Lääketieteellinen tiedekunta

 

Recent Submissions

  • Holmlund-Suila, Elisa (Helsingin yliopisto, 2016)
    Vitamin D deficiency in infancy and childhood impairs normal bone development and growth: defective bone mineralization leads to rickets. For many decades in Finland, vitamin D supplementation in infants has been successful in preventing rickets. However, along with increasing knowledge of non-skeletal vitamin D actions, optimal serum 25-hydroxyvitamin D (25OHD) concentration, has been under debate. The optimal vitamin D status and the dose of supplemental vitamin D in different populations may differ considerably. This doctoral thesis aimed to define the prevalence of vitamin D deficiency (serum 25OHD less than 50 nmol/l) in Finnish children, focusing on individuals with high risk of vitamin D deficiency, and studying in chronically ill children the factors that further increase the prevalence of low serum 25OHD concentration. Vitamin D interventions in infants and young adults enabled examination of the effect and safety of higher than currently recommended vitamin D supplementation, and exploring vitamin D and mineral metabolism in more detail. The study populations comprised 113 healthy term newborns and 42 young adults who participated in vitamin D intervention, and 1,335 children followed at Children s Hospital Helsinki between 2007 and 2010 for a chronic illness. The vitamin D interventions were double-blinded controlled randomized trials. The newborns received either 10, 30 or 40 µg of vitamin D3 daily from 2 weeks to 3 months, and blood samples were obtained at baseline and at 3 months. The young adults were either normal weight (n=24), or suffered from severe childhood-onset obesity (n=18). Both groups received either placebo or 50 µg of vitamin D3 daily for 12 weeks. Blood samples were taken at baseline, 6 and 12 weeks. Data on chronically ill children were collected in a retrospective manner from the hospital laboratory database. The prevalence of vitamin D deficiency exceeded 40% in the study cohorts: especially adolescents and obese individuals were at risk for low 25OHD concentration. Seasonal variation was evident in school-age children, with the lowest prevalence of vitamin D deficiency in summer, and the highest prevalence in winter and spring. In younger children, vitamin D deficiency was less prevalent, and seasonal variation was lacking. Daily vitamin D3 supplementation with 30 to 40 µg in infants, and 50 µg in young adults was safe in short-term follow-up. With good adherence to intervention, both 30 and 40 µg dosing increased infant 25OHD concentration to more than 80 nmol/l. The vitamin D3 intervention did not affect serum intact fibroblast growth factor 23 (FGF23) concentrations, but a distinct sex difference was observed, girls having higher concentration than boys at three months of age. Obesity associated with inferior response to vitamin D3 supplementation. Obese individuals receiving 50 µg vitamin D3 daily achieved similar 25OHD concentrations as normal-weight subjects who received placebo. Chronically ill children and obese subjects need individualized vitamin D supplementation and follow-up.
  • Janér, Cecilia (Helsingin yliopisto, 2016)
    Respiratory distress is a major contributor to morbidity in newborn infants. Insufficient clearance of lung liquid at birth causes maladaptation (transient tachypnea of the newborn, TTN) primarily in late preterm (delivery at 34 0/7 to 36 6/7 gestational weeks) and term infants. In small preterm infants excess lung liquid further complicates respiratory distress syndrome (RDS) caused by a lack of pulmonary surfactant. Importantly, the risk of respiratory morbidity remains elevated in early term infants (i.e. delivery at 37 0/7- 38 6/7 gestational weeks), especially if delivered by elective cesarean section (CS). Thus, major risk factors for respiratory morbidity include preterm delivery or delivery by elective CS. In both prematurity and CS, the hormonal milieu of the fetus, important in adaptation to air-breathing, differs from that of the term vaginally delivered infants. In particular, glucocorticoids (GCs) play an important role in the maturation of the fetal lung. GCs are often administered antenatally to mothers at risk of preterm delivery as an attempt to reduce respiratory morbidity in their preterm infants. Although relevant to preventive and treatment strategies for respiratory distress in newborn infants, data on airway ion and liquid transport and their hormonal regulation in human infants are limited. Therefore, the aim of this thesis was to acquire new data on lung liquid clearance and its molecular mechanisms in newborn infants. In particular, we studied their hormonal regulation during pulmonary adaptation. We used ultrasound to estimate the amount of lung liquid in term newborn infants, and compared infants delivered vaginally (VD) and by elective CS. The gene expression of the epithelial sodium channel (ENaC), Na-K-ATPase and serum- and glucocorticoid-inducible kinase 1 (SGK1) were determined in airway epithelial cells from nasal epithelium, used as a surrogate for the lower airway epithelium. Gene expression was quantified by measuring mRNA levels of these genes with a real-time reverse-transcription polymerase chain reaction. We determined the concentration of cortisol hormone in umbilical cord blood and saliva with liquid-chromatography tandem-mass spectrometry or enzyme-linked immunosorbent assay. The relationship of ENaC, Na-K-ATPase and SGK1 gene expression with cortisol was studied in cohorts of late preterm and term infants. In preterm infants, we studied the effect on airway ENaC expression of a repeated antenatal dose of the glucocorticoid betamethasone to the mother. The term infants and the majority of the preterm infants in the studies were born at the Women’s Hospital, Helsinki University Hospital. The lung liquid content of the term, healthy infants at three hours postnatally was significantly higher in infants delivered by elective CS than in VD infants. The umbilical cord cortisol concentration was lower in CS than in VD infants, and CS was associated with lower SGK1 expression at 1.5 hours after delivery. At this time point, the airway expression of ENaC, Na-K-ATPase, and SGK1 was lower in infants delivered late preterm and early term than in infants delivered ≥ 39 weeks. In addition, ENaC expression of late preterm and term infants correlated positively with umbilical cord blood and salivary cortisol concentrations. However, a repeat antenatal dose of betamethasone to the mother had no significant effect on ENaC expression in the preterm infants. This thesis work confirms that compared with infants delivered vaginally the amount of lung liquid remains more abundant after CS, and that ultrasound is a useful tool for evaluating lung liquid content in newborns. Low SGK1 expression after CS delivery and lack of labor could contribute to the insufficient activation of transmembrane sodium transport causing defective liquid absorption. In infants delivered < 39 weeks of gestation airway epithelial amounts of ENaC, Na-K-ATPase, and SGK1 mRNAs were all lower than in those delivered ≥ 39 weeks. These physiological data are in line with the epidemiologic evidence of a higher risk for respiratory morbidity in infants delivered before 39 weeks of gestation, especially when delivered by CS. Thus, our data support the practice of postponing elective CS until 39 weeks. Our findings provide valuable information for planning strategies aimed at preventing or treating respiratory distress in preterm and term infants.
  • van Adrichem, Arjan (Helsingin yliopisto, 2016)
    Small guanosine triphosphatases (GTPases) are a family of low molecular weight guanosine diphosphate (GDP)-/guanosine triphosphate (GTP)- binding proteins that act as molecular switches , regulating key cellular processes, such as cell proliferation and differentiation. The activity of small GTPases is controlled by guanine nucleotide exchange factors (GEFs), which facilitate nucleotide exchange from GDP to GTP, and GTPase activating proteins (GAPs), which stimulate intrinsic GTP hydrolysis of the GTPase. Complex, yet unresolved, mechanisms maintain the precise spatiotemporal regulation of these proteins, which is essential for accurate cellular signaling. Accordingly, aberrant function of small GTPases is associated with a large number of human diseases, such as cancer, neurodegenerative diseases and inflammatory disorders. The availability of small molecule modulators of GTPase activity would be highly valuable to improve the understanding of small GTPases of the Ras superfamily and their regulators. However, very few probes of this kind currently exist. To address this gap, I have focused on discovering different small GTPase-modulating probes through different screening approaches. MgcRacGAP is a GAP protein for Rho family small G-proteins and a key regulator of cytokinesis. After almost two decades of research, the detailed functional role, its relevant target GTPase and its GAP activity in cytokinesis are not yet fully resolved. Furthermore, like many other regulators of GTPase activity, its overexpression has been linked to different malignant properties, such as epithelial-to-mesenchymal transition, cell polarity and (invasive) migration, as well as correlated to a poor clinical prognosis in many types of cancers. To be able to investigate the biological role of MgcRacGAP, we first addressed the lack of chemical tools to probe its function by developing a high throughput screening strategy to identify compounds inhibiting its GAP function. The discovery of MgcRacGAP Inhibitor Compound 1 (MINC1), a selective MgcRacGAPRac1 inhibitor, represents the first described selective small molecule inhibitor of a Rho GTPase activating protein and shows that development of inhibitors of small GTPase activating proteins is possible. We utilized MINC1 to study the function of MgcRacGAP in cell division as well as the role in the events that regulate signal transducer and activator of transcription 3 (STAT3) signaling. Notably, we showed that MINC1- mediated inhibition of MgcRacGAP caused impaired mitotic spindle formation during the metaphase, which suggests that the GAP activity of MgcRacGAP plays an important role in mitosis. To address the role of MgcRacGAP in the events that regulate STAT3 phosphorylation and subsequent nuclear translocation, we used both MINC1 treatment and small interference RNA (siRNA)-mediated gene silencing of MgcRacGAP. With these complementary techniques we showed that inhibition of MgcRacGAP triggers STAT3 phosphorylation caused by a Rac1-PAR3-IL6- IL6R-JAK2 mediated autocrine/paracrine mechanism. Due to their key role in different essential cellular processes, non-specific inhibition of GTPases or their regulators is expected to result in significant risk of side effects. Compounds that interfere with specific protein-protein interactions are expected to circumvent this problem, yet most current screening methods fail to detect these. To address this, we have developed a protein-protein interaction inhibitor screening strategy for the oncoprotein Ras and identified ten compounds that inhibited GTP hydrolysis in a concentration dependent manner. Four of these compounds could not be detected with the established method. Of the three compounds that showed efficacy in Ras signaling dependent cell lines, one compound had a direct effect on the activation status of Ras. In summary, this thesis describes i) the discovery of the first RhoGAP inhibitor named MINC1, ii) the application of MINC1 to elucidate the role of MgcRacGAP in the activation and nuclear translocation events of STAT3 and iii) the development and exploration of a new screening strategy to discover Ras protein-protein inhibitors.
  • Peltola, Mikko (Helsingin yliopisto, 2016)
    This thesis analysed the impact that the introduction of new technology may have on the quality of care in a hospital setting. Technological change in medicine is rapid, as new devices are constantly taken into use in patient care, and new methods of delivering the care to patients are introduced, replacing existing routines and creating new practices all with the aim of benefitting patients. The present thesis focused on total hip and knee replacement, by analysing the introduction of implants, and by assessing the effects that a change in the production network (technology) via the launch or closure of an entire unit performing arthroplasty had on the quality of surgery as assessed by revision risk. Data for this study came from the Finnish Arthroplasty Register data on total joint replacements, Finnish administrative data on hospital discharges, prescribed medication, special reimbursements to medication and cause-of-death statistics. All total hip and knee replacements performed because of primary osteoarthritis in the period 1998 to 2011 were included. Survival analysis, with the revision of the joint in the follow-up as the outcome measure, was applied in the studies. For both total hip and knee replacement, the introduction of an implant in a hospital increases the risk of revision within three to five years, respectively, after the primary operation. The risk was higher for knee implants. For both surgeries, this learning effect was found to be implant specific. Of the analysed ten most common total hip and knee implant models, one hip and four knee implant models showed a learning curve, with the risk for reoperation decreasing with the greater number of surgeries performed with the implant model. Launching of arthroplasty surgery in a unit was not associated with a learning curve, but in units terminating total hip replacement surgery, the last 100 patients had worse short-term implant survivorship compared to patients who had had surgery in these units before the closure stage. The introduction of total hip and knee implants in a hospital was associated with increased revision risk in the introductory phase for some implant models. The introduction of new medical devices should be carefully considered in hospitals. The personnel acquiring new devices for use in a hospital should base their decisions on solid evidence of the efficiency of the technology and practice beforehand with the new devices. In addition, patients should be informed when new technology is introduced in their treatment and given the possibility to decline its use. On the health system level, health policy makers should take into account the result that unit closures may affect the quality of care in the closure stage, and try to guarantee stable operation settings for patients.
  • Ylikoski, Ari (Helsingin yliopisto, 2016)
    The diagnosis of Parkinson's disease has remained essentially a clinical one. The diagnostic criteria consist of cardinal motor symptoms and signs, such as bradykinesia and at least one of the following: rest tremor, muscular rigidity or postural instability. However, non-motor symptoms (i.e. cognition, mood, sleep, pain, dysautonomy) constitute a major clinical challenge. The total burden of non-motor symptoms is likely to be more important than the motor symptoms in determining the quality of life across all stages of the disease. The current study aims to evaluate, by means of a structured questionnaire approach, the occurrence of sleep disorders, sleeping difficulties, health-related quality of life and other comorbidities in a non-selected population of Finnish Parkinson patients. The response rate was 59% (N=854). The occurrence of rapid eye movement sleep behavior disorder was 39.0%, restless legs syndrome 20.3%, chronic insomnia disorder 36.9%, narcolepsy like symptoms 11.0%, sleep disordered breathing 22.1%, respectively. Low quality of life occurred in 45.0% of the participants, depression in 20.9%, excessive daytime sleepiness in 30.2%, fatigue in 43.9%.
  • Kyhälä, Lea (Helsingin yliopisto, 2016)
    Acute pancreatitis (AP) is a common abdominal disease with no specific treatment. The clinical manifestation of AP can vary widely from a mild self-limiting disease to a potentially life-threatening severe form. Severe acute pancreatitis (SAP) develops in about 20-25% of patients with AP, and the severe disease is associated with the development of such complications as pancreatic necrosis and distant organ failure (OF). A complicated inflammatory reaction precedes development of OF and is accompanied by disturbances in the coagulation system. The patients who will develop SAP need to be identified early in the course of the disease and aggressively treated (i.e. vigorous fluid resuscitation in intensive care unit) to prevent mortality. The aim of this study was to investigate whether ecto-5 nucleotidase (CD73) predicts the development of SAP and whether recombinant activated protein C (APC) treatment alleviates the course of SAP. We evaluated also the effects of recombinant APC treatment on occurrence of bleeding complications, course of systemic inflammation, and coagulation cascade disturbances in patients with SAP. The clinical study consists of four parts. All of the patients investigated had AP when admitted to Helsinki University Central Hospital. The first study comprised 161 patients with AP. Their serum levels of soluble CD73 (sCD73) were analyzed by three different methods (activity, protein concentration, and mRNA levels) and the association of the levels with different severity grades of AP was evaluated. In addition, the ability of sCD73 to predict SAP was examined. The second study was a pilot, double-blind clinical trial in which 32 patients with SAP were randomized to receive recombinant APC treatment or placebo, and the effects of APC on evolution of organ failure and and the safety of APC treatment were analyzed. In the third study, the effects of recombinant APC treatment on markers of systemic inflammation were examined. The changes on humoral and cellular markers of systemic inflammation were determined. In the fourth study, the effects of recombinant APC treatment on coagulopathy in SAP were evaluated. The plasma levels of selected biomarkers of coagulation and physiological anticoagulation were determined. The results showed that the sCD73 levels on admission to hospital correlated inversely with the severity of AP and the sCD73 activity is able to predict the severe form of AP among patients with no signs of OF on admission. We found no beneficial effects of recombinant APC treatment on evolution of organ failures or mortality in SAP. No significant bleeding complications occurred. Recombinant APC treatment did not alter the course of systemic inflammation, but we could monitor the changes in parameters of systemic inflammation reaction produced by SAP. We found that the SAP patients showed coagulopathy and the recombinant APC treatment slowed normalization of the levels of coagulation and anticoagulation markers in SAP. Based on these studies, sCD73 activity predicts the development of SAP. The recombinant APC treatment does not have a significant effect on evolution of OFs or on mortality in SAP. The effects of APC treatment on parameters of systemic inflammatory reaction are minor. The coagulation in SAP is hindered and APC-treated patients achieve normal homeostasis of coagulation slower than patients receiving placebo.
  • Leskinen, Katarzyna (Helsingin yliopisto, 2016)
    Y. enterocolitica is a human pathogen that cause mostly food-borne yersiniosis, usually a diarrheal disease sometimes followed by post-infectious reactive arthritis. Yersiniosis is considered to be the third most common cause of gastroenteritis in Europe. In Finland both Y. pseudotuberculosis and Y. enterocolitica cause hundreds of human infections annually. The aim of the study is to characterize the intricate regulatory networks of Yersinia especially those that control the expression of the virulence factors. To achieve that goal three regulators (YbeY, RfaH and Hfq) were initially selected. The absence of ybeY gene in Y. enterocolitica serotype O:3 resulted in misprocessing of 16S rRNA, severe decrease of growth rate, disturbed regulation of the Yersinia virulence plasmid genes expression and affected the expression of small RNAs. Furthermore, the ybeY mutant displayed impairment of many virulence-related features, and decreased infectivity in the cell infection model. The study revealed that RfaH of Y. enterocolitica O:3 acts as a highly specific regulator that enhances the transcription of selected operons involved in biosynthesis of lipopolysaccharide. Furthermore, the transcriptomics study indicated that some changes seen in the rfaH mutant strain were actually due to indirect responses to the loss of O-antigen. Moreover, the lack of RfaH resulted in attenuated stress response and lower resistance to selected chemical compounds. The loss of Hfq caused impairment in growth, elongation of the bacterial cells, and decreased the resistance of bacteria to heat, acid and oxidative stresses, as well as attenuation in mouse infection experiments. Moreover, this study revealed that several alterations typical for the hfq-negative phenotype were due to derepression of the transcriptional factor RovM. In conclusion, all the studied mutations caused different alterations in gene regulatory networks and eventually led to a compromise in the virulence of Y. enterocolitica O:3. The studied mutants showed significant decrease in resistance to different environmental conditions and alterations in the bacterial physiology that contribute to vitality and ability to establish infection in host organism.
  • Polvinen, Anu (2016)
    This study examines the socioeconomic inequalities in disability retirement due to various diseases in the period 1988-2009. It also outlines the contribution of diseases, health behaviours and working conditions to the socioeconomic differences in disability retirement. In addition, this study portrays the socioeconomic differences in mortality from various causes of death after disability retirement due to different diseases. In this study, we used administrative register data from various sources linked together by Statistics Finland. We also made use of the nationally-representative Health 2000 survey, to which register-based data have been linked. From the early 1990s, the incidence of disability retirement has decreased in all socioeconomic classes. We found large socioeconomic differences in disability retirement due to musculoskeletal diseases, psychoactive substance use and cardiovascular diseases. The socioeconomic differences due to depression were less significant. Musculoskeletal diseases contributed most to the overall socioeconomic differences in disability retirement. Socioeconomic inequalities due to all causes, and especially due to musculoskeletal diseases, were largely mediated through working conditions and health. The socioeconomic inequalities in mortality after disability retirement were smaller than in the general population. Small socioeconomic inequalities in mortality after disability retirement due to mental disorders and cardiovascular diseases were found. The importance of working conditions should be noted when attempting to lower the number of disability retirements. More attention should be paid to the importance of adverse physical working conditions among manual workers. Small socioeconomic differences in mortality after disability retirement may indicate that disability retirees are a select group displaying lowered work ability and health.
  • Salonen, Kirsi (Helsingin yliopisto, 2016)
    The incidence of type 1 diabetes has been increasing over recent decades. The reason for the increase, and the initial trigger for the autoimmune process remain unknown. Children with significantly increased risk for type 1 diabetes can be recognized from the general population based on their HLA-genotype and a panel of autoantigen-specific antibody assays. Recently a novel autoantibody specificity was discovered, the ZnT8 autoantibodies (ZnT8A). The advanced glycation end products (AGEs) are produced via glycation reactions between reducing sugars and proteins. AGEs and their interaction with the receptor for AGEs (RAGE), have been implicated to play a role in the pathogenesis and complications of diabetes. The soluble form of RAGE (sRAGE) is counteracting the effects of proinflammatory, membrane-bound RAGE. A decrease in sRAGE has been observed in various studies on acute inflammation. Polymorphisms of the gene encoding RAGE, AGER, are associated with the risk and the complications of diabetes according to previous studies. This thesis set out to define the characteristics of ZnT8A and RAGE in clinical and pre-clinical type 1 diabetes. Among the newly diagnosed children with type 1 diabetes, 63% tested positive for ZnT8A. ZnT8A were associated with age and metabolic state at diagnosis as well as to HLA genotype. ZnT8A-assay did not improve the detection rate of beta-cell autoimmunity. Concentrations of sRAGE correlated positively with age in children with newly diagnosed type 1 diabetes, but not in controls. Diabetic ketoacidosis at diagnosis and the high-risk HLA-genotype were related to lower sRAGE. Two polymorphic variants of the AGER gene, associated with increased risk for type 1 diabetes, correlated with reduced sRAGE. Prediabetic children had higher sRAGE than autoantibody-negative controls. A reduction in sRAGE coincides with the appearance of autoantibodies in children progressing to overt type 1 diabetes, but not in healthy controls, or in children seroconverting to autoantibody positivity later in childhood. After the seroconversion, the sRAGE concentrations remained stable. The RAGE/AGE ratio was higher in the cases than in the controls. To conclude, sRAGE, which has been considered cytoprotective in previous studies, is positively associated with older age at disease onset, protection from metabolic decompensation at diagnosis and AGER genotypes with a lower risk for type 1 diabetes. Children, who seroconvert to humoral islet cell autoimmunity early in childhood, experience a drop in sRAGE coinciding with the appearance of the first autoantibodies. They have higher sRAGE and sRAGE/AGE-ratio than the controls before seroconversion. These observed associations might be a result of an intrinsic protective mechanism, which fails at seroconversion.
  • Hyvärinen, Satu (Helsingin yliopisto, 2016)
    The complement system is a network of over 30 plasma proteins that act in, for example, protecting the body against invading microbes and removing damaged self cells. Complement activation proceeds in a cascade-like fashion, resulting in deposition of certain protein fragments on cell surfaces and liberation of others to the fluid nearby. Due to the destructive nature of complement attacks, it is imperative that this system be stringently controlled. When regulation fails, deleterious complement activation on self cells follows. This is the case for example in atypical hemolytic uremic syndrome (aHUS), a rare thrombotic microangiopathy characterized by hemolysis, thrombocytopenia and renal impairment. In roughly two thirds of aHUS patients, abnormalities in complement genes can be detected. A frequent observation is a mutation in the regulator factor H (FH). The majority of FH mutations in aHUS cluster in the two C-terminal domains of the protein, i.e. FH19-20. These are critical for directing FH onto cell surfaces under complement attack. Several important aspects of the syndrome have been uncovered, but the mechanisms underlying aHUS pathogenesis remain incompletely understood. The general aim of this work was to better explain the molecular events leading to aHUS. First it was asked, whether disturbances in the recently described interactions between FH19-20 and malondialdehyde (MDA) adducts could be involved in aHUS. To study this, binding assays employing MDA-modified proteins and wt and mutant FH19-20 fragments were performed. FH19-20 binding was observed only if a high density of MDA adducts on the surface was present. Since such extensive MDA modification of cell surface proteins is improbable in vivo, it was concluded that disturbed recognition of MDA adducts on cells by FH is unlikely to be relevant in aHUS. Next, the hypothesis of plasminogen to regulate propagation of the complement cascade on cell surfaces was tested. Serum complement was activated on erythrocytes, endothelial cells, and platelets with or without added plasminogen, and complement activation determined as cell lysis or deposition of C5b-9 or C3b. Plasminogen was observed to cause only a very minor inhibition of complement activation on platelets. It was concluded that reduced complement control by plasminogen does not properly explain the recent association of plasminogen deficiency with aHUS. The final aim of this work was to determine the role of sialic acid in FH-mediated regulation on not only erythrocytes, but also on endothelial cells and platelets. To this end, the abilities of several FH19-20 fragments carrying aHUS-associated mutations to antagonize FH function on different types of cells was compared. Flow cytometry detection of FH binding and C3b deposition revealed identical patterns of FH19-20 function on all types of cells. Removal of sialic acid from cells impaired FH function on them. With the help of recent structural data, it was concluded that mutations impair simultaneous binding of the C-terminus of FH to surface sialic acid and C3b, providing a unifying explanation for association between the C-terminus mutations of FH and the clinical disease aHUS. In conclusion, the work presented in this thesis improves our understanding of the pathogenic mechanisms involved in aHUS. Most importantly, the results show that in aHUS, the underlying common defect of various FH C-terminus mutations is the inability to simultaneously bind sialic acid and C3b on cells under complement attack.
  • Barreto, Goncalo (Hansaprint Oy, 2016)
    Osteoarthritis (OA), the most common form of arthritis, is estimated to be in the top 5 leading causes of disability worldwide. Yet OA incidence is estimated to keep growing partly due to the overall worldwide trend of increased obesity and ageing population. Cartilage erosion, a hallmark of OA, has its onset in the traumatic events caused by incorrect biomechanical loading of the joint and the consequent biological response. Currently we still poorly comprehend the molecular pathophysiology of preclinical and clinical symptomatic OA, which consequently results in no current available therapy to prevent OA progression. We hypothesize that innate immunity and its receptor, in particularly toll-like receptors (TLRs), could be major drivers of OA disease progression and onset. The process could be initiated as a proinflammatory reaction against extracellular matrix (ECM)-derived damage-associated molecular patterns (DAMPs). DAMPs accumulate in avascular articular cartilage as a result of traumatization and degeneration, leading directly at their source to a reactive chondrocyte-mediated and TLR-dependent production of proinflammatory and algogenic secondary mediators, which then cause a secondary synovitis with consequent joint pain. For this propose, we collected cartilage and isolated primary chondrocytes from a total of 27 OA patients. Synovial fluid was obtained from knee meniscectomy, total knee arthroplasty (TKA) due to OA, and rheumatoid arthritis (RA) patients generating a total of 30 patient samples. HEK (human embryonic kidney)-blue TLR4 reporter cell line, primary OA chondrocytes, and cartilage explants were used for functional studies. Our results confirmed that TLR1, TLR2 and TLR9 expression is present in healthy primary chondrocytes isolated from articular cartilage, and derived from chondroprogenitors. During our chondrogenesis differentiation studies initial high expression of TLR1, TLR2 and TLR9 was significantly reduced to baseline levels. We demonstrated that proinflammatory cytokine tumour necrosis factor alpha (TNF-α) is able to increase the expression of TLR2 in both healthy primary chondrocytes and mesenchymal stem cells (MSC) derived chondrocytes cultured for 21 days. TNF-α stimulation was demonstrated to induce cartilage degradation in de novo ECM matrix from pellet cultures of MSC-derived chondrocytes cultured for 21 days. This implicates TNF-α as an inducer of matrix degradation, with wide implications in the use of MSCs strategies in cartilage repair strategies for OA. Our study also added further evidence of a role for TNF-α in TLR-innate immunity in the OA synovial joint. TLRs protein expression in cartilage between knee and first carpometacarpal (CMC-I) joints from OA patients was shown to be strikingly different. Our study demonstrated for the first time all TLRs being expressed at protein levels in articular cartilage from knee OA patients. Moreover, we demonstrated that their expression is up-regulated in a cartilage zone-dependent fashion accordingto the histological progression of knee OA. TLRs expression in cartilage from CMC-I OA patients was highly heterogeneous although it followed an expression pattern according to TLRs cellular organization. This indicates that TLR-mediated innate immune response between the two joints may be significantly different. Decorin (DCN), a known small structural proteoglycan with leucine-rich repeats (SLRP) ligand able to activate TLR2 and TLR4, was discovered in knee synovial fluid from OA and RA patients. We confirmed the ability of soluble DCN (sDCN) to activate to TLR4 signaling. However, the observed low and stable concentration levels across the studied groups mean that this may not be of clinical relevance in OA pathogenesis and the associated TLR-mediated inflammatory events. Biglycan (BGN), another known SLRP ligand able to activate TLR2 and TLR4, was discovered in knee synovial fluid from OA and RA patients. Interestingly, we discovered that soluble BGN (sBGN) is upregulated in synovial fluid from OA and RA patients. sBGN ability to activate TLR-innate immunity as confirmed to be essentially activated through TLR4 signaling by studies in articular chondrocytes and human HEK-blue TLR4 reporter cell line. The sBGN stimulation lead to the upregulation and release of proinflammatory cytokines, matrix-degrading enzymes and the release of ECM degradation products. Overall, the results of this thesis demonstrate that TLRs are markedly present in articular cartilage from OA patients at different progression stages of the disease. The detection of BGN and DCN in synovial fluid, and their ability to activate TLR4-mediated proinflammatory cellular responses gives new knowledge of proinflammatory molecules present in the OA synovial joint. An enhanced molecular understanding of the triggering mechanisms by which TLRs are activated and regulated during OA progression stages may help find therapeutic options in the treatment of OA.
  • Ylösmäki, Leena (Helsingin yliopisto, 2016)
    Src homology 3 (SH3) domains are small modular protein structures that recognize and bind to short proline-rich sequence motifs in their ligand proteins. Viral proteins may also harbor such binding motifs and thereby serve as SH3 ligands in order to regulate the host cell signaling to support virus growth and replication, and to modulate virulence. The aim of this study was to examine if influenza A virus (IAV) might also use this strategy to take control of its host cells, and to characterize possible SH3 domain-containing host cell binding partners of IAV to establish their role in the cell biology of IAV infection. IAVs cause seasonal epidemics and occasional pandemics that pose a major threat to human health. The nonstructural protein 1 (NS1) is an important virulence factor of IAV. It is a multifunctional protein that suppresses the host interferon response via multiple mechanisms. Another function of NS1 is to activate phosphatidylinositol-3 kinase (PI3K) signaling in the host cell through direct binding to the p85β regulatory subunit of PI3K. The NS1-induced activation of PI3K is required for efficient replication of many IAV strains. We found that NS1 proteins from some IAV strains contain an SH3 binding site that mediates strong and selective binding to the N-terminal SH3 (nSH3) domain of Crk-family proteins, an important class of adaptor proteins involved in the coordination of cellular signal transduction. This Crk SH3 binding motif was present in the NS1 of infamous 1918 Spanish Flu pandemic virus as well as in many contemporary avian IAV strains. In contrast, it is not found in most NS1 proteins of seasonal human IAV strains. We found that the capacity of avian and Spanish Flu NS1 proteins to interact with Crk SH3 domains provided them with a greatly enhanced capacity to activate PI3K signaling. The molecular mechanism underlying this potentiation was found to be due to a reorganization of the natural PI3K-Crk complex by the SH3-binding competent NS1 protein. Of note, Crk proteins were found to indirectly (via p85β binding) contribute also to PI3K regulation by NS1 proteins of common human IAV strains that lack an SH3 binding motif and a capacity for direct Crk recruitment. Moreover, we found that the role of the NS1/Crk interaction is not limited to PI3K regulation. We observed that binding of NS1 to the Crk SH3 domain induced a robust nuclear accumulation of the predominantly cytoplasmic Crk proteins. This nuclear translocation of Crk proteins was shown to lead to a change in tyrosine phosphorylation pattern of nuclear proteins. In summary, our studies establish Crk adaptor proteins as important cellular co-factors exploited by the IAV virulence factor NS1 to manipulate host cell signaling. These results increase our understanding of the role of NS1 in IAV cell biology, and reveal possible new targets for future antiviral drug development aimed against critical host cell interactions rather than highly mutable viral proteins.
  • Muona, Mikko (Helsingin yliopisto, 2016)
    Epilepsies are a heterogeneous group of central nervous system diseases characterised by recurrent epileptic seizures. They are one of the most common neurological diseases with a lifetime prevalence of ~4%. Epileptic seizures are also a common comorbidity of various neurobiological disorders where epilepsy is not the primary diagnosis. Most epilepsies have a genetic origin, either monogenic or polygenic, however, the causal genetic variants have remained unknown in a substantial proportion of individuals with epilepsies. Over the past decade, technological advances in DNA sequencing have allowed the characterisation of the genetic basis of human disorders rapidly and efficiently. One of the most widely used methods is whole-exome sequencing (WES) where genetic variants in the protein coding regions of the genome, the exome, are captured. Even though the exome constitutes only ~1.5% of the genome, the majority of disease-causing variants underlying severe, monogenic diseases are located in the protein coding regions. Here, we aimed to decipher the molecular genetic basis of severe epilepsy syndromes by utilising WES to identify disease-causing genetic variants in patients without a genetic diagnosis. We studied patients with progressive myoclonus epilepsy (PME, n=84) or severe infantile-onset epileptic syndromes (n=30), which are one of the most devastating forms of genetic syndromes with epilepsy and characterised by frequent, pharmacoresistant seizures and poor prognosis. Given that the patients had undergone genetic testing to varying extent prior to this study, we specifically aimed to establish novel genes and molecular biological mechanisms underlying these syndromes. We made substantial progress in understanding the genetic architecture and molecular basis of the studied syndromes. For PMEs, we established a new major genetic cause and also expanded the genotypic and phenotypic spectrum of previously established disease genes. For severe infantile-onset epileptic syndromes, we identified one new, definite causal gene and one that requires identification of additional patients to confirm the causal role. The three newly identified disease genes represent three different molecular functions that together give new insight on epileptogenic mechanisms. The new PME subtype is caused by a heterozygous missense variant c.959G>A (p.Arg320His) in KCNC1 that was identified in 11 unrelated patients (13%) in the PME exome sequencing cohort. We have subsequently identified six additional patients. The gene encodes a potassium ion channel KV3.1 that has an important role in generating action potentials in the central nervous system, with the mutation disrupting the ability to transport potassium ions across the cell membrane. This mutation occurs in most families de novo, that is, it is a newly arising mutation. Based on the estimated mutation rate, the recurrent KCNC1 mutation is a worldwide cause of PME with likely hundreds of affected individuals globally. In five families with altogether nine affected siblings, we identified compound heterozygous variants in UBA5 as the cause of an infantile-onset syndrome characterised initially by irritability, followed by epilepsy, dystonic movements, moderate to severe intellectual disability, microcephaly and stagnation of development. The gene encodes an activating enzyme for UFM1, which is a small ubiquitin-like protein that is conjugated to its target proteins. The function of the highly conserved UFM1 conjugation system is still largely unknown. Functional analysis of the UBA5 mutants suggest that the identified variants cause reduced enzymatic activity of UBA5. Symptoms of the UBA5 patients and our findings in the central nervous system specific knockout mice for Ufm1 together indicate that UFM1-cascade is essential for normal development and function of the central nervous system. Finally, we identified compound heterozygous variants in ADAM22 as the likely cause of the disease in a patient with an infantile-onset rapidly progressing encephalopathy with epilepsy and cortical atrophy. The gene encodes a postsynaptic protein that functions as a receptor for LGI1, and we show that the identified variants abolish the ability of ADAM22 to bind to LGI1. The LGI1-ADAM22 complex is an antiepileptogenic factor regulating synaptic transmission throughout life. Highlighting the important role of this complex, knockout of Adam22 and Lgi1 in mice causes lethal epilepsy. Autosomal dominant LGI1 variants also cause epilepsy in humans. Identification of a patient with loss-of-function variants in ADAM22 suggest that also this gene is linked to epilepsy in humans. This connection should be confirmed through identification of additional affected individuals with ADAM22 variants. Altogether, this thesis demonstrates the power of WES in identification of causal genetic variants even in phenotypically heterogeneous patient cohorts subjected to prior genetic screenings. The findings improve diagnostics of these syndromes, increase knowledge of the underlying molecular mechanisms and potentially aid in developing new therapeutic interventions. Finally, for these families, establishment of the genetic diagnosis ends years of uncertainty and frustration of not knowing the cause of the disease and prevents need for unnecessary diagnostic testing.
  • Salo, Kati Hannele (Helsingin yliopisto, 2016)
    This work deals with the paleopathology of 555 skeletons from nine cemeteries in southern Finland dating from the 11th to the beginning of the 19th century. This is the first large-scale osteological investigation of Finnish human remains, since skeletal material in Finland is not usually preserved well. A greater number of younger individuals were identified from coastal sites than from inland sites, which may be due to real health differences, or to fertility differences between coastal towns and inland sites, or simply to taphonomy. Previous bioarchaeological studies have shown that early towns were unhealthy environments, which may also be a factor. Dental diseases were found to be more common in females, and trauma in males, as in previous bioarchaeological studies. Dental diseases, joint diseases, and trauma are age-progressive diseases, and cribra orbitalia and metabolic diseases were found more often in subadult individuals, as expected. No association between stature and any of the health-related parameters could be observed. Dental diseases are more common in more modern populations than in more ancient ones, as expected. These questions should, however, be studied further with the help of paleodietary/paleogenetic analyses. Most of the pathological lesions that were statistically significantly associated with each other were expected to be so, but some unexpected relationships also appeared. For example, skeletal trauma and entheseal changes were statistically significantly found more often in individuals with more dental calculus, whereas periapical lesions were statistically significantly associated with vertebral osteoarthritis. These unexpected co-ocurrences should be studied further, not only in bioarchaeology, but also in modern medicine, to discover whether these are just rare coincidences or whether these pathologies really do/did co-occur for some reason.
  • Arvilommi, Petri (Helsingin yliopisto, 2016)
    This study is part of a collaborative bipolar research project between the Unit of Mental Health of the National Institute for Health and Welfare, Helsinki (the former Department of Mental Health and Alcohol Research of the National Public Health Institute, Helsinki) and the Department of Psychiatry, Jorvi Hospital, Helsinki University Central Hospital (HUCH), Espoo, Finland. The Jorvi Bipolar Study (JoBS) is a prospective, naturalistic cohort study of 191 secondary-level care psychiatric in- and outpatients with a new episode of DSM-IV bipolar disorder (BD). Overall, the study involved screening 1,630 adult patients (aged 18-59 years) using the Mood Disorder Questionnaire (MDQ) for symptoms of bipolar disorder in the Department of Psychiatry, Jorvi Hospital, from January 1, 2002, to February 28, 2003, for a possible new episode of bipolar disorder. A clinical diagnosis of ICD-10 schizophrenia was an exclusion criterion for screening. The 490 consenting patients were interviewed with a semi-structured interview (SCID-I/P). Thereby, 191 patients were diagnosed with an acute phase of DSM-IV BD and included in the study. The patients participating were interviewed again 6 and 18 months after baseline. The course of the disease, with timing and durations of different phases, was examined by gathering all available data, which were then combined in the form of a graphical life chart. Observer- and self-reported scales were included at baseline and at both follow-up assessments. Also, the treatments provided were investigated at baseline and at both follow-up interviews. The aim in the first study was to investigate the adequacy of acute phase pharmacotherapy received by psychiatric in- and outpatients with a research diagnosis of BD I or BD II, including patients with and without a clinical diagnosis of BD. Information about treatments received during the index acute episode was gathered in the interview and from psychiatric records. Definitions of adequate acute-phase pharmacotherapy were based on published treatment guidelines. Only 42% of all 191 patients and 65% of those diagnosed with bipolar disorder received adequate treatment for the acute index phase. Clinical diagnosis of bipolar disorder was the factor most strongly independently associated with adequate treatment. In addition, rapid cycling, polyphasic index episode, or depressive index phase independently predicted inadequate treatment. Outpatients received adequate treatment markedly less often than inpatients. Lack of attention to the longitudinal course of the illness was another major problem area of treatment. Next, our aim was to investigate the adequacy of the maintenance-phase pharmacotherapy received during the first maintenance phase after an acute episode, following the same patients as in the first study. We defined adequate maintenance-phase pharmacotherapy based on published treatment guidelines. Of the patients with a maintenance phase in follow-up, adequate maintenance treatment was received by 75% for some time, but by only 61% throughout the maintenance phase and for 69% of the total maintenance time. Having adequate maintenance treatment throughout the maintenance phase was most strongly independently associated with having a clinical diagnosis of BD. In addition, inpatient treatment, rapid cycling, and not having a personality disorder predicted receiving adequate maintenance treatment throughout the maintenance phase. In addition, we investigated the continuity of attitudes toward and adherence to various types of psychopharmacological and psychosocial treatments among psychiatric in- and outpatients with BD I or II. During the 18-month follow-up, a quarter of the patients using mood stabilizers or atypical antipsychotics discontinued medication by their own decision, and of the medications continued, a third were not used regularly enough to provide a benefit. Overall, more than half of BD patients either discontinued pharmacotherapy or used it irregularly. The highest risk for discontinuing pharmacotherapy was present when the patients were depressed. Also, a quarter of the patients receiving psychosocial treatments did not adhere to the treatment. The main reasons patients gave for nonadherence toward pharmacological treatment were side-effects, lack of motivation, and a negative attitude toward the offered treatment; for individual/supportive psychotherapy, the reasons included practical barriers to coming to sessions and lack of motivation. Rates of nonadherence to mood stabilizers and antipsychotics did not differ, but the predictors did. Last, we investigated the prevalence and clinical factors predicting the granting of a long-term disability pension for patients with BD. We used register data to gather precise information on the pensions granted and their timing. During the 18-month follow-up after an acute episode, a quarter of the patients belonging to the labor force were granted a disability pension. Higher age, male gender, depressive index episode, comorbidity with generalized anxiety disorder (GAD) or avoidant personality disorder, and a higher number of psychiatric hospital treatments all independently predicted the granting of a disability pension. Moreover, patients subjective estimations of their vocational ability were surprisingly accurate in forecasting the granting of a future disability pension. In addition, the depression-related cumulative burden and the proportion of time spent in depression during the follow-up were important predictors. However, the predictors may vary depending on the subtype of illness, gender, and age group of the patient.