines are expected to confer seroprotection against all major JE virus genotypes circulating. Study III evaluated long-term seroprotection after JE-VC primary and booster vaccinations. Two years after primary immunization, 93% of vaccinees were seroprotected against the vaccine strain but only 73% against the emerging genotype GI. JE-MB primed vaccinees were seroprotected against both vaccine (100%) and non-vaccine (89%-95%) genotype strains two years after the heterologous JE-VC booster dose, further supporting the use of a single JE-VC dose for boosting JE-MB immunity. Study IV explored the diagnostic markers of dengue in 93 Finnish traveler patients. The duration of viremia (9 days, 95% CI: 8-10) and NS1 antigenemia (15 days, 95% CI: 12-20) were longer than reported in endemic settings, presumably due to a high proportion of primary infections among travelers. The data support use of test combinations, e.g. antibody and NS1 detection, for efficient diagnostics. The relative levels of viremia and NS1 antigenemia were associated with some central clinical parameters, suggesting these virologic markers as predictors of the clinical manifestations in travelers dengue.
  • Heikkilä, Annukka (Helsingin yliopisto, 2013)
    Follicular thyroid carcinoma (FTC) is the second most common malignancy of the thyroid gland, with predisposing genetic alterations such as rat sarcoma (RAS) mutation and paired box gene 8-peroxisome proliferator-activated receptor γ (PAX-PPARγ) alteration, as well as suggested risk factors such as iodine insufficiency and female gender. Distinguishing FTC from the most common neoplasm of the thyroid, follicular thyroid adenoma (FTA), or even from a non-neoplastic goitrous nodule, is often impossible preoperatively, leading to unnecessary surgery and exposing patients to surgical complications. In this study, 127 follicular thyroid neoplasia patients (83 FTAs and 44 FTCs) treated at Helsinki University Central Hospital (HUCH) in Finland between 1990 and 2009 were examined to find methods for differential diagnosis between follicular thyroid lesions. Tissue markers were investigated by immunohistochemistry in follicular neoplasms and non-neoplastic control tissues, and were correlated with clinical parameters, such as with metastatic disease and survival. Additionally, cancer registry data were gathered, concerning the diminishing incidence of FTC accompanied by an increase in the incidence of papillary thyroid carcinoma. Carcinomas were reanalysed according to the new World Health Organization classification of endocrine tumours, in which a new tumour entity, poorly differentiated carcinoma of the thyroid, was introduced. Markers with possible clinical utility were found; e.g. in an attempt to differentiate between non-neoplastic and neoplastic follicular lesions of the thyroid (HES5) as well as between FTA and FTC (MIB-1, Cyclin D1, TLR-2, ERβ), a marker with prognostic value in carcinomas (ERβ), as well as a marker correlating with the presence of metastatic disease (TLR-4). These results aid in the challenging field of diagnostics in follicular thyroid lesions. Measuring the expression of HES5 may help in differentiating between neoplastic and non-neoplastic follicular thyroid lesions. Markers, such as MIB-1 and ERβ, are partly able to differentiate between benign and malignant follicular thyroid neoplasias, whereas ERβ and TLR-4 have prognostic value in FTC.
  • Paalanen, Laura (Helsingin yliopisto, 2013)
    The Republic of Karelia in north-western Russia and North Karelia in eastern Finland are situated right next to each other. Part of the Republic of Karelia, including the district of Pitkäranta, was part of Finland until the Second World War, when it was annexed to the Soviet Union. The aim of this study was to explore trends and educational differences in food habits and related biomarkers in Pitkäranta, Russia, and North Karelia, Finland, over a 15-year period, which encompasses the early transition years from a centrally planned economy towards a market economy after the dissolution of the Soviet Union in 1991. Two population-based repeated cross-sectional datasets from the two areas were used: 1) health behaviour surveys from 1994, 1996, 1998, 2000 and 2004 (total n=3599 in Pitkäranta, total n=3652 in North Karelia) and 2) risk factor surveys from 1992, 1997, 2002 and 2007 (total n=2672 in Pitkäranta, total n=5437 in North Karelia). The data were collected by the National Public Health Institute (KTL) (the current National Institute for Health and Welfare, THL) in Finland. In Pitkäranta, the data were collected by the National Public Health Institute in collaboration with the Central Hospital of Pitkäranta and the Ministry of Health and Social Development in the Republic of Karelia. The trends and overall prevalence in food habits were very different between Pitkäranta and North Karelia. Food habits changed remarkably in Pitkäranta between 1992 and 2007. The proportion of those who used butter in cooking plunged from 50% to less than 10%. The proportion of those who used butter on bread decreased as well, although not consistently. The proportion of persons who consumed fat-containing milk fluctuated. The prevalence of daily consumption of fresh vegetables and fruit increased notably. In North Karelia, the changes were smaller. A small decrease in the proportion of those who used butter or consumed fat-containing milk was observed. The prevalence of daily consumption of fresh vegetables and fruit also decreased slightly in North Karelia. The educational differences in food habits were somewhat more notable in North Karelia than in Pitkäranta. In general, food habits were less favourable in the lowest compared to the highest education group in both areas. For example, the use of butter in cooking and the consumption of fat-containing milk were more common among subjects in the lowest education group, whereas the daily consumption of vegetables and fruit was more common among their more highly educated counterparts. The education gradient in the quality of spread used on bread was the opposite in the two areas. In Pitkäranta, using butter on bread tended to be more common among men in the highest education group, whereas in North Karelia, men with a low education used butter on bread more often. The mean serum total cholesterol did not differ by education in Pitkäranta. In North Karelia, it tended to be higher among subjects in the lowest education group. The mean plasma vitamin C concentration was strikingly low in Pitkäranta throughout the study period. In North Karelia, the overall level of plasma vitamin C was higher. The plasma vitamin C concentration tended to be higher among subjects with a higher education in both areas. The study demonstrated that food habits may change quite rapidly if the local circumstances change. In Pitkäranta, the availability and prices of foods are possible underlying factors that are related to the remarkable changes in food habits. In North Karelia, active health policy and the health consciousness of the population are probably more important in directing food choices.
  • Lempiäinen, Anna (Helsingin yliopisto, 2012)
    Testicular cancer is the most common malignant disease in young men. In order to avoid long-term toxicity of adjuvant therapy, treatment of testicular cancer is often limited to surgery, which leads to higher relapse rates. Thus, there is a need for sensitive and specific markers that enable early detection of relapse, and ideally, identify high risk patients needing adjuvant therapy. Human chorionic gonadotropin (hCG) is an extremely sensitive marker for testicular cancer. However, hCG is a heterogeneous protein with several different isoforms, of which the free β-subunit (hCGβ) has prognostic value in many nontrophoblastic cancers while hyperglycosylated form (hCG-h) has been suggested to be the very key to malignant transformation of testicular cancer. Our aim was to study the clinical and prognostic utility of hCG, hCGβ and hCG-h as serum markers for testicular germ cell tumors. We first confirmed the validity of our archival samples by re-determining hCG after storage at -20 °C in 152 serum and 74 urine samples. We developed an immunofluorometric assay for hCG-h, since no commercial ones are available. We determined hCG, hCGβ in 3802 and hCG-h in 176 serum samples from testicular cancer patients and analyzed the association between serum concentrations and known prognostic factors, progression free survival time and disease course. We found that serum hCG is stable for years at -20 °C, but in most urine samples hCG immunoreactivity is lost during storage at this temperature. Urea probably plays a role in the degradation of hCG, but other mechanisms are likely to participate in the process. Complement causes interference in the determination of serum hCG-h when the monoclonal antibody B152 is used in the assay. The interference was eliminated by using EDTA plasma rather than serum, or by inactivating complement in serum with EDTA before the assay. hCGβ is a sensitive marker for all types of testicular cancer and in seminomas it is superior to hCG. Separate determination of hCGβ provides clinically valuable information, since approximately one third of marker-positive seminomas and of relapses would have been missed by an assay measuring hCG and hCGβ together. Most of the hCG in testicular cancer patients was shown to be hyperglycosylated. Thus assays used for diagnosis and monitoring of this disease should recognize hCG and hCG-h equally. However, separate measurement of hCG-h does not seem to provide additional information as compared to assays of hCG and hCGβ. We also describe a case with increasing hCG levels due to hypogonadism causing suspicion of a relapse. Treatment of testicular cancer can be initiated on the basis of marker elevation alone, and therefore it is important to understand the behaviour of tumor markers under various physiological conditions.
  • Mäyränpää, Mervi (Helsingin yliopisto, 2012)
    Fractures are common in childhood. Repeated fractures, and especially vertebral fractures, in children may be a sign of impaired bone health, but it remains unestablished when and how fracture-prone children should be assessed. Bone mineral density (BMD) affects bone strength, and it can be measured with dual-energy X-ray absorptiometry (DXA). However, DXA has limitations in growing children, as have the biochemical markers of bone metabolism. In this work, we studied epidemiology of fractures in children under 16 years in Helsinki, 2005. Special attention was given to those children with repeated fractures or vertebral fractures; patients were further evaluated for skeletal characteristics and predisposing factors. To evaluate the clinical use of two rarely used methods in children, we assessed the accuracy and advantages of vertebral fracture assessment (VFA) by densitometry, and histomorphometry from bone biopsy in children with suspected osteoporosis. In a prospective study a total of 1396 fractures in 1373 children were recorded: the annual overall incidence of fractures in children under 16 years was 163/10 000. Boys sustained 63% of all fractures. Fracture incidence was highest in puberty: in boys at 14 years and in girls at 10 years. Forearm fracture was the most common fracture type (37%). There had been an 18% decrease in the overall fracture incidence over the preceding 22 years, mainly due to the decrease in hand and foot fractures (-39% and -48%, respectively). The greatest decrease was seen for children aged 10 to 14 years (-30%). For the same period, a 31% increase of forearm fractures and 39% increase of upper arm fractures was observed. One fourth of the children with acute fracture reported a prior fracture, but only in 5% was the fracture history regarded as significant. These fracture-prone children (n=66) were found to have impaired bone health: on average, they had lower calcium intake, physical activity level, and BMD than age- and sex-matched controls did. Vitamin D was below recommended level in more than half of the patients and controls; low levels were associated with lower BMD in both groups. Asymptomatic vertebral compressions, and more hypercalciuria and hyperphosphaturia were also observed in the fracture-prone patients. The visibility and detection rate of compressed vertebrae by VFA was assessed in 65 children; standard radiographs were used for comparison. The poor resolution of low-radiation VFA compromised the accuracy in younger children and in those with low BMD. In older children close to their skeletal maturation and adult height, the visibility of VFA was mostly good. Transiliac bone biopsy was performed on 24 children with suspected primary osteoporosis based on frequent fractures and/or low BMD. Findings were variable; only 29% were found to have low trabecular bone volume. Bone turnover was low for age in one third and high in one third. Histomorphometric findings correlated poorly with DXA measurements or clinical data, underscoring the importance of this method in severe pediatric osteoporosis.
  • Karhemo, Piia-Riitta (Helsingin yliopisto, 2013)
    The most deadly aspect of cancer is its ability to spread from its original location to other sites in the body and grow as distant metastases. Formation of metastases is a multistep process and metastases can form even decades after the removal of the primary tumor. Cell surface proteins are known to play central roles in the adhesive contacts and molecular interactions between the tumor cell and the stroma during various stages of metastasis. In addition, they mediate important signals to intracellular proteins. As the detailed mechanisms of metastasis are still unclear, the aim of this thesis was to discover novel metastasis-associated cell surface proteins for further investigation. This thesis established an optimized method for the isolation of biotinylated cell surface proteins for proteomic identification. This method was applied to compare the cell surface proteins isolated from an isogenic pair of human MDA-MB-435 cancer cell line with opposite metastatic phenotypes. We found 29 differentially expressed proteins and analyzed the molecular pathways they were involved in. Of these proteins expression of CD109 was shown to mark metastatic melanoma cells and invasive breast cancer cells. Nucleophosmin (NPM) is a multitasking protein with both oncogenic and tumor suppressive functions. In our comparative proteomics analysis we discovered NPM to be expressed on the surface of the non-metastatic subclone of the MDA-MB-435 cells. We showed that NPM was detected at different localizations in the non-metastatic and metastatic cells most likely due to the expression of novel NPM splice variants discovered in this thesis work. In addition, we showed that expression level of NPM is one mechanism affecting its localization. In regards to patient prognosis, we revealed that high levels of NPM were expressed in the luminal epithelial cells of histologically normal breast tissue and that high levels independently associated with good prognosis in the luminal A breast cancer subtype. On the contrary, novel granular staining pattern and Threonine199 phosphorylation of NPM (NPMpThr199) correlated with aggressive characteristics, basal subtype and poor prognosis of human breast cancer. In brief, this study provides several novel metastasis associated cell surface proteins for future investigation. By using breast cancer tumor microarrays from two large breast cancer patient cohorts and cellular models this thesis demonstrates for the first time, that different NPM forms play divergent and opposite roles in breast cancer.
  • Laisi, Jaana (Helsingin yliopisto, 2012)
    The From Home To Operation (FHTO) process was developed to respond to the dilemma of increasing patient load but limited resources. In the FHTO process, the patients are admitted on the morning of operation through a specialised preoperative unit immediately adjacent to the operation rooms (OR) regardless of the type of anaesthesia and surgical procedure, and of the possible postoperative need for hospitalisation. To maintain the anaesthesia safety in the process, some of the patients need to be referred to an anaesthesia preoperative evaluation clinic (APEC) according to predefined criteria. The aims of the study were to investigate the FHTO process, focusing on cost-effectiveness, the effect of process transition on surgery outcome, the role of anaesthesia preoperative evaluation clinic, and the rates and reasons for surgery cancellation. The study was conducted in Helsinki and Uusimaa Hospital District Hyvinkää Hospital, and it included 13 000 surgical patients. The FHTO process was cost-effective compared to traditional preoperative process in laparoscopic cholecystectomy patients. The process transition from the combination of the FHTO and the traditional process to only the FHTO process did not have any effect on surgical outcome. Over 90% of elective patients can be admitted straight to the OR on the morning of operation. Only 25% of patients were referred to the APEC evaluation. As expected, these patients suffered from more additional health issues and underwent more difficult operation than the patients who were not evaluated at the APEC. Other postoperative complication rates did not differ between these two patient groups. Not all patients require a preoperative APEC evaluation. The cancellation rate was 4.5% in the FHTO process. Over 70% of all cancelled operations were related to patient-related reasons, and the most common single reason was that the operation was no longer necessary. The highest rates were in hand surgery and orthopaedic surgery. The overall cancellation rate in the FHTO process was at a reasonable level, however, to improve OR usage it should still be decreased. The intention should be to admit elective patients on the morning of the operation without any preoperative visit to the surgical ward. The preoperative processes should be developed as a whole, and the traditional process allowed to pass into history.
  • Tiippana, Elina (Helsingin yliopisto, 2013)
    Postoperative pain may persist for months and it is crucial to identify those patients at risk, treat their pain and to develop methods that decrease the incidence of persistent pain. This study investigated the intensity of acute postoperative pain, the incidence of chronic postsurgical pain, and the possibilities of influencing these by focusing on thoracotomy and laparoscopic cholecystectomy (LCC) as examples. This research also assessed the efficacy of perioperative opioid-sparing drugs (gabapentinoids, dexamethasone, NSAIDs and paracetamol), and analyzed whether tropisetron abolishes the analgesic action of paracetamol. Acute and chronic pain after thoracic surgery was investigated in two clinical studies. Study I included 111 patients, and Study IV included 30 intervention patients + a control group of 111 standard care patients. Pain was treated with thoracic epidural analgesia (TEA) or intravenous patient controlled analgesia with morphine or oxycodone (IV-PCA) + NSAIDs. Study II included 160 day-case LCC patients who received pare/valdecoxib or paracetamol, with or without dexamethasone. Study III was a systematic review with a meta-analysis including 22 randomized, controlled trials on the perioperative administration of gabapentin (21) and pregabalin (1) for postoperative pain relief. Study V consisted of 2 randomized, double-blind, crossover studies with 18 healthy male volunteers in each. The cold pressor test, contact heat pain and electrical stimulation were used, and the volunteers received tropisetron or saline, followed by IV paracetamol. TEA was effective in alleviating movement-related pain and the duration of pain after coughing in thoracotomy patients. The extended protocol for pain management in hospital, which also covers the sub-acute phase at home, was found to be important in preventing acute and persistent post-thoracotomy pain. One week after discharge, 92-100% of the patients needed daily pain medication and 71-77% required weak opioids. In Study I, the incidence of disturbing chronic pain at 6 months was 12%, and in Study IV, these numbers were 3% in the intervention group versus 24% in the control group (p<0.01). Dexamethasone significantly reduced the need for oxycodone after LCC, and shoulder pain was long-lasting. Multimodal pain treatment enabled smooth outpatient LCC. The systematic review indicated that pain relief was significantly better in the gabapentin groups. The consumption of opioids 24 h after a single dose of preoperative gabapentin was reduced by 20-62%, and opioid-related adverse effects were also reduced. In Study V, paracetamol did not display a statistically significant analgesia on the thermal or electrical pain stimulation tests. In fact, tropisetron seemed to amplify the analgesic action of paracetamol.
  • Vanharanta, Sakari (Helsingin yliopisto, 2006)
    Germline mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell cancer (HLRCC). FH is a nuclear encoded enzyme which functions in the Krebs tricarboxylic acid cycle, and homozygous mutation in FH lead to severe developmental defects. Both uterine and cutaneous leiomyomas are components of the HLRCC phenotype. Most of these tumours show loss of the wild-type allele and, also, the mutations reduce FH enzyme activity, which indicate that FH is a tumour suppressor gene. The renal cell cancers associated with HLRCC are of rare papillary type 2 histology. Other genes involved in the Krebs cycle, which are also implicated in neoplasia are 3 of the 4 subunits encoding succinate dehydrogenase (SDH); mutations in SHDB, SDHC, and SDHD predispose to paraganglioma and phaeochromocytoma. Although uterine leiomyomas (or fibroids) are very common, the estimations of affected women ranging from 25% to 77%, not much is known about their genetic background. Cytogenetic studies have revealed that rearrangements involving chromosomes 6, 7, 12 and 14 are most commonly seen in fibroids. Deletions on the long arm of chromosome 7 have been reported to be involved in about 17 to 34 % of leiomyomas and the small commonly deleted region on 7q22 suggests that there might be an underlying tumour suppressor gene in that region. The purpose of this study was to investigate the genetic mechanisms behind the development of tumours associated with HLRCC, both renal cell cancer and uterine fibroids. Firstly, a database search at the Finnish cancer registry was conducted in order to identify new families with early-onset RCC and to test if the family history was compatible with HLRCC. Secondly, sporadic uterine fibroids were tested for deletions on 7q in order to define the minimal deleted 7q-region, followed by mutation analysis of the candidate genes. Thirdly, oligonucleotide chips were utilised to study the global gene expression profiles of uterine fibroids in order to test whether 7q-deletions and FH mutations significantly affected fibroid biology. In the screen for early-onset RCC, 214 families were identified. Subsequently, the pedigrees were constructed and clinical data obtained. One of the index cases (RCC at the age of 28) had a mother who had been diagnosed with a heart tumour, which in further investigation turned out to be a paraganglioma. This lead to an alternative hypothesis that SDH, instead of FH, could be involved. SDHA, SDHB, SDHC and SDHD were sequenced from these individuals; a germline SDHB R27X mutation was detected with loss of the wild-type allele in both tumours. These results suggest that germline mutations in the SDHB gene predispose to early-onset RCC establishing a novel form of hereditary RCC. This has immediate clinical implications in the surveillance of patients suffering from early-onset RCC and phaeochromocytoma/paraganglioma. For the studies on sporadic uterine fibroids, a set of 166 fibroids from 51 individuals were collected. The 7q LOH mapping defined a commonly deleted region of about 3.2 mega bases in 11 of the 166 tumours. The deletion was consistent with previously reported allelotyping studies of leiomyomas and it therefore suggested the presence of a tumour suppressor gene in the deleted region. Furthermore, the high-resolution aCGH-chip analysis refined the deleted region to only 2.79Mb. When combined with previous data, the commonly deleted region was only 2.3Mb. The mutation screening of the known genes within the commonly deleted region did not reveal pathogenic mutations, however. The expression microarray analysis revealed that FH-deficient fibroids, both sporadic and familial, had their distinct gene expression profile as they formed their own group in the unsupervised clustering. On the other hand, the presence or absence of 7q-deletions did not significantly alter the global gene expression pattern of fibroids, suggesting that these two groups do not have different biological backgrounds. Multiple differentially expressed genes were identified between FH wild-type and FH-mutant fibroids, and the most significant increase was seen in the expression of carbohydrate metabolism-related and hypoxia inducible factor (HIF) target genes.
  • Katz, Anna (Helsingin yliopisto, 2012)
    The Uukuniemi virus (UUKV) is a member of the Bunyaviridae family (genus Phlebovirus). The virus was isolated from Ixodes ricinus ticks from Uukuniemi, Finland in 1959 and was found to be non-pathogenic for humans. UUKV has served for more than four decades as an excellent model to study the molecular and cellular biology of the serious human pathogens that reside within this group. UUKV has a segmented, single-stranded RNA genome of negative polarity. The three RNA segments (S, M, and L) encode four structural proteins: a nucleocapsid (N) protein, two glycoproteins (Gn and Gc), and an RNA-dependent RNA polymerase (L protein). In addition, a non-structural protein (NSs) is encoded from the S segment using an ambisense coding strategy. At the termini of the RNA segments, there are non-coding regions, which contain regulatory elements for viral transcription and replication. The very terminal 5' and 3' ends within all non-coding regions are complementary to each other, and highly conserved within the genus. In order to function as templates for transcription and replication, all three RNA segments must be encapsidated by the N protein. The N protein forms oligomers, in which N protein molecules are bound to each other; this oligomer associates with RNA. This study focused on analyzing the function of the non-coding regions in the termini of the RNA segments, and on locating amino acid residues or domains of the UUKV N protein, which could potentially be involved in the oligomerization or RNA-binding. The function of the non-coding regions was studied using a minigenome system developed for UUKV, where the viral protein coding sequence is replaced by sequences encoding a reporter protein. The cells are transfected with the minigenomes and helper plasmids, and after replication and transcription of the minigenomes, the reporter protein expression can be measured. The non-coding regions of all three RNA segments were analyzed and promoter strengths and packaging efficiencies were compared. The results showed that the non-coding regions in all three RNA segments contain all the necessary signals for initiation of transcription and replication and encapsidation and packaging of the RNA segments. The strongest promoter strength was observed in M segment, followed by L and S segments. The role of the intergenic region, which is located between the N and NSs genes in the UUKV S segment was also analyzed and was found to regulate termination of transcription. To study the oligomerization and RNA-binding of UUKV N protein, a set of N protein mutants were generated based on 2D and 3D predictions of the N protein. The functionality of these mutants was analyzed using mammalian two-hybrid-, minigenome-, and virus-like particle-assays, which showed that both the N- and C-termini of the N protein are needed for the oligomerization. A specific structure in the N-terminal region plays an important role in the N-N interactions. Some putative RNA-binding residues were found, which severely affected the N protein functionality in all three assays. These residues were located within the proposed RNA-binding cavity in the predicted UUKV N protein models. These results are in agreement with observations with other bunyaviruses, and could help to better understand the molecular biology of bunyaviruses. Moreover, understanding the details of the oligomerization and RNA-binding of the N protein could help in design of potential antivirals for the pathogenic phleboviruses.
  • Boldt, Robert (Helsingin yliopisto, 2014)
    Hearing is a versatile sense allowing us, among other things, to avoid danger and engage in pleasurable discussions. The ease with which we follow a conversation in a noisy environment is astonishing. Study I in this thesis used functional magnetic resonance imaging to explore the large-scale organization of speech and non-speech sound processing during a naturalistic stimulus comprised of an audio drama. Two large-scale functional networks processed the audio drama; one processed only speech, the other processed both speech and non-speech sounds. Hearing is essential for blind subjects. Anatomical and functional changes in the brains of blind people allow them to experience a detailed auditory world, compensating for the lack of vision. Therefore, comparing early-blind subjects brains to those of sighted people during naturalistic stimuli reveals fundamental differences in brain organization. In Study II, naturalistic stimuli were employed to explore whether one of the most distinguishing traits of the auditory system the left-lateralized responses to speech changes following blindness. As expected, in sighted subjects, speech processing was left-hemisphere dominant. Curiously, the left-hemisphere dominance for speech was absent or even reversed in blind subjects. In early-blind people, the senses beyond vision are strained as they try to compensate for the loss of sight; on the other hand, the occipital cortices are devoid of normal visual information flow. Interestingly, in blind people, senses other than vision recruit the occipital cortex. Additional to changes in the occipital cortex, the sensory cortices devoted to touch and hearing change. Data presented here suggested more inter-subject variability in auditory and parietal areas in blind subjects compared with sighted subjects. The study suggested that the greater the inter-subject variability of the network, the greater the experience-dependent plasticity of that network. As the prefrontal areas display large inter-subject spatial variability, the activation of the prefrontal cortex varies greatly. The variable activation might partly explain why the top-down influences of the prefrontal cortex on tactile discrimination are not well understood. In the fourth study, anatomical variability was assessed on an individual level, and transcranial magnetic stimulation was targeted at individually-chosen prefrontal locations indicated in tactile processing. Stimulation of one out of two prefrontal cortex locations impaired the subjects ability to distinguish a single tactile pulse from paired pulses. Thus, the study suggested that tactile information is regulated by functionally specialized prefrontal subareas.
  • Rytsälä, Heikki (Helsingin yliopisto, 2006)
    This study is one part of a collaborative depression research project, the Vantaa Depression Study (VDS), involving the Department of Mental and Alcohol Research of the National Public Health Institute, Helsinki, and the Department of Psychiatry of the Peijas Medical Care District (PMCD), Vantaa, Finland. The VDS includes two parts, a record-based study consisting of 803 patients, and a prospective, naturalistic cohort study of 269 patients. Both studies include secondary-level care psychiatric out- and inpatients with a new episode of major depressive disorder (MDD). Data for the record-based part of the study came from a computerised patient database incorporating all outpatient visits as well as treatment periods at the inpatient unit. We included all patients aged 20 to 59 years old who had been assigned a clinical diagnosis of depressive episode or recurrent depressive disorder according to the International Classification of Diseases, 10th edition (ICD-10) criteria and who had at least one outpatient visit or day as an inpatient in the PMCD during the study period January 1, 1996, to December 31, 1996. All those with an earlier diagnosis of schizophrenia, other non-affective psychosis, or bipolar disorder were excluded. Patients treated in the somatic departments of Peijas Hospital and those who had consulted but not received treatment from the psychiatric consultation services were excluded. The study sample comprised 290 male and 513 female patients. All their psychiatric records were reviewed and each patient completed a structured form with 57 items. The treatment provided was reviewed up to the end of the depression episode or to the end of 1997. Most (84%) of the patients received antidepressants, including a minority (11%) on treatment with clearly subtherapeutic low doses. During the treatment period the depressed patients investigated averaged only a few visits to psychiatrists (median two visits), but more to other health professionals (median seven). One-fifth of both genders were inpatients, with a mean of nearly two inpatient treatment periods during the overall treatment period investigated. The median length of a hospital stay was 2 weeks. Use of antidepressants was quite conservative: The first antidepressant had been switched to another compound in only about one-fifth (22%) of patients, and only two patients had received up to five antidepressant trials. Only 7% of those prescribed any antidepressant received two antidepressants simultaneously. None of the patients was prescribed any other augmentation medication. Refusing antidepressant treatment was the most common explanation for receiving no antidepressants. During the treatment period, 19% of those not already receiving a disability pension were granted one due to psychiatric illness. These patients were nearly nine years older than those not pensioned. They were also more severely ill, made significantly more visits to professionals and received significantly more concomitant medications (hypnotics, anxiolytics, and neuroleptics) than did those receiving no pension. In the prospective part of the VDS, 806 adult patients were screened (aged 20-59 years) in the PMCD for a possible new episode of DSM-IV MDD. Of these, 542 patients were interviewed face-to-face with the WHO Schedules for Clinical Assessment in Neuropsychiatry (SCAN), Version 2.0. Exclusion criteria were the same as in the record-based part of the VDS. Of these, 542 269 patients fulfiled the criteria of DSM-IV MDE. This study investigated factors associated with patients' functional disability, social adjustment, and work disability (being on sick-leave or being granted a disability pension). In the beginning of the treatment the most important single factor associated with overall social and functional disability was found to be severity of depression, but older age and personality disorders also significantly contributed. Total duration and severity of depression, phobic disorders, alcoholism, and personality disorders all independently contributed to poor social adjustment. Of those who were employed, almost half (43%) were on sick-leave. Besides severity and number of episodes of depression, female gender and age over 50 years strongly and independently predicted being on sick-leave. Factors influencing social and occupational disability and social adjustment among patients with MDD were studied prospectively during an 18-month follow-up period. Patients' functional disability and social adjustment were alleviated during the follow-up concurrently with recovery from depression. The current level of functioning and social adjustment of a patient with depression was predicted by severity of depression, recurrence before baseline and during follow-up, lack of full remission, and time spent depressed. Comorbid psychiatric disorders, personality traits (neuroticism), and perceived social support also had a significant influence. During the 18-month follow-up period, of the 269, 13 (5%) patients switched to bipolar disorder, and 58 (20%) dropped out. Of the 198, 186 (94%) patients were at baseline not pensioned, and they were investigated. Of them, 21 were granted a disability pension during the follow-up. Those who received a pension were significantly older, more seldom had vocational education, and were more often on sick-leave than those not pensioned, but did not differ with regard to any other sociodemographic or clinical factors. Patients with MDD received mostly adequate antidepressant treatment, but problems existed in treatment intensity and monitoring. It is challenging to find those at greatest risk for disability and to provide them adequate and efficacious treatment. This includes great challenges to the whole society to provide sufficient resources.
  • Stenbacka, Linda (Helsingin yliopisto, 2010)
    Visual information processing in brain proceeds in both serial and parallel fashion throughout various functionally distinct hierarchically organised cortical areas. Feedforward signals from retina and hierarchically lower cortical levels are the major activators of visual neurons, but top-down and feedback signals from higher level cortical areas have a modulating effect on neural processing. My work concentrates on visual encoding in hierarchically low level cortical visual areas in human brain and examines neural processing especially in cortical representation of visual field periphery. I use magnetoencephalography and functional magnetic resonance imaging to measure neuromagnetic and hemodynamic responses during visual stimulation and oculomotor and cognitive tasks from healthy volunteers. My thesis comprises six publications. Visual cortex forms a great challenge for modeling of neuromagnetic sources. My work shows that a priori information of source locations are needed for modeling of neuromagnetic sources in visual cortex. In addition, my work examines other potential confounding factors in vision studies such as light scatter inside the eye which may result in erroneous responses in cortex outside the representation of stimulated region, and eye movements and attention. I mapped cortical representations of peripheral visual field and identified a putative human homologue of functional area V6 of the macaque in the posterior bank of parieto-occipital sulcus. My work shows that human V6 activates during eye-movements and that it responds to visual motion at short latencies. These findings suggest that human V6, like its monkey homologue, is related to fast processing of visual stimuli and visually guided movements. I demonstrate that peripheral vision is functionally related to eye-movements and connected to rapid stream of functional areas that process visual motion. In addition, my work shows two different forms of top-down modulation of neural processing in the hierachically lowest cortical levels; one that is related to dorsal stream activation and may reflect motor processing or resetting signals that prepare visual cortex for change in the environment and another local signal enhancement at the attended region that reflects local feed-back signal and may perceptionally increase the stimulus saliency.
  • Viertiö, Satu (Helsingin yliopisto, 2011)
    There is substantial evidence of the decreased functional capacity, especially everyday functioning, of people with psychotic disorder in clinical settings, but little research about it in the general population. The aim of the present study was to provide information on the magnitude of functional capacity problems in persons with psychotic disorder compared with the general population. It estimated the prevalence and severity of limitations in vision, mobility, everyday functioning and quality of life of persons with psychotic disorder in the Finnish population and determined the factors affecting them. This study is based on the Health 2000 Survey, which is a nationally representative survey of 8028 Finns aged 30 and older. The psychotic diagnoses of the participants were assessed in the Psychoses of Finland survey, a substudy of Health 2000. The everyday functioning of people with schizophrenia is studied widely, but one important factor, mobility has been neglected. Persons with schizophrenia and other non-affective psychotic disorders, but not affective psychoses had a significantly increased risk of having both self-reported and test-based mobility limitations as well as weak handgrip strength. Schizophrenia was associated independently with mobility limitations even after controlling for lifestyle-related factors and chronic medical conditions. Another significant factor associated with problems in everyday functioning in participants with schizophrenia was reduced visual acuity. Their vision was examined significantly less often during the five years before the visual acuity measurement than the general population. In general, persons with schizophrenia and other non-affective psychotic disorder had significantly more limitations in everyday functioning, deficits in verbal fluency and in memory than the general population. More severe negative symptoms, depression, older age, verbal memory deficits, worse expressive speech and reduced distance vision were associated with limitations in everyday functioning. Of all the psychotic disorders, schizoaffective disorder was associated with the largest losses of quality of life, and bipolar I disorder with equal or smaller losses than schizophrenia. However, the subjective loss of qualify of life associated with psychotic disorders may be smaller than objective disability, which warrants attention. Depressive symptoms were the most important determinant of poor quality of life in all psychotic disorders. In conclusion, subjects with psychotic disorders need regular somatic health monitoring. Also, health care workers should evaluate the overall quality of life and depression of subjects with psychotic disorders in order to provide them with the basic necessities of life.
  • Markkanen-Leppänen , Mari (Helsingin yliopisto, 2006)
    Oral cancer ranks among the 10 most common cancers worldwide. Since it is commonly diagnosed at locally advanced stage, curing the cancer demands extensive tissue resection. The emergent defect is reconstructed generally with a free flap transfer. Repair of the upper aerodigestive track with maintenance of its multiform activities is challenging. The aim of the study was to extract comprehensive treatment outcomes for patients having undergone microvascular free flap transfer because of large oral cavity or pharyngeal cancer. Ninety-four patients were analyzed for postoperative survival and complications. Forty-four patients were followed-up and analyzed for functional outcome, which was determined in terms of quality of life, speech, swallowing, and intraoral sensation. Quality of life was assessed using the University of Washington Head and Neck Questionnaire. Speech was analyzed for aerodynamic parameters and for nasal acoustic energy, as well as perceptually for articulatory proficiency, voice quality, and intelligibility. Videofluorography was performed to determine the swallowing ability. Intraoral sensation was measured by moving 2-point discrimination. The 3-year overall survival was over 40%. The 1-year disease-free survival was 43%. Postoperative complications arose in over half of the patients. Flap success rate was high. Perioperative mortality varied between 2% and 11%. Unemployment and heavy drinking were the strongest predictors of survival. Sociodemographic factors were found to associate with quality of life. The global quality of life score deteriorated and did not return to the preoperative level. Significant reduction was detectable in the domains measuring chewing and speech, and in appearance and shoulder function. The basic elements necessary for normal speech were maintained. Speech intelligibility reduced and was related to the misarticulations of the /r/ and /s/ phonemes. Deviant /r/ and /s/ persisted in most patients. Hoarseness and hypernasality occurred infrequently. One year postoperatively, 98% of the patients had achieved oral nutrition and half of them were on a regular masticated diet. Overt and silent aspiration was encountered throughout the follow-up. At 12-month swallow test, 44% of the patients aspirated, 70% of whom silently. Of these patients, 15% presented with pulmonary changes referring to aspiration. Intraoral sensation weakened but was unrelated to oral functions. The results provide new data for oral reconstructions and highlight the importance of the functional outcome of the treatment for an oral cancer patient. The mouth and the pharynx encompass a unit of utmost functional complexity. Surgery should continue to make progress in this area, and methods that lead to good function should be developed. Operational outcome should always be evaluated in terms of function.
  • Pehrsson, Minja (Helsingin yliopisto, 2011)
    Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.