Lääketieteellinen tiedekunta

 

Recent Submissions

  • Helin, Tuukka (Helsingin yliopisto, 2017)
    Direct oral anticoagulants (DOACs), the thrombin inhibitor dabigatran and the anti-Xa inhibitors rivaroxaban and apixaban are are simple to use as standard dosing is recommended and no routine monitoring of coagulation is advocated. However, in certain clinical situations, e.g. acute thrombosis or bleeding, emergency surgery and renal or hepatic failure, assessing anticoagulant bioactivity is essential. The aims of this study were to firstly assess the effects of these DOACs in commonly used coagulation screening tests (PT, INR and APTT) using both spiked samples and samples collected from patients on treatment. Secondly, the availability of specific methods for drug effect assessment, namely drug-calibrated thrombin time (dabigatran) and anti-Xa (rivaroxaban, apixaban) assays were assessed in laboratory surveys. Thirdly, the effects of DOACs on these specific assays were explored using patient samples. Finally, the effect of DOACs on the global coagulation assay thrombin generation (TG) was assessed in patients using dabigatran, rivaroxaban or apixaban. The spiked sample laboratory surveys included a total of 86 European laboratories. The effects of all DOACs on INR were modest, but large reagent variability was noted in the responses (p<0.001). In APTT, rivaroxaban and apixaban prolonged the APTT only modestly, but with dabigatran there was a clear prolongation. Only about a fourth of the laboratories were able to provide more specific methods, TT or anti-Xa assays for drug quantification. Dabigatran effects in patient samples were studied 241 unselected patient samples. The PT was rarely prolonged and the APTT was in curvilinear relationship (R2 = 0.71) with dabigatran. However, the sensitivity effects among patients varied, with some patients having a normal APTT at dabigatran levels up to 160 ng/mL. Several specific assays, (diluted thrombin time, ecarin clotting assay and anti-IIa assay) accurately quantified dabigatran concentrations. In TG, a paradoxical increase was observed in the endogenous thrombin potential (ETP) and Peak TG, while lag time also prolonged with increasing concentrations of dabigatran. Rivaroxaban and apixaban effects on coagulation in patient samples were further assessed in a well-characterised group of patients using these drugs for thromboprophylaxis after orthopaedic surgery. Rivaroxaban peak drug levels were higher and trough levels lower than with apixaban, reflecting the once daily vs twice daily dosing of the drugs. In TG, the ETP response at peak drug levels was strong with rivaroxaban, whereas with the trough drug levels, the TG parameters were close to the baseline levels. With apixaban the responses were more stable. The RVVT, used as a qualitative test, detected prolongation only at rivaroxaban peak levels. In conclusion, DOACs present significant challenges in the clinical laboratory, as a wide variety of reagents are used in coagulation assays, and the drug responses also vary widely between individual patients. Many factors other than drug concentration influence the responses in coagulation assays and TG. In clinical practice, care and diligence is required when assessing the effects of DOACs on patients.
  • Parkkonen, Olavi (Helsingin yliopisto, 2017)
    Takotsubo cardiomyopathy (TTC) is an acute cardiac condition resembling in symptoms acute coronary syndrome (ACS), but without obstructive coronary artery disease. TTC develops almost solely in post-menopausal women and usually after preceding stress. Of all patients with ACS symptoms, TTC incidence is 2%. Due to similar symptoms and findings, differential diagnosis requires coronary angiography (CAG). The pathophysiology of TTC is unknown. Even though the accumulated evidence suggests a causative role for a catecholamine surge, other theories exist. Aborted myocardial infarction (MI) produces similar electrocardiography (ECG) and biochemical findings as in TTC. In such cases, because of non-stenotic coronary artery plaques, a dissolved coronary thrombus might show no any signs in the CAG, which could lead to an assumption of non-atherothrombotic etiology for the heart attack. In ACS, altered levels of proteolytic enzyme called matrix metalloproteinase 8 (MMP-8), and its inhibitor, the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), associate to plaque rupture. Their direct comparison between ACS and TTC remains unknown. The purpose of this thesis is to test whether either of two non-invasive methods: the ECG (I) or MMP-8 and TIMP-1 (III), could differentiate TTC from ACS. We also set out to find whether, after all, what is responsible for the TTC is transient thrombosis in the coronary circulation (II). Both our prospective and retrospective collection of patients resulted in 92 TTC cases. The demographics of our material were similar to those of other TTC reports worldwide. In the ECG study (I), a review and comparison of 57 TTC and ACS acute ECGs resulted in criteria differentiating the two with 63% sensitivity and 93% specificity. In cases of suspected ST-elevation myocardial infarction, such accuracy is insufficient for a decision against coronary intervention. Blood samples from 45 TTC patients and matching numbers of ACS patients and controls were analyzed for coagulation markers. Despite the similar acute-phase reaction in TTC and ACS patients, the TTC d-dimer levels matched those of controls, and were lower and less frequently above reference level than with those in ACS. The blood samples were further analyzed for MMP-8 and TIMP-1 levels showing, on admission, better differentiation between TTC and ACS by TIMP-1 than by troponin T (TnT). In TTC, low ejection fraction (EF) correlated with low MMP-8/TIMP-1 ratio. In conclusion, ECG lacked the ability to differentiate TTC from ACS that would have allowed avoidance of invasive diagnostics. Secondly, the coagulation results supported catecholamine and argued against the thrombosis theory. Finally, TIMP-1 emerged as a potential future biomarker in differentiation between ACS and TTC. Furthermore, in some TTC patients MMP-8 and TIMP-1 levels may explain more severe left-ventricle (LV) impairment.
  • Lorey, Martina (Helsingin yliopisto, 2017)
    The innate immune system is the first line of defence against microbial infections. Macrophages are the key cells of the innate immune system. Activated macrophages release inflammatory mediators through conventional and extracellular vesicle (EV)-mediated protein secretion. These protein secretion pathways are major components of intercellular communication in the immune system. Protein secretion is activated by inflammasomes in response to microbial components and endogenous danger signals. Inflammasomes are molecular platforms of the innate immune system that are critical to both local and systemic inflammation: they are involved in the pathogenesis of immune-mediated inflammatory diseases. Inflammasomes can be activated by both microbial stimuli as well as by endogenous danger signal molecules. The secretome of the cell is the pattern of all secreted molecules at a given time; it can provide important insights about the cell’s status as well as information about intercellular communication. The secretome can be studied by mass spectrometry (MS)-based proteomics; this technique provides a system-level overview of the current state of the cell. While traditional assays like ELISA or Luminex can quantify selected proteins in low pg/mL levels, they can only analyse a limited number of proteins. Proteomics offers the same sensitivity but supplemented with the capability of multiplex quantitative analysis of thousands of proteins in the same sample. The main goal of this work was to unravel the innate immune response of human macrophages activated by microbial stimuli with a focus on protein secretion using proteomics combined with bioinformatics and functional studies. The microbial stimuli used included Influenza A viruses (IAVs) and lipopolysaccharide (LPS), the cell wall components of Gram-negative bacteria. Both IAVs and Gram-negative bacteria are important human pathogens associated with aggressive infections. Influenza A virus and LPS are known to activate canonical NLRP3 inflammasome and non-canonical caspase-4/5 inflammasome, respectively. Inflammasome activation is followed by protein secretion which was studied in detail, with a special emphasis on unconventional protein secretion. In both studies, mediators of inflammation were released through EVs in response to influenza A virus infection and intracellular LPS stimulation. These included many danger signal proteins, cytokines and components of translational machinery. Our system-level characterization using modern mass spectrometry-based proteomics approach provides novel information how macrophages communicate to other cells through protein secretion. The results suggest that unconventional protein secretion is an essential part of the innate immune response to different activation stimuli.
  • Pussila, Marjaana (Helsingin yliopisto, 2017)
    Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. Yet, the mechanisms by which diet impacts colorectal tumorigenesis remain largely unknown. Colorectal cancer evolves as a multistep process, which requires a series of genetic and epigenetic alterations in growth regulatory genes. The process is accelerated in individuals with inherited cancer predisposition such as Lynch syndrome (LS) which is one of the most common inherited cancer susceptibility syndromes and caused by inherited mutation in one of the DNA mismatch repair (MMR) genes. CRC is thought to develop via the so called adenoma-carcinoma sequence. However, the early events that occur in colon mucosa prior to polyp formation remain unknown. The research presented here investigates the gene expression changes arising in histologically normal colonic mucosa as putative cancer-preceding events available for early detection. This was achieved by pursuing a long-term feeding experiment in the mouse model for human Lynch syndrome (Mlh1+/-), and the wild type (Mlh1+/+) littermates, fed with either Western-style (WD) diet or healthy AIN-93G control diet. Carcinomas developed mainly in WD fed mice. WD also accelerated the progression of carcinogenesis. In the first study, the expression of 94 growth-regulatory genes previously linked to human CRC was studied for 5 weeks and 12 months old mice. Promoter CpG island methylation status was also studied for the genes which showed reduced expression. In mice fed for 12 months with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in several tumor suppressor genes. Furthermore, a reduced mRNA expression was accompanied by an increased CpG dinucleotide promoter methylation of the respective genes suggesting a cause for the mRNA down regulation. The strongest expression decrease together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seemed to predispose to neoplasias in the proximal colon. The findings suggest that the inactivation of Dkk1 is a prominent early marker for colon oncogenesis. In study 2 the aim was to comprehensively clarify the role of Mlh1 expression during colon tumorigenesis, which is usually associated with Lynch syndrome and MSI. Here, the same mouse model and diets were used to study cancer-preceding expression changes in the colon mucosa of 12 and 18-month-old mice. The Mlh1 protein expression and MSI status were studied in the colon carcinomas, and the effect of inherited predisposition (Mlh1+/) and Western-style diet on those. CRC development always includes a lack of genomic integrity and the different types of genomic instabilities, such as chromosomal instability and MSI are thought to reflect distinct cancer initiating mechanisms. In the present study neither wildtype Mlh1+/+ nor heterozygote Mlh1+/- mice lacked the Mlh1 protein or showed MSI in their CRCs, while Mlh1 RNA expression was already significantly decreased in their normal mucosa. Instead, CRC mice showed a distinct expression profile with shortage of Mlh1 and several other chromosomal segregation gene-specific transcripts in mucosa and aberrant mitosis in tumors. The genome wide expression profiling experiment demonstrated that cancer-preceding changes are already seen in histologically normal colon mucosa and a that decreased expression of Mlh1 together with other chromosomal segregation genes may form a field-defect in mucosa and trigger MMR-proficient, chromosomally unstable CRC.
  • Jiang, Ping (Helsingin yliopisto, 2017)
    The high-level cognitive abilities such as attention and working memory develop throughout childhood and adolescence. Neuroimaging studies have shown that several brain regions including areas in the prefrontal (PFC) and parietal cortices play an important role in cognitive control. The prolonged maturation of the PFC and related networks may underlie the immature cognitive control abilities in children. In this thesis, functional magnetic resonance imaging and 1-back WM tasks were used to investigate 1) top-down modulation of brain activity in cortical areas related to visual information processing and 2) functional connectivity of resting state and task-related brain networks in healthy 7-11-year-old children and young adults. The tasks required the subjects to attend to either face or scene stimuli and to ignore distracting scene or face images, respectively. Studies I and II of this thesis found weaker or otherwise immature top-down modulation of the face processing-related visual association cortices that could partially be explained by the observed weaker functional connectivity between the PFC and the visual association cortex in the typically developing 7–11-year-old children compared to the young adults. Moreover, there were age-dependent differences in the recruitment of the PFC during visual working memory tasks. These age-dependent differences between the two groups are in line with the observed differences in the performance of the working memory tasks that was poorer in children than young adults. Study III showed that the 7-11-year-old children have already established an adult-like pattern of resting state networks, but especially during task performance, the functional connectivity within and between these networks differed from that in young adults. The group differences observed in the brain activation and functional connectivity are likely partly related to the morphological developmental state of the grey- and white matter in the 7–11-year-old children (i.e., the ongoing synaptic pruning and myelination of axons that continue up to young adulthood). The findings of this thesis conform to the suggestion that during development, the function of brain regions, especially the PFC, and the functional connectivity of brain networks, undergo dynamic changes, and that the same cognitive function may rely on different brain networks at different ages.
  • Tarkiainen, Katriina (Helsingin yliopisto, 2017)
    Carboxylesterases (CESs) catalyze the hydrolysis of a variety of ester- and amide containing compounds to their respective free acids. The main CES isozymes involved in drug metabolism are carboxylesterases 1 and 2 (CES1 and CES2). CES1 contributes to an estimated 80 to 95% of the total hydrolytic activity in the human liver. A CES1 c.428G>A (p.G143E, rs71647871) single nucleotide variation (SNV) markedly decreases the catalytic efficiency of CES1 in vitro. Possible effects of this variant on drug pharmacokinetics in vivo in humans, however, have not been systematically investigated. Furthermore, only a few studies have investigated the effects of CES1 variants on its expression and/or activity. Therefore, this thesis aimed to investigate genetic variability in the CES1 gene in the Finnish population and their effects on drug pharmacokinetics and pharmacodynamics in humans. The frequency distribution of the CES1 c.428G>A SNV was investigated in 860 healthy Finnish volunteers. The effects of the CES1 c.428G>A SNV on the pharmacokinetics of oseltamivir, clopidogrel, quinapril, and enalapril were investigated in 40 healthy volunteers. The CES1 gene and its flanking regions were sequenced in 192 healthy volunteers to identify previously unknown variants affecting CES1 whole blood expression. The findings were replicated in another set of 88 healthy volunteers. Furthermore, the effects of the detected variants on CES1 liver expression were investigated in 177 liver samples and on clopidogrel pharmacokinetics in 106 healthy volunteers from previous pharmacokinetic studies on clopidogrel. The CES1 c.428G>A variant allele was found with a minor allele frequency of 2.2%. The c.428G>A SNV reduced the hydrolysis of oseltamivir to the active oseltamivir carboxylate. The oseltamivir carboxylate to oseltamivir area under the plasma concentration-time curve (AUC) ratio was 23% smaller in heterozygous carriers than noncarriers. The c.428G>A SNV reduced the hydrolysis of clopidogrel to the inactive carboxylic acid metabolite. Consequently, the AUC of the parent clopidogrel was about 120% higher and that of the active metabolite about 70% higher in carriers than in noncarriers. Consistently, the c.428G>A SNV markedly enhanced the platelet inhibitory effect of clopidogrel. The average percentage inhibition of platelet aggregation at 0-12 hours was 19% higher in carriers than in noncarriers. The c.428G>A SNV significantly reduced the hydrolysis of enalapril to active enalaprilat. The AUC of enalaprilat was 20% lower in carriers than in noncarriers. The c.428G>A SNV had no observable effect on the pharmacokinetics of quinapril. Two intronic CES1 rs12443580 and rs8192935 SNVs were discovered to have a major effect on CES1 expression in whole blood, but not the liver. Moreover, these two SNVs had no effect on clopidogrel pharmacokinetics. In conclusion, the CES1 c.428G>A SNV reduces the bioactivation of oseltamivir, markedly increases the clopidogrel active metabolite plasma concentrations and antiplatelet effects, and reduces the bioactivation of enalapril in vivo in humans. The two intronic CES1 rs12443580 and rs8192935 SNVs have tissue-specific effects on CES1 expression. This could lead to substrate-dependent effects of these SNVs on drug biotransformation.
  • Hotta, Jaakko (Helsingin yliopisto, 2017)
    Complex regional pain syndrome (CRPS) causes disabling and severe limb pain that is difficult to treat. The pain typically increases during motor actions, but is present also at rest. The pathophysiology of CRPS is incompletely understood. Some of the symptoms suggest involvement of the central nervous system, and accordingly, patients have been shown to display alterations in, for instance, the primary sensorimotor cortex (SM1) and indications of neuroinflammation. More thorough pathophysiological knowledge of CRPS would help to develop better treatments and diagnostics. My thesis includes four studies exploring the cerebral basis of CRPS. All patients participating in these studies suffered from unilateral upper-limb CRPS. Studies I and II explored the complex couplings between pain, motor actions and visual information in CRPS. In healthy persons, observation and imagery of motor actions are known to elicit brain activation in similar brain areas as does action execution. CRPS patients experience pain while they execute or imagine actions and at the same time their brain activity is abnormal. However, these phenomena have not previously been studied for action observation in CRPS. In Study I, ratings of subjective experiences (e.g. pain) during observation of others’ actions were compared between 19 CRPS patients and 19 healthy control subjects. We found that CRPS patients experienced observation of motor actions as unpleasant and painful, and they overestimated the force applied in the observed actions. In Study II, the brain responses to action observation, measured with functional magnetic resonance imaging (fMRI), were compared between 13 CRPS patients and 13 healthy control subjects using multivariate pattern analysis (MVPA). During painful action-observation, the patients displayed abnormal activity in, for example, the representation area of the painful limb in the SM1 as well as in other brain areas important for pain experience and motor actions. With MVPA, these abnormal activations reliably discriminated CRPS patients from healthy subjects, indicating potential of fMRI responses as biomarkers for CRPS. Together Studies I and II build the grounds for future studies on using action observation as an add-on treatment for CRPS. Study III elucidated alterations of brain functioning during spontaneous pain in CRPS. Comparison of resting-state fMRI between 12 CRPS patients and 17 healthy control subjects showed disrupted functional connectivity of the SM1 in CRPS, especially for the representation areas of the painful and contralateral non-painful limb. These results suggest that pain alters SM1 functions even during rest, disrupting normal brain processes that could e.g. support motor learning. In Study IV, volumes of brain structures, measured with magnetic resonance imaging, were compared between 12 CRPS patients, 8 non-CRPS pain patients and 12 healthy control subjects. In the CRPS patients, the right-ventricle choroid plexus was about one-fifth larger than in healthy subjects and about one-eight larger than in non-CRPS patients. Choroid plexus is important in e.g. neuroinflammation and this finding may open new lines in research for the pathogenesis and treatment of CRPS. In conclusion, these studies revealed novel brain-derived symptoms and signs of CRPS. Brain changes involved especially areas that have already previously been studied in CRPS (e.g. the representation areas of the painful and contralateral non-painful limb in the SM1) but also other areas, such as choroid plexus. The findings further corroborate central nervous system involvement in CRPS.
  • Sjöstedt, Noora (Helsingin yliopisto, 2017)
    Transport proteins embedded in the cell membranes of many organs can affect the absorption, distribution and elimination of numerous drugs. This can lead to the enhanced or restricted uptake or distribution of the drugs, nonlinear pharmacokinetics, transporter-mediated drug-drug interactions (DDIs) and inter-individual variability. Transporters may therefore alter the safety and efficacy of drugs, thus it is important to study drug-transporter interactions in drug development. The breast cancer resistance protein (BCRP, ABCG2) is one of the transporters involved in drug disposition. It belongs to the ATP-binding cassette (ABC) transporter family and uses ATP to expel drugs and other substrates out from cells. BCRP was initially found to cause drug resistance in cancer cell lines, but it is also expressed in healthy tissues such as the intestine, liver and blood-brain barrier, where it is one of the transporters limiting the uptake of many structurally diverse compounds. Despite interest in BCRP and other ABC transporters, it remains poorly understood how they recognize their substrates and which chemical structures are liable to interaction. In this thesis, a vesicle-based in vitro method was used to study the ligand preferences of BCRP. The results were compared to those obtained for the multidrug resistance associated protein 2 (MRP2, ABCC2), which is also implicated in drug transport. The results show that a range of natural compounds and their derivatives are able to inhibit BCRP transport and among these, flavonoids were identified as the most important group for inhibition. Conversely, MRP2 transport was affected by only few of the tested compounds. However, a more similar pattern of inhibition was seen for the two transporters when selected food additives were studied, where several food colourants were identified as inhibitors. In addition, the effect of one assay component (bovine serum albumin, BSA) on the in vitro transport kinetics of BCRP and MRP2 was evaluated. The inclusion of BSA in the vesicle assay lead to moderate changes (up to 2-fold) in transport activity, but the effects on in vitro − in vivo extrapolation are expected to be minor, at least based on the tested compounds. Finally, the vesicle assay was used to study the functionality of selected BCRP variants with polymorphisms in the transmembrane helices and they were found to have significantly impaired transport activity and expression compared to wild type BCRP. In summary, the vesicle-based transport assay was successfully applied to identify and evaluate the effects that BCRP interactions may have on the pharmacokinetics of BCRP substrates.
  • Ronkainen, Riitta (Helsingin yliopisto, 2017)
    Speech and language therapy for children with cochlear implants focuses on promoting the children's spoken language skills. In this dissertation, the professional practices of one speech and language therapist in promoting language learning are examined using a conversation analysis methodology. The research data consist of video recordings from speech and language therapy sessions, totalling approximately 36 hours. The total duration of analysed sequences is 3 hours, 52 minutes. The participants in the study are seven children with profound congenital hearing impairment who have received a cochlear implant and their speech and language therapist. The general aim of the dissertation is to increase knowledge of speech and language therapists' professional practices in supporting spoken language learning of children with cochlear implants. The therapist's practices are examined in both play and task interactions at three different stages of the therapy. First, the dissertation examines the ways in which the therapist enhances the children's listening and imitation skills in the early stages of therapy and cochlear implant use (Study I). Second, it analyses the therapist's professional practices of involving the parents in multiparty therapy interaction (Study II). Third, it demonstrates how the therapist promotes lexical learning in children with cochlear implants in the later stages of therapy (Study III). The dissertation offers new insights into the institutional nature of interaction in the speech and language therapy for children with cochlear implants. It demonstrates the therapist's professional practices and pinpoints techniques and strategies used in the intervention. Primarily, the children are provided with a repetitive and prosodically emphasised spoken language model to enhance their listening skills and spoken language learning. In addition multimodal elements such as gestures, signs and body movements are systematically used. The dissertation shows how the therapist supports the children's participation and fosters their competence, which is seen in the form of enhanced collaboration. Furthermore, the dissertation provides information about the ways in which the therapist involves parents in the therapy. The findings reported here contribute to research on speech and language therapy interaction, as well as more broadly to the study of institutional interaction. The findings expand and specify the professional stock of interactional knowledge about speech and language therapy. The dissertation provides detailed and concrete descriptions of therapeutic practices and suggests practical guidelines for supporting the spoken language learning of children with cochlear implants. These may be useful for clinicians and students working both with children with cochlear implants and children who have other communication disabilities.
  • Neuvonen, Anu (Helsingin yliopisto, 2017)
    Finland’s unusual history and resulting genetic structure have a number of consequences for the practical applications of genetic testing in Finland today, including forensic analysis. The objective of this study was to characterize coding and noncoding genetic variation in the Finnish gene pool using forensic markers, in order to improve the efficiency of forensic testing in Finland while simultaneously broadening our understanding of its history. Finland is characterized by a clear genetic delineation between Eastern and Western regions of the country, the origins of which have heretofore remained undetermined. Here, patterns of distribution observed in markers of prehistoric association suggest this delineation represents the vestiges of an ancient border between Mesolithic hunter-gatherer and Neolithic farmer populations, undetectable in other regions of Europe. This study provides further insight into the development of the current population structure and clarifies the resolution of uniparental marker variation in contemporary Finland, with implications for forensic applications such as ancestry- informative testing. Along with the aforementioned population stratification, Finland’s unusual history has also left its mark on the population in the form of reduced diversity, visible especially in the Y-chromosome. In order to improve the efficiency of Finnish Y-profiling, novel multiplex panels of highly polymorphic Y-microsatellite markers were developed and evaluated. The new 7- and 24-locus Y-STR panels demonstrate improved suitability for practical forensic applications, with enhanced discrimination power and a reduction in regional subdivision compared to commercial sets. In order to assess the applicability of a novel commercial panel of insertion-deletion markers in Finnish forensic profiling, the Investigator DIPplex kit was evaluated in the Finnish population. Earlier studies of the applicability of insertion/deletion polymorphisms as a tool of forensics had indicated that they were likely to be beneficial for casework analysis both in individual identification as well as the testing of familial relationships. The results suggest that while these markers were well suited for individualization purposes, they were inefficient for paternity testing in the Finnish population. These two studies highlight the need for careful population-specific validation of commercial marker sets widely in use in forensics. Historical population bottlenecks can result in the enrichment of mutations, including those with clinical effects. The assessment of metabolic gene ABCB1 polymorphisms in Finns found increased frequency of these mutations in comparison to other populations, suggesting that Finns may demonstrate an increased susceptibility to drug intoxication. A further investigation performed on post-mortem samples revealed a positive correlation between mutation frequency and level of blood digoxin, confirming previous results of the adverse effect of ABCB1 mutations on metabolic processes. These findings will aid forensic medicine by providing valuable additional evidence for molecular autopsies. A thorough understanding of underlying patterns of genetic variation and the history that created them is vital in recognizing the factors affecting practical forensic analysis today. In these studies, the deep genetic delineation between Eastern and Western regions of Finland was observed in a variety of forensic loci, and shown for the first time to extend also to mitochondrial markers, giving further evidence of its ancient history. The results of this thesis thus reveal new information about the history and demographics of the Finnish population while offering globally applicable improvements to forensic typing. The end result is more straightforward analysis and improved reliability for a spectrum of forensic applications ranging from individualization to cause of death determinations.
  • Oshukova, Svetlana (Helsingin yliopisto, 2017)
    The construct of psychopathy is formed by a constellation of specific interpersonal (lying, manipulation and grandiosity), affective (callousness, low emotion) and behavioral (irresponsibilty, impulsivity) traits. Current conceptualizations view psychopathy as a developmental neuropsychological disorder and as a malicious version of the extremes of normal personality traits. Psychopathic traits are relatively stable over time, from childhood through adolescence to adulthood. Thus, there has been an increase in interest to apply the construct of psychopathy to children and adolescents. The present study aimed:1)to study the psychometric properties of the Finnish versions of two self-assessments for psychopathic traits, 2)to explore gender differences in the traits, 3)to investigate relation of the traits to other forms of psychopathology, 4)to assess the prevalence of limited prosocial emotions (LPE) and the ability of this specifier to reveal adolescents high on psychosocial problems, 5)to compare self-assessed psychopathic traits between Finnish and Dutch community boys and girls, 6)to compare self-assessed psychopathic traits between female adolescent psychiatric outpatients and community girls, 7)to explore how those traits relate to psychiatric disorders in female outpatients, and 8)to assess psychopathic traits and related background variables in forensic psychiatry sample of girls charged with severe violent offenses in comparence with age- and offense-matched boys. The Finnish community data comprised 370 9th graders from secondary schools and 155 female students from vocational and high schools. Dutch data comprised 776 adolescents from the upper grades of secondary schools in the Netherlands. The outpatient data consisted of 163 female patients of adolescent psychiatric policlinics. The forensic psychiatric data comprised 25 girls and their age- and offense-matched male counterparts. The Youth Psychopathic traits Inventory (YPI), Antisocial Process Screening Device - Self Report (APSD-SR), Youth Self Report and Psychopathy Checklist - Youth Version were used. Forensic psychiatric examination reports and patient files were reviewed. Both the YPI and the APSD-SR are promising screening tools for psychopathic traits in community, though somewhat weak to catch the affective deficits; the YPI showed slightly better properties than the APSD-SR. Community boys scored significantly higher than girls in overall psychopathic traits. The traits correlated with externalizing and internalizing problems, similarly in boys and girls. LPE were common in community youth, and they did not designate those with psychosocial problems from those without. Culture-related differences in juvenile psychopathic traits seem to exis, but the research should be replicated in other cross-national samples before generalizing the conclusions. Psychiatric outpatient girls showed more impulsive and irresponsible lifestyles than community girls. Girls with externalizing psychopathology excibited more affective deficits than did girls in the community. Psychopathic traits in girls were associated with psychopathology and especially with externalizing psychiatric disorders. The psychiatric examination of outpatient girls would likely benefit from screening for psychopathy. In the forensic sample, approximately every third girl showed high traits of psychopathy, and no significant gender difference in the prevalence was observed. With regard to the underlying factor and item scores, girls were less antisocial than boys, but their interpersonal relationships were more unstable. Compared to boys, girls more often had a history of child sexual abuse, and their victims were more often family members or current or ex-partners. Interventions should take into account these special features of severely violent offending girls.
  • Yuryev, Mikhail (Helsingin yliopisto, 2017)
    Embryonic brain development is a highly dynamic period in human life. Any disturbances at this stage can cause life-long negative consequences, such as developmentally related diseases, including autism, schizophrenia, and bipolar spectrum disorders. During development the mother-embryo interface plays a crucial role in supplementing the growing organism with oxygen and nutrients, regulating chemical cues, and protecting it from infections and potentially hazardous compounds. However, most of the studies of embryonic brain development have utilized ex vivo systems such as slices and neuronal cultures. Despite the great value of the data obtained from ex vivo studies, they usually completely ignore the significance of the mother-embryo interaction. Thus, there is desperate need for novel preclinical models of embryonic development. In this thesis, we developed a novel technique for in vivo two-photon imaging in mouse embryos connected to the mother via umbilical cord. We developed the special chamber with polymer membrane allowing to keep embryos separately in the heated physiological solution while the umbilical connection to the anesthetized mother is preserved. We developed the protocol for stimulation of calcium activity using high-power laser radiation and studied the propagation of the resulting calcium waves in the mouse embryonic cortex in vivo under ketamine/xylazine anesthesia. We confirm the enhancing effect of caffeine on the evoked activity and the suppressing effect of the adenosine triphosphate (ATP) -receptor blockade, known from previous ex vivo studies. We analyzed the patterns of wave propagation and show the non-uniform spreading, which suggests the presence of differing connectivity patterns in the cortex already during the early stage of development. Further, we studied spontaneous calcium activity and cellular motility in the mouse embryonic cortex in vivo under light isoflurane anesthesia. We demonstrate two various patterns of ongoing activity: sporadic activation of single cells and correlated activity in the form of calcium waves. We show that blockade of N-methyl-D-aspartate (NMDA) receptors with ketamine inhibits the calcium activity in vivo, corresponding with the arrest of cellular motility. In the last part of the thesis, we studied the dynamics of the externally introduced substance to the mouse embryonic brains in vivo. We used porous silicon nanoparticles, which are a promising drug delivery platform, as they can be loaded with poorly water-soluble drugs. We show that the nanoparticles can breach the placental barrier and accumulate in the brains of the embryo. To study the dynamics of nanoparticles when already in the cortex, we injected the embryonic brains intraventricularly. Nanoparticles, including ones 3-4 um in size, were distributed in 80% of the cortex already 4 hours following the injection, thus demonstrating high motility in the brain tissue of embryos. We confirmed the motility of nanoparticles in real time using the in vivo two-photon imaging of embryos connected to the mother under ketamine/xylazine anesthesia. The results emphasize the susceptibility of the embryonic cortex at the early stage of development to external particles, which should be taken into account in nanomedicine development. In summary, the developed in vivo imaging technique allowed functional studies in the embryonic cortex in real time. This will allow preclinical pharmacological investigations of the compounds while maintaining the physiological mother-embryo interface.
  • Nylund, Karita (Helsingin yliopisto, 2017)
    ORAL HEALTH IN PATIENTS WITH CHRONIC KIDNEY DISEASE - EMPHASIS ON PERIODONTITIS Background: Periodontitis is a common bacteria-induced chronic inflammatory disease with mild symptoms. It leads to destruction of the periodontium and finally to tooth loss in a susceptible patient. Periodontitis is associated with many systemic diseases such as diabetes, atherosclerosis, cardiovascular diseases, and chronic kidney disease (CKD) through low-grade systemic inflammation. However, no causality can be drawn. CKD is defined as a deficiency in kidney structure or function lasting over three months. Its prevalence is over 10% globally. The main risk factors of CKD are diabetes mellitus, hypertension, and obesity although periodontitis has been proposed to be a non-traditional risk factor. When CKD is progressed to end-stage renal disease (ESRD), renal replacement therapy (dialysis or kidney transplantation) is needed. Prior to entering kidney transplantation, the candidate must be screened for infections, including oral infections, before immunosuppressive medication can be administered. Patients with ESRD have higher mortality rates compared with general population. Aim and hypotheses: The general aim of this thesis was to examine the oral health of 144 CKD patients with emphasis on periodontal disease; first at predialysis and secondly at post-transplantation stage. Other purposes were to investigate the association between periodontal inflammatory burden and salivary matrix metalloproteinase -8 (MMP-8) concentration, and further, to determine whether oral inflammatory burden associates with mortality. Oral health of diabetic nephropathy patients were compared with other CKD patients. The main hypotheses were that oral health, periodontal disease in particular, is more severe among diabetic nephropathy patients than among other CKD patients. Furthermore, we expected oral health being worse at predialysis compared with post-transplantation stage. Methods: CKD patients were followed up for 157 months (over 13 years). They underwent a full clinical and radiographic oral examination. Salivary samples were collected both at predialysis and post-transplantation stages for detection of MMP-8 by immunofluorometric assay (IFMA). Results: Patients with diabetic nephropathy indeed had worse oral health. Higher salivary MMP-8 concentrations associated with worse oral health at prediaysis stage. The follow-up study showed that the 10-year survival rate of diabetic nephropathy patients was 28% compared to 62% of the other CKD patients. The overall 10-year survival rate was 50%. The most common cause of death was a major cardiovascular event, followed by infection and malignant disease. In the multivariable Cox regression model, older age, diabetic nephropathy diagnosis and having fewer teeth were significant independent risk factors for death. Oral health was better at the follow-up than at the predialysis stage when oral infection treatment had been given. Conclusion: The present results support the existing protocol of the Helsinki University Hospital, where oral examination, accurate diagnosis and proper treatment of oral infection foci are mandatory at the predialysis stage for reducing systemic inflammation among CKD patients. Salivary MMP-8 assessment could help clinical decision making in the future. The association of missing teeth and mortality could be explained by long lasting low grade systemic oral inflammation. Since diabetic nephropathy is associated with poorer oral health, this patient group needs particular attention.
  • Hällfors, Jenni (Helsingin yliopisto, 2017)
    Cigarette smoking is a worldwide health burden causing several diseases. Chronic obstructive pulmonary disease, heart disease and several cancers remain the main avoidable causes for premature deaths. Among smokers, tobacco use is largely motivated by nicotine dependence (ND). Smoking is influenced by the complex interplay between genetic vulnerability, environmental risk factors and individual characteristics. Despite the high heritability estimates of ND, the underlying genetic factors remain largely unknown. This work aimed to identify the contribution of genes and specific genetic variants predisposing individuals to smoking behavior and ND, and to further assess their ability to explain other smoking-related comorbidities and traits, such as alcohol use. A Finnish twin family sample enriched for heavy smoking was used to map susceptibility genes for smoking-related phenotypes. Using a dense set of markers on chromosome 20, the previous linkage finding (Loukola et al., 2008) was replicated with an ND measure diagnosed by the Diagnostic and Statistical Manual of Mental Disorders (DSM), 4th edition (DSM-IV) criteria. The finding was extended using a larger sample. Sex-stratified analyses provided a clear distinction between genetic elements on chromosome 20 that influence ND in adult male and female smokers. This study highlights linkage between chromosome 20 and ND in Finnish adult smokers. The same Finnish twin family sample was used in a GWAS for smoking behavior. The data was imputed using the 1000 Genomes Phase I reference panel together with a whole genome sequence-based Finnish reference panel obtained from the Sequencing Initiative Suomi (SISu) project. The study yielded genome-wide significant association on 16p12.3 with smoking quantity defined as self-reported cigarettes smoked per day (CPD). Several highly correlating single nucleotide polymorphisms (SNPs) within the association region map to an intergenic locus near gene CLEC19A. In addition, association was detected on 11p15.5 with nicotine withdrawal symptoms assessed by the DSM-IV criteria. The finding pinpointed genes AP2A2 and MUC6. These findings on chromosomes 16 and 11 highlight the role of neurotrophin signaling pathway in smoking behavior. The most robust smoking behavior locus to date has been mapped to chromosome 15q24-q25, which harbors a gene cluster (CHRNA5-CHRNA3-CHRNB4) encoding nicotinic acetylcholine receptor (nAChR) subunits α5, α3 and β4. Three of the most intensively studied SNPs on the locus were analyzed by combining two large and individual Finnish population-based samples, The National FINRISK Study and The Health 2000 Survey, into one sample. The study replicated an earlier finding reporting association between three distinct loci on 15q25.1 and CPD (Saccone et al., 2010), and confirmed that the alleles at this strongest smoking behavior locus explain approximately 1% of the variance in CPD. Plausible pleiotropic effects of nAChRs were examined in two studies using SNPs tagging the robust smoking behavior locus on chromosome 15. First, we provided novel evidence of association between a genetic variant on 15q25.1 and alcohol use in the National FINRISK Study and the Health 2000 Survey. Second, we participated in an international collaboration work aiming to estimate the causality between smoking and body mass index (BMI). The National FINRISK study was included in the Mendelian Randomization meta-analyses, which demonstrated that variants on 15q25.1 may contribute to BMI: never smokers are associated with higher BMI, whereas current smokers are associated with lower BMI. Taken together, these results improve our knowledge of the genetic factors affecting smoking behavior and ND. They also provide further evidence on the pleiotropic effects of the nAChRs. However, bigger samples and large collaborations are needed to observe the many small effects of yet unidentified variants affecting complex traits.
  • Kauttu, Tuuli (Helsingin yliopisto, 2017)
    Esophageal adenocarcinoma (EAC) is a cancer with a poor prognosis that has increased in Western countries in the recent decades. Among its predisposing factors are gastroesophageal reflux disease, Barrett´s esophagus (BE), obesity, increasing age, and male gender. To decrease EAC incidence and to improve its prognosis, preventive methods and enhanced knowledge of its pathogenesis are essential. We examined possible EAC prevention by antireflux surgery at the population level, and by laser ablation of BE combined with antireflux surgery. When those 53 patients with EAC developed after antireflux surgery and diagnosed in Finland in 1980-2006 where thoroughly surveyed, we found that endoscopic esophagitis preoperatively, BE, and failure of antireflux surgery were frequent findings. When the EAC patients with previous antireflux surgery (starting from 1987 when antireflux surgery register was established) were compared with those without antireflux surgery by means of standardized incidence ratio, we found that EAC occurred 9.2 times more often after antireflux surgery. To explore if the removal of Barrett’s epithelium with concomitant reflux inhibition achieved by antireflux surgery would yield better results, the effect of laser ablation for BE in 18 patients with antireflux surgery was determined at a 7.9 years’ follow-up: no sign of malignant development was seen, but one-third of the patients had BE. The oxidative stress in both the neosquamous mucosa and in the proximal esophagus remained partly increased. To gain insight into the molecular mechanisms in GERD and EAC, the expression of a disintegrin and metalloproteinases (ADAMs) 9, 10, 12, 17, and 19 was studied in patients with GERD, BE, or EAC, and that of the oncogenes CIP2A and c-MYC was studied in EAC patients. The ADAM expression was increased in the various stages of GERD, although no single ADAM showed a steady progressive increase in the esophagitis – BE – dysplastic BE– EAC – sequence. Particularly, ADAM9 was increased in EAC, and its mRNA was increased also in BE and dysplastic BE. CIP2A and c-MYC, although abundantly expressed in EAC, were not associated with prognosis.