Faculty of Medicine

 

Recent Submissions

  • Facciotto, Chiara (Helsingin yliopisto, 2021)
    Overcoming drug resistance in cancer is one of the most pressing issues in oncology. The last century saw a dramatic increase in the discovery of new cancer therapies, so much so that chemotherapeutic agents and immunotherapies are now, alone or in combination, the backbone of treatment for many cancers. Despite the increased rate of treatment success brought by these regimens, cancer patients can become resistant to these drugs. This leads to disease relapse, hindering patient survival. Drug resistance remains the primary cause of death in most advanced-stage cancer patients. The molecular mechanisms responsible for the development of a resistance phenotype in cancer cells are complex and include both genetic and epigenetic alterations. Since drug resistance is a multifactorial phenomenon, we used a systems biology approach to investigate it on different fronts. Specifically, we developed a high-throughput drug screening method to test new drug combinations, identifying epigenetic inhibitors able to sensitize lymphoma cells to doxorubicin. We also implemented a bioinformatic pipeline which combines multiple omics data to identify genes and pathways driving platinum response across multiple cancers. We then developed a method to compute differential methylation between cancer samples with varying and unknown tumor purity, which we used to investigate DNA methylation changes linked to drug resistance in ovarian cancer and lymphoma. Finally, we created a workflow management system to build complex bioinformatic pipelines and aid researchers in the analysis of high-throughput biomedical data. By combining laboratory biology experiments and computational analyses, we gained a broader understanding of the cellular mechanisms behind immunochemotherapy failure. Moreover, we were able to identify novel biomarkers associated with platinum response in multiple cancers, as well as new drug combinations able to overcome immunochemotherapy resistance in lymphoma cells. The in vitro and in silico methods presented in this thesis can not only assist researchers in the cancer field, but are broadly applicable to other fields of biomedical research. Overall, this work is an important stepping stone in both understanding and overcoming drug resistance in cancer, and has great potential to improve outcomes for cancer patients in the future.
  • Ruohtula, Terhi (Helsingin yliopisto, 2021)
    Type 1 diabetes (T1D) and allergies are considered autoimmune diseases. The exact mechanisms leading to these diseases are mostly unknown, but genetic and environmental factors are involved. Regulatory T cells (Tregs) have a crucial role in initiating and maintaining tolerance, limiting harmful autoantigen-specific inflammation processes. This doctoral thesis explores the effect of environmental factors on the development of the immune system by studying peripheral immunological responses in healthy infants at genetic risk of T1D in two follow-up studies. The first study investigated the development of peripheral Treg cells in infants from Estonia and Finland, neighboring countries with differing living standards, and the incidence of T1D and allergies. We demonstrated a two-step maturation process in the circulating Treg cell population in the first two years of life. First, a decrease in the proportion of Treg cells followed by an increase in the highly activated Treg cells (TregFOXP3high), with enhanced suppressive activity. The Treg cell maturation process coincided with the microbiota composition development from a Bifidobacteria dominated to one dominated by butyrate-producing species by 36 months. The abundance of Bifidobacteria and the relative abundance of B.longum alone at three months showed an inverse association with atopic sensitization. The switch in the microbiota happened earlier in Estonia and was associated with more highly activated TregFOXP3high cells with FOXP3 and CTLA-4 expression. This earlier maturation of the circulating Treg population is associated with a lower risk of allergic diseases in Estonian children. Additionally, we analyzed the common enteral virus infections in the first year of life. We observed a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with enterovirus infection during the preceding 30 or 60 days had a significantly decreased FOXP3 expression in TregFOXP3high cells. In addition, we observed upregulation of Th1- and Th17 -related cytokines and a decreased activation of CCL22, associating with activation of proinflammatory pathways and reduced immune regulation. In the second study, we examined whether exposure to bovine insulin is a trigger of autoimmunity by studying the appearance of diabetes autoantibodies. We studied exclusively breastfed infants or infants after exposure to different infant formulas; cow-milk formula (CMF) with bovine insulin, whey-based hydrolyzed formula, or a virtually bovine insulin-free whey-based hydrolyzed FINDIA formula during the first six months of life, whenever breast milk was not available. The follow-up was up to at least three years of age. We showed that weaning to the FINDIA formula reduced the incidence of autoantibody positivity when compared with CMF-formula. This result might reflect a dysfunctional immune system. Bovine insulin peptides from the infant formula may activate autoreactivity against the primary autoantigen, i.e., human insulin, resulting in non-tolerogenic T cells. To conclude, the study shows that environmental factors may influence regulatory T cells and immunotolerance. Also, nutrition may influence the development of the immune system or autoantibodies to T1D in infants genetically at risk. Keywords: type 1 diabetes, allergy, regulatory T cells, autoantibodies, viruses, diet
  • Rämö, Lasse (Helsingin yliopisto, 2021)
    Introduction Humeral shaft fractures account for 1–3% of all adult fractures. They are usually caused by simple falls, traffic accidents, and sports injuries. Historically, the treatment of these injuries has been mainly nonsurgical. However, there has been a marked increase in the rate of surgical treatment for humeral shaft fractures in recent years without high-quality evidence supporting this trend. Aim The Finnish Shaft of the Humerus (FISH) randomized clinical trial was planned to compare the effectiveness of surgery versus nonsurgical care in the treatment of humeral shaft fractures in patients traditionally deemed suitable for nonsurgical care with functional bracing (Study I). Patients and methods Patient recruitment was conducted at the Helsinki and Tampere University hospitals between November 2012 and January 2018. Consenting adult patients with displaced, closed, unilateral humeral shaft fractures were randomized to either surgical care using open reduction and plate fixation or nonsurgical care using functional bracing. Patients with a history or condition affecting the function of the injured upper limb, pathological fracture, other concomitant injury affecting the same upper limb, other trauma requiring surgery (e.g., fracture, internal organ, brachial plexus, or vascular injury), polytrauma, multimorbidity with high anesthesia risk, or inadequate cooperation for any reason were excluded. Altogether, 321 patients were assessed for eligibility and of these 140 were eligible for randomization. After informed consent, 82 were willing to undergo randomization. The primary outcome was the Disabilities of the Arm, Shoulder, and Hand (DASH) score (range, 0 to 100 points; 0 = no disability, 100 = extreme disability). In Study II, the patients were analyzed according to the initially allocated treatment method (surgery group and bracing group). In Study III, the patients were analyzed in three groups according to their final treatment method: 1) initial surgery group, 2) bracing group with successful healing, and 3) secondary surgery group including patients randomized to functional bracing but who underwent late surgery due to fracture healing problems. Results Study II. The mean DASH score was 8.9 (95% confidence interval [CI], 4.2 to 13.6) in the surgery group and 12.0 (95% CI, 7.7 to 16.4) in the bracing group at 12 months. The between-group difference was -3.1 (95% CI, -9.6 to 3.3). This difference was not statistically significant, and it was below the predefined minimal clinically important difference of 10 points. Of the patients allocated to functional bracing, 13/44 (30%) underwent late surgery due to healing problem during the 12-month follow-up. In the post hoc analysis, the results of those with initial surgery were superior to those with late surgery due to healing problem (between-group difference, -11.1; 95% CI, -20.1 to -2.1) at 12 months. Study III. The mean DASH score was 6.8 (95% CI, 2.3 to 11.4) in the initial surgery group (n=38), 6.0 (95% CI, 1.0 to 11.0) in the bracing group (n=30), and 17.5 (95% CI, 10.5 to 24.5) in the secondary surgery group (n=14) at the 2-year follow-up. The between-group difference was -10.7 (95% CI, -19.1 to -2.3) between the initial and secondary surgery groups and -11.5 (95% CI, -20.1 to -2.9) between the bracing and secondary surgery groups. Conclusions Study II. Surgery with plate fixation, compared with functional bracing in the treatment of adult patients with closed humeral shaft fractures, did not significantly improve functional outcomes at 12 months. However, 30% of the patients allocated to functional bracing underwent late surgery due to healing problem. Study III. Shared decision-making between the clinicians and patients with closed humeral shaft fractures should weigh the prospect that 2/3 of patients undergo successful healing and have good functional outcomes using functional bracing against the 1/3 risk of fracture healing problems leading to secondary surgery and inferior outcomes even at 2 years after the injury.
  • Abushahba, Ahmed (Helsingin yliopisto, 2021)
    Bone tissue engineering (BTE) has shown a great promise in providing the next generation medical bioimplants for treating bone defects. However, BTE faces many obstacles which need to be addressed for promoting translatability. The objective of this thesis work was to explore clinically translatable tissue engineering approaches for the management of craniomaxillofacial bone defects. The role of the employed cells has witnessed a critical turning point towards an increased appreciation of the cellular paracrine effects. This paracrine effect is mediated via secreted proteins and released membrane-bound vesicles called extracellular vesicles (EVs). For advancing our knowledge about the biological roles of EVs, we employed RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs). Our findings revealed distinctive small non-coding RNA profiles from hPSCs and AT-MSCs EVs. The regulatory miRNAs of stem cells at cellular level are also present in their EVs, indicating an important regulatory role which is mediated via EVs. Vascularization is the key challenge for BTE applications in large bone defects. The local delivery of growth factors leads to short lived effects. Small molecule chemicals feature alternative cost-effective bioactive agents with better stability. We assessed the ability of two small molecules; DMOG and baicalein, in triggering the proangiogenic secretome of AT-MSCs in vitro. Additionally, other effects, such as proliferation and osteogenic differentiation of AT-MSCs were assessed. DMOG and baicalein efficiently stabilized the hypoxia-inducible factor (HIF-1α) and upregulated proangiogenic cytokines, e.g., vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB) of AT-MSCs in normoxic conditions. These effects were further associated with upregulated stemness-related gene expression, slowed proliferation, and reduced osteogenic potential. Chemically-induced hypoxia maintained the stemness and self-renewal properties of AT-MSCs, while enhancing their proangiogenic potential. The in vivo bioreactor (IVB) concept combines the potential of BTE and reconstructive surgery by employing the patient body for prefabricating new prevascularized tissues. Ideally, IVB should minimize the need for exogenous growth factors or cells and harness the native regenerative potential of employed tissues. Using acellular alloplastic bone blocks, we compared muscle-IVB with and without periosteal/pericranial grafts and flaps for prefabricating tissue engineered bone (TEB) flaps. We also assessed their functional outcomes in reconstructing a mandibular defect in an ovine model. The employment of vascularized periosteal flaps did result in more robust vascularization as compared to other IVB techniques. Both the periosteal grafts and periosteal flaps enhanced the performance of the prefabricated TEB flaps after transplantation into a mechanically stimulated bony microenvironment. However, more new bone formation and biomaterial remodeling was associated with the vascularized periosteal flaps.
  • Panduru, Nicolae Mircea (Helsingin yliopisto, 2021)
    Introduction: Diabetic nephropathy (DN) is a devastating diabetes complication. DN is associated with a competitive risk of either progression to a worse stage of DN or premature mortality. Mortality is mainly due to cardiovascular events. Tubular dysfunction is involved in the loss of kidney function. Therefore, tubular biomarkers could add significant benefit to early identification of DN, prediction of DN progression and to screening for risk of cardiovascular events. Aims: The aims of this thesis were to investigate, in individuals with T1DM, if three urinary biomarkers [liver-type fatty acid binding protein (L-FABP), kidney injury molecule 1 (KIM-1) and urinary adiponectin (ADP)] could outperform the currently available biomarkers for the prediction of DN development and progression or even be causally related to the loss of kidney function. In addition, the thesis aimed to investigate if these biomarkers could predict cardiovascular disease (CVD) and premature mortality. Subjects and methods: All individuals with T1DM included in this thesis were enrolled between January 1998 and December 2002. The studies were part of the Finnish Diabetic Nephropathy Study (FinnDiane), a nationwide cohort of individuals with T1DM followed prospectively at more than 80 centres throughout Finland. The participants were followed for a median of 5.8 to 14.1 years, and clinical outcomes were prospectively evaluated. Results: In study I, L-FABP was shown to be an independent predictor of progression of DN irrespective of the disease stage and did not improve the risk prediction of DN progression. In study II, urinary ADP was an independent predictor of progression to end-stage renal disease (ESRD) and performed even better than AER and as good as eGFR. In study III, KIM-1 did not predict the progression to ESRD independently of AER. However, the Mendelian randomization (MR) analysis showed that KIM-1 is very likely to play a causal role in the loss of eGFR. In study IV, L-FABP was an independent predictor of stroke and premature mortality but did not add any predictive benefit on top of AER and eGFR. It is of note that urinary ADP and AER were common determinants of all tested biomarkers. Discussion and conclusions: By predicting the progression of DN, these biomarkers proved that tubular dysfunction is an important part of DN progression. However, judging by their baseline determinants, the studied tubular biomarkers represent much more than tubular injury, capturing also glomerular damage as well as systemic factors. L-FABP was an as good predictor as eGFR or AER of stroke or premature mortality, while the other biomarkers predicted various other cardiovascular outcomes. A causal relationship between these biomarkers and loss of kidney function could be demonstrated only for KIM-1, but this particular observation confirms a causal role of tubular dysfunction for the DN progression.
  • Mesihää, Samuel (Helsingin yliopisto, 2021)
    Legal medicines and illegal drugs play a significant role in today’s society. The laboratory analysis of these substances is central not only in therapeutic monitoring but also in clinical and forensic toxicology, which aims to provide evidence-based information on the abuse and harmful effects of these substances. The rapid emergence of newly abused drugs known as new psychoactive substances (NPS) has challenged the conventional drug testing concept. On the European illicit drug market, approximately fifty new substances are identified each year, many of which are poorly characterized for their pharmacological effects or toxicity. Moreover, the lack of authentic primary reference standards (PRS) bottlenecks the development of analytical assays for NPS, which hinders both detection and toxicological evaluation of these drugs. High-resolution mass spectrometry (HRMS) has recently been applied to tentative identification of unknown compounds by targeting the precursor ions with high mass accuracy, resolution and speed. This capability has permitted a breakthrough in facile screening for suspected NPS, but until now, no practical method has been described for quantitative bioanalysis of NPS in the absence of PRS. In this thesis, a new analytical platform was developed and exploited for the simultaneous qualitative and quantitative analysis of NPS and metabolites without using PRS. In this platform, the gas chromatographic (GC) flow was divided between an atmospheric pressure chemical ionization - quadrupole time-of-flight mass spectrometer (APCI-QTOFMS) for tentative identification and a nitrogen chemiluminescence detector (NCD) for quantitative estimation based on the detector’s equimolar response to nitrogen-containing substances. Replacement of the ordinary electron ionization (EI) source with APCI was proven to be useful in substance identification by QTOFMS because it allowed the preservation of the precursor ion for tentative identification. The GC-APCI-QTOFMS tandem mass spectrometry (MS/MS) experiments using 29 NPS showed that all substances shared the same major fragments as in the commercial spectral library created by using liquid chromatography (LC) electrospray (ESI) MS/MS instrumentation. Consequently, these findings promote the usability of external soft-ionization compound libraries with the new GC-APCI-QTOFMS platform. The accuracy and precision of the N-equimolar quantification by GC-NCD were tested using several NPS in four separate studies using post-spiked sheep blood (n = 5), post-mortem blood (n = 38) and urine (n = 3), and in seized powdery material (n = 28). The combined results from these studies, excluding the post-spiked samples, showed that NPS could be quantified with a grand mean accuracy of 91.7% and with a grand mean imprecision (CV) of 9.5% in the absence of PRS for compensating sample preparation and analysis. In conclusion, the GC-NCD-APCI-QTOFMS platform proved feasible for analyzing suspected nitrogen-containing drugs, such as stimulant-type NPS and metabolites, in situations where appropriate reference standards are not readily accessible. Simultaneous tentative identification by HRMS and quantitative estimation by NCD has unparalleled potential for fast preliminary analysis until a certified PRS becomes available. The possibility for high-throughput retrospective identification and quantification is an additional advantage of this platform, providing a means for further research on emerging NPS.
  • Ignatenko, Olesia (Helsingin yliopisto, 2021)
    Mitochondria are organelles critical for cellular energy metabolism and homeostasis. Pathogenic DNA variants that disrupt organelle function manifest as a heterogeneous group of diseases. These include severe brain encephalopathies that lack curative treatments, leading to early childhood lethality. Typical findings in brain samples of patients with mitochondrial encephalopathies include neuronal degeneration and histopathological changes of non-neuronal cells, referred to as reactive gliosis. The severe manifestations of mitochondrial encephalopathies have thus far been explained by the vulnerability of neurons to mitochondrial dysfunction, while reactive gliosis is considered a secondary response to the neuronal pathology. In my thesis research, I used genetically modified mouse models to investigate the cell-specific contribution to the pathogenesis of mitochondrial dysfunction in the central nervous system. Using Cre-Lox recombination, the gene encoding the mitochondrial DNA helicase Twinkle was conditionally disrupted in postnatal astrocytes or neurons. In neurons, we observed the well-established vulnerability to mitochondrial dysfunction. Whereas in astrocytes, our data show reactive astrogliosis as a cell-autonomous response to mitochondrial dysfunction. Furthermore, the formation of microscopic vacuoles in the brain characteristic of spongiotic encephalopathies was only observed upon mitochondrial dysfunction in astrocytes. Collectively, these findings shift the paradigm on the contribution of individual cell types to the brain pathology of mitochondrial disorders. Next, I used these mouse models to test two therapeutic approaches that act to remodel cellular metabolism for modulating mitochondrial dysfunction. The first intervention used rapamycin to inhibit activity of the key nutrient sensor mTORC1; while the second used dietary intervention by shifting the carbon source to generate ketone bodies as an alternative energy source for the brain. Neither of the treatments improved the spongiotic pathology or attenuated reactive astrogliosis, and moreover the ketogenic diet exacerbated these phenotypes. Since rapamycin and ketogenic diet have been used successfully in treating other mouse models of mitochondrial dysfunction, it emphasizes the importance of using disease-specific models in preclinical studies. In the final part of my thesis, astrocyte responses to mitochondrial dysfunction were investigated. We found that lipid biosynthesis was downregulated in astrocytes, which was paralleled by changes in brain lipid composition and accumulation of lipid droplets. We also discovered an induction of a motile ciliogenesis program as an astrocyte response to pathological stimuli. Mitochondrial dysfunction resulted in anomalous expression of motile cilia components and abnormal morphology of cilia in astrocytes. Astrocytes are normally devoid of motile cilia but possess a primary cilium, which has signalling functions. Our findings raise the possibility of the remodelling of cilia function in astrocytes in response to mitochondrial dysfunction, which may contribute to pathogenesis. Altogether, the research presented in this thesis has implicated astrocytes as a critical contributor to mitochondrial disease manifestations, and provided a solid base for the future efforts to target astrocyte responses to mitochondrial dysfunction.
  • Dang, Kien (Helsingin yliopisto, 2021)
    The aim of this doctoral thesis was to utilize molecular biology techniques to detect expressed KRAS and BRAF mutations in samples at high sensitivity and specificity and evaluate the clinical role of the RNA expression of these mutations in certain MAPK/ERK-driven cancers. In the early phase of the study, we developed a novel method named extendable blocking probe reverse transcription (ExBP-RT) for detecting expressed mutations at the mRNA level. With this method we were able to detect mutations expressed in mRNA with a very high sensitivity and specificity. The ExBP-RT assay was optimized to detect expressed BRAF and KRAS mutations in a 1000-fold – 6000-fold excess of wild-type mRNA. A further improvement of the method allowed detection of expressed BRAF mutations, in thyroid cancer (TC) tissue, in a 10000-fold excess of wildtype mRNA. This novel strategy not only reveals the presence or absence of low-abundance mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of the mRNA levels of the mutated genes in different settings, such as quantification of allele-specific expression. We used the ExBP-RT technique to measure the mRNA levels of all seven KRAS mutations at codon 12 and 13 in primary tumour tissue samples of 571 patients with colorectal cancer (CRC). Survival data was analysed to determine the prognostic potential of this novel mRNA-based biomarker. A high level of mutated KRAS mRNA was associated with an inferior 5-year diseasespecific survival (DSS). This association was highly significant, but only in left-sided CRC (P < 0.001). Thus, the mRNA level of the mutated KRAS allele in primary tumour tissue was found to be highly prognostic in left-sided CRC, but not in right-sided disease. To study the RNA expression of BRAF mutations as a diagnostic marker in thyroid cancer (TC), we analysed 62 formalin fixed paraffin embedded (FFPE) samples from TC patients. We detected BRAF V600E mutations at the mRNA level in 56,3% (18/32) and on the DNA level in 40,6% (13/32) of thyroid cancer patients, which is in concordance with the reported prevalence of these mutations. In order to evaluate whether sensitive detection of KRAS and BRAF mutations in mRNA could serve as a biomarker for early detection of malignant transformation in patients at risk of developing colorectal cancer, we analysed esophageal atresia (EA) patient cohorts. The ExBP-RT technique was used to evaluate tissue expression of KRAS and BRAF mutations in endoscopic biopsies from 61 adults, who had been surgically treated for EA in infancy. Despite the presence of histological findings indicating an increased risk of developing cancer, we found no detectable tissue expression of KRAS or BRAF mutations in this cohort. In conclusion, we successfully established a novel technique – ExBP-RT to detect KRAS and BRAF mutations at the mRNA level with very high sensitivity and selectivity. We found a strong association between the tumour tissue expression of KRAS mutations and prognosis in left-sided, stage III CRC. We also successfully detected and quantified the level of BRAF V600E mRNA in FFPE tissue from thyroid cancer. We could not, however, detect neither KRAS nor BRAF mutations in either of the cohorts of EA patients representing potential pre-malignant conditions.
  • Murtoniemi, Katja (Helsingin yliopisto, 2021)
    Pre-eclampsia is a placenta-driven pregnancy complication causing systemic inflammation and endothelial dysfunction. It is characterised by increased blood pressure and proteinuria and affects vital organs of the body. Therefore, without surveillance and treatment it threatens the health and life of the mother and the foetus. Every year approximately 70,000 mothers die due to pre-eclampsia and its complications globally. There is still no cure for pre-eclampsia other than delivery. The aim of this thesis was to investigate associations of biomarkers to pre-eclampsia, and to find better ways to predict the disease and its subtypes, and accordingly to also increase the understanding of the mechanisms and pathways of the pathophysiology of pre-eclampsia, which eventually may lead to innovations of new therapeutic targets. This thesis includes data from the multidisciplinary PREDO (Prediction and prevention of pre-eclampsia and fetal growth restriction) project, which consists of 988 pregnant women with and 117 without known risk factors for pre-eclampsia. These women were recruited between September 2005 and December 2009 in ten maternity clinics in Finland. The usefulness of serum hyperglycosylated human chorionic gonadotropin (hCG-h) or the proportion of hCG-h to hCG (%hCG-h) was assessed for the prediction of pre-eclampsia in the first trimester (Study I). These were combined with maternal risk factors, known biomarkers (placental growth factor (PlGF), free human chorionic gonadotropin beta (hCGβ) and pregnancy-associated plasma protein A (PAPP-A)), and biophysical measurements (mean arterial pressure and mean uterine artery pulsatility index). The study was conducted in a subcohort of 257 women with known risk factors for pre-eclampsia. We found that prior pre-eclampsia and low serum %hCG-h were associated with late-onset and non-severe pre-eclampsia, whereas low serum PlGF was associated with early-onset and severe pre-eclampsia. Free hCGβ was higher in women who developed severe pre-eclampsia than in women who did not develop severe pre-eclampsia, whereas low serum PAPP-A was associated with non-severe pre-eclampsia. Multivariate models constructed with regularised logistic regression provided only modest prediction rates for all pre-eclampsia (36% sensitivity with 90% specificity) and its subtypes (20% sensitivity for early-onset pre-eclampsia and 32% sensitivity for late-onset pre-eclampsia with 90% specificity). The effect of low-dose aspirin (100mg/d) on maternal serum concentrations of PlGF during pregnancy from the first trimester to the late second trimester was investigated (Study II). Blood samples were collected at 12–14, 18–20 and 26–28 weeks of gestation. The main finding was that high-risk women who had low-dose aspirin treatment (N=61) started before 14 weeks of gestation for prevention of pre-eclampsia had higher concentrations of serum PlGF than high-risk women who had placebo treatment (N=62). The difference was evident from mid-gestation onwards. We investigated the differences of haemoglobin scavenger proteins haemopexin (Hpx) and alfa-1-microglobulin (A1M) in high risk women and low risk women at 26–28 weeks of gestation (Study III). It was a case–control study with high-risk women who subsequently developed pre-eclampsia (n=42), high-risk women who did not develop pre-eclampsia (n=49) and low-risk women (n=51). We found higher plasma Hpx concentration in high risk women, who developed pre-eclampsia compared to low risk women. Plasma A1M was higher in the group of high-risk women, who did not develop pre-eclampsia than in other groups. We investigated the changes in maternal plasma Hpx and A1M concentrations from the first trimester to late second trimester (blood sampling at 12–14, 18–20 and 26–28 weeks of gestation) (Study IV). We used the same study cohort than in the third study. It appeared that high-risk women, who did not develop pre-eclampsia had a unique profile of haemoglobin scavenger proteins during pregnancy. Firstly, unlike in other groups, plasma Hpx concentration did not change during the study period, and it was lower during the first half of the pregnancy than in other groups. Secondly, the A1M concentration increased during the first half of the pregnancy and stayed at the higher level compared to the other two study groups. During the first half of the pregnancy the change in A1M concentration in high-risk women who did not develop pre-eclampsia was opposite to the change seen in those women who developed pre-eclampsia, while there was no change of plasma A1M concentration in low-risk women. We also found that women who subsequently developed pre-eclampsia and gave birth to a small-for-gestational-age newborn had consistently higher plasma levels of A1M than women who developed pre-eclampsia and gave birth to an appropriate-for-gestational-age newborn. The difference was significant from mid-gestation onwards. In conclusion, the serum PlGF concentration of women who started low dose aspirin before 14 weeks of gestation was higher from mid-gestation onwards compared to the other groups. This may provide one mechanism by which low-dose aspirin prevents pre-eclampsia. This thesis sheds light on the serum haemoglobin scavenger protein dynamics during the first and second trimester of pregnancy in women who have low or high risk for pre-eclampsia, as it was found that higher plasma concentrations of A1M in high-risk women, who do not develop pre-eclampsia may be associated with a reduced risk of developing pre-eclampsia. Furthermore, high-risk women who do not develop pre-eclampsia may have unique, protective dynamics of serum haemoglobin scavenger proteins. These findings suggest that not only clarification of the pathophysiology of pre-eclampsia, but also protective factors should be a focus of future research. Additionally, it was shown that the change in plasma A1M concentration in women who subsequently develop pre-eclampsia may be inversely related with foetal growth from mid-gestation onwards until the late second trimester. Multivariate models constructed with regularised logistic regression provided only modest prediction rates for all pre-eclampsia and its subtypes. This thesis strengthens the theory that pre-eclampsia is a complex multi-factorial syndrome, in which many biochemical pathways are affected.
  • Markkanen, Helene (Helsingin yliopisto, 2021)
    The measurement of serum growth hormone (GH) is the cornerstone of the diagnosis and management of GH-related disorders, acromegaly, and GH deficiency (GHD). GH secretion from the anterior pituitary gland is pulsatile and influenced by several factors (e.g. exercise, stress, glucose). Therefore, the GH result of a single blood sample is not diagnostic. Thus, the diagnosis is based on the suppression and stimulation tests of pituitary GH secretion. GH measurement is challenging due to the heterogenous structure and binding proteins of GH. The first assays for GH were based on radioimmunoassay (RIA) using polyclonal antibodies. The old International Standard (IS) reference preparations for GH assay calibration were of pituitary origin and contained a pooled cadaver-derived mixture of GH isoforms. During the 1990s, a significant methodological change in serum GH assays occurred, when RIAs were gradually replaced by more specific sandwich assays with monoclonal antibodies and highly sensitive labels. The use of monoclonal antibodies and IS preparations containing different GH isoforms caused significant between-assay and between-laboratory variation of GH levels. During the 1990s, the standard of recombinant 22-kDa GH became available. At the beginning of the 2000s, the WHO IS 98/574 (22 kDa) was established. During the last 10 years, GH immunoassay manufacturers have adopted this new standard for calibration of GH assays. Together with monoclonal antibodies, this has resulted in a significant reduction of GH concentrations obtained by recent GH assays. The aim of this study was to evaluate the interlaboratory variation of different GH assays used in Finland between 1998 to 2003 on the basis of an external quality assessment round of GH. Moreover, we wanted to compare the clinical use of two automated GH assays, AutoDELFIA and Immulite 2000 assays (calibrated against the same standard), with old polyclonal RIA with older standardization. The utility of consensus cut-offs of the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) in clinical practice was also studied by using those two automated assays. At the beginning of this study, the clinical use of a new GHRH+ARG test was assessed in control subjects and GHD patients by using the AutoDELFIA GH assay. Later, the cut-offs for the GHRH+ARG test were evaluated by using the Immulite 2000 XPi GH assay, which is calibrated against the current WHO IS 98/574. Our results showed that the international cut-off values for GH in consensus statements and guidelines cannot be directly adopted in diagnostic use without a clinical validation of the particular GH assay in use. We also showed that male control subjects in the OGTT and GHRH+ARG test had significantly lower GH values than women, suggesting that gender-specific cut-offs are needed. In our study, the ITT was an unreliable test for GHD. Almost half of apparently healthy adults have peak GH values <3 μg/L, which is the cut-off for GHD recommended in the consensus statement of 2011. Our present Immulite 2000 XPi assay yielded sufficiently similar GH results as the former AutoDELFIA assay. Thus, awareness of the differences between GH assays is important. It is crucial to use the same GH assay in the follow up of each patient with acromegaly or GHD.
  • Salo, Juho (Helsingin yliopisto, 2021)
    Chest wall resection and reconstruction pose unique surgical challenges given the complex anatomy and important functional role of the chest wall and its protective function for vitally important organs. Yet, a paucity of literature has reported large patient series, likely attributable to the rarity of cases and the challenges posed by this complex surgical procedure. This thesis summarises a retrospective analysis of chest wall resections and reconstructions resulting from malignant disease. Here, the focus lies on the surgical technique, short-term outcomes, survival and quality of life amongst patients. Study I consists of a retrospective review of patients who underwent oncological chest wall resection and reconstruction from 1997 through 2015 in the Department of Plastic Surgery at Töölö Hospital (Helsinki, Finland). The primary indications for resections were breast cancer, soft-tissue sarcoma and bone or chondrosarcoma. Amongst these resections, 53% were full- and 47% were partial-thickness resections, primarily located anterolaterally. Clear histological margins were reached in 82% of the resections. Reconstruction of the chest wall was warranted in 87% of cases and with 48% of cases involving stabilisation with a concurrent soft-tissue flap. The remaining patients underwent either chest wall stabilisation or soft-tissue flap coverage. This coverage most commonly consisted of pedicled or local flaps. Free flaps were necessary in 21% of cases, and no flaps were lost. Amongst 135 patients, 29 (21%) experienced complications. The most common complications included pneumonia and partial flap loss. We observed a 0% mortality rate. With a 4-year median follow-up, the 5-year overall survival rate reached 70%. Study II describes our surgical technique for diaphragm and thoracoabdominal wall reconstruction following oncological resection, focusing here on surgical outcomes. The most common indication for surgery was sarcoma. A thoracoabdominal wall reconstruction was performed using mesh in 14 cases and 7 cases relied on mesh and a flap. A diaphragm reconstruction with a second mesh was warranted in 6 cases. In 15 cases, the diaphragm was reattached using an acceptable tension. Our method of thoracoabdominal wall and diaphragm reconstruction proved safe without abdominal wall hernias or paradoxical chest wall movement. Study III evaluated the short-term outcomes, survival and tumour recurrence following chest wall resection amongst 49 soft-tissue sarcoma patients. Amongst these, 63% were high-grade and 37% were low-grade tumours. Surgery required 19 full- and 30 partial-thickness chest wall resections. The resection margins were wide or marginal in 86% of cases. The chest wall was stabilised and covered with soft tissue in 13 patients, reconstructed with a flap in 11, stabilised in 13and closed primarily in 12. In total, 11 patients experienced complications. During follow-up, local recurrence developed in 8 patients and 9 patients developed metastasis. The 1-, 5- and 10-year survival rates were 93.8%, 76.0% and 71.6%, respectively. Recurrence-free rates were 84.4%, 70.7% and 70.7%, respectively. Positive prognostic factors consisted of being under 50 years old (p = 0.01), a wide margin (p = 0.02) and radical treatment (p = 0.04) consisting of either resection with a wide margin or a marginal resection combined with adjuvant radiotherapy. Patients undergoing nonradical treatment experienced a 3.1-fold reduction in survival compared to patients who underwent radical treatment (95% confidence interval [CI] 0.96–10.12; p = 0.06). Cross-sectional study IV aimed to assess the long-term health-related quality of life (HRQoL) following chest wall reconstruction after oncological resection. In total, 78 patients who underwent surgery between 1997 and 2015 were invited to complete the 15D and Core Quality of Life for Cancer questionnaire (QLQ-C30) HRQoL instruments. Altogether, 55 patients (71%) completed the questionnaires, a median 66 ([IQR] 38–141) months after surgery. Indications for surgery included soft-tissue sarcoma (n = 16), advanced breast cancer (n = 15), bone or chondrosarcoma (n =14) or other tumour (n = 10). Following chest wall resection and reconstruction, the mean 15D score (0.878, standard deviation [SD] ±0.111) was comparable to that amongst the age- and gender-standardised general population (0.891, SD ±0.041). Patients were statistically significantly worse off on the dimensions of ‘breathing’ and ‘usual activities’. The QLQ-C30 cancer-specific HRQoL was 72 (maximum 100) and scores for the QLQ-C30 functional scales ranged from 78 (physical) to 91 (social). Within specific reconstruction subgroups, no statistically significant differences in HRQoL were detected after analyses were adjusted. In conclusion, chest wall resection and reconstruction represents a safe therapeutic modality when accompanied by careful patient selection, appropriate perioperative and postoperative care and selection of the proper surgical technique both in sarcoma and advanced breast cancer patients. Resection with wide margins remains the primary aim for treatment of chest wall soft-tissue sarcoma patients. If wide margins are not achieved, treatment should be combined with adjuvant radiotherapy. In locally advanced breast cancer, surgical chest wall resection and reconstruction play a role in the treatment of these patients. Following chest wall reconstruction after tumour resection, patients’ HRQoL is comparable to that amongst the age- and gender-standardised general population.
  • Mäkelä, Kati (Helsingin yliopisto, 2021)
    Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease of a dismal prognosis. While IPF is a rare disease, it is still the most common of idiopathic interstitial pneumonias. The radiological and histopathological manifestation of the disease is the usual interstitial pneumonia (UIP) pattern. The etiology of IPF is unknown, but inorganic dust is one of the known risk factors for IPF. The diagnosis of IPF is usually based on clinical and radiological data, but a surgical lung biopsy is required for a minority of patients. Confirming the diagnosis can be challenging as many interstitial lung diseases share similar features, and interobserver variation between radiologists and pathologists is significant. Separating IPF from other interstitial lung diseases is crucial due to differences in treatment and prognosis. In this doctoral thesis, we hypothesized that histopathological features in IPF lung tissue would be associated with survival and lung function. In addition, we aimed at investigating interobserver agreement among pathologists, inorganic particulate matter (PM) in the lung tissue of patients with IPF, and the use of artificial intelligence (AI) in analyzing lung tissue samples of IPF patients. Our study cohort originated from a prospective, multicenter registry study, namely the FinnishIPF registry. We searched for patients with available histological lung tissue samples and compared the histopathological features to the registry data. In Study I, four pathologists experienced in pulmonary pathology re-evaluated 60 lung tissue samples using the 2011 diagnostic criteria of IPF. They also recorded atypical histopathological features for IPF. Most of the samples were re-evaluated as definite UIP (38/60, 63%). The most common atypical feature for IPF was abundant inflammation (15/60, 25%). Using Cohen’s κ coefficient, the interobserver agreement varied from slight to substantial (κ=0.04-0.78); the variation might be partly causative of differences in the interpretation of the presence of giant cells. Radiologically definite UIP associated with a poor survival. However, the histopathological UIP pattern or atypical features for IPF were not associated with survival. In Study II, we focused on inorganic PM in 73 IPF lung tissue samples. We developed a semiquantitative scoring method (0-5) for coal dust pigment and inorganic PM using polarizing light microscopy. PM scores were compared to clinical, population density, and air quality data. An energy dispersive spectrometry with a field emission scanning electron microscope was used to analyze the elemental compositions of six IPF lung tissue samples. There were high scores of inorganic PM in the samples from southern Finnish university hospital districts compared to the samples from northern districts (31/50, 62% vs. 7/23, 30%, p=0.02). The highest scores of 4 and 5 were connected to an exposure to inorganic dust (n=15, p=0.004). Aluminum, silicon, and potassium were found in all six samples. In Study III, we tested AI in the analysis of histopathological features in IPF samples. With 20 different IPF samples, we developed an AI model using a convolutional neural network in Aiforia® platform. The AI model was taught to recognize alveolar parenchyma from the lung tissue, fibroblast foci (FF), interstitial mononuclear inflammation, and intra-alveolar macrophages. The samples of 71 IPF patients were analyzed with the model. The high area of FF was associated with a poor survival (p=0.01), and we found that high amounts of interstitial mononuclear inflammation and intra-alveolar macrophages were associated with a prolonged survival (p=0.01 and p=0.01, respectively). FF and intra-alveolar macrophages also had a link with lung function. High numbers of FF were associated with a low diffusing capacity for carbon monoxide (p=0.03), whereas a high intra-alveolar macrophage density was associated with a high forced vital capacity of predicted (p=0.03). In conclusion, FF seem to be the most potent single histological prognostic markers of survival in IPF. Of the other markers, inflammatory cells appeared to predict a prolonged survival. The interobserver agreement on the histopathological features of IPF varied, and especially the interpretation of giant cells seemed to cause a discrepancy. Inorganic PM in the lung tissue of IPF patients was not associated with the survival. Instead, the histological PM could reflect the level of exposure to air pollution. In the prognostic evaluation of the histopathological features in IPF lung tissue samples, AI could function as a future tool.
  • Tolmacheva, Aleksandra (Helsingin yliopisto, 2021)
    Spinal cord injury (SCI) is a debilitating condition with a considerable socioeconomic impact on healthcare resources and on the injured individuals. Since acute management of SCI has considerably improved, rehabilitation of SCI is in high demand to improve patient quality-of-life. SCI is characterized with an interruption of neuronal relay from the brain to the efferent organs and back to the brain, resulting in paralysis. Currently, there is no cure for SCI. Widely used conventional rehabilitation programs do not enable restoration of motor function in a severe SCI. Paired associative stimulation (PAS) is a relatively new non-invasive method that applies two-site stimulation within the motor system. A single PAS session results in a transient increase of motor output in neurological patients. However, the potential of long-term PAS on functionally meaningful recovery in SCI patients has not been explored. The goal of this dissertation was to investigate the efficacy of long-term PAS on hand motor recovery in chronic SCI patients (studies IV, V, VI). The altered physiology of the motor system in SCI individuals had to be considered regarding the feasibility of the PAS protocol (studies I, II, III). PAS was implemented with transcranial magnetic stimulation (TMS) and peripheral nerve stimulation (PNS). TMS- and PNS-induced pulses were timed to coincide in the spinal cord as determined by the value of an interstimulus interval (ISI). This neuronal interaction supposedly resulted in long-term potentiation (LTP)-like plasticity in the corticomotoneuronal synapses. A classical PAS protocol requires accurate determination of an ISI to induce LTP-like plasticity in the targeted synapses. In our laboratory, the PAS protocol was modified (a single-pulse high-intensity TMS and a high-frequency PNS train) to increase the feasibility of PAS in SCI individuals. The exact determination of ISI is not always possible in SCI patients. Study I mimicked this clinically possible scenario. PAS protocols with different ISIs that provide non-synchronized arrival of TMS- and PNS-induced pulses were examined. All tested PAS protocols were effective, suggesting that this PAS protocol is feasible for neurological patients. Study II consisted of two experiments. Experiment 2.1 sought to determine more effective PAS settings. PAS protocols with different frequencies of PNS train (25 Hz, 50 Hz, 100 Hz) were tested. Although all protocols increased motor-evoked potential (MEP) amplitudes, PAS with 100-Hz PNS exhibited the strongest and most sustainable MEP potentiation. Experiment 2.2 addressed the challenge of accurate motor cortex mapping in neurological patients. We observed demonstrated efficacy of our PAS protocol even when TMS was administered to a suboptimal spot in the primary motor cortex (M1). Study III consisted of three experiments. Experiment 3.1 sought to determine a more convenient PAS protocol for SCI patients. PAS with increased frequency of PNS-TMS pairings (0.4 Hz PAS) that allowed a half-duration PAS session was tested. The shortened protocol was less effective compared to our original PAS protocol (0.2 Hz PAS). Experiment 3.2 continued exploring the impact of PNS frequency (100 Hz, 200 Hz, 400 Hz) on the effectiveness of the PAS protocol. PAS with a 100-Hz PNS train remained the most reliable protocol. Experiment 3.3 sought to enhance PAS efficacy by increasing collision of neuronal events in the corticomotoneuronal synapses by employing 20-Hz paired-pulse TMS in PAS. This PAS protocol induced a significant MEP suppression. Study IV assessed the efficacy of the novel PAS protocol in two subjects with SCI. Study V explored the efficacy of PAS in a group of traumatic SCI patients. After a 4-week PAS, the manual muscle testing (MMT) score improved in the PAS-treated hand. Sham PAS stimulation of the contralateral hand (sham TMS and actual PNS) induced a significantly smaller MMT score increase compared with the hand activated by PAS. Study VI applied long-term PAS with the most effective settings (100-Hz PNS) in a group of SCI patients with different neurological origins. In addition to considerable improvement in MMT scores, daily functioning of the patients improved. The observed improvement persisted at least 6 months after the PAS treatment. Our PAS protocol was designed to potentiate spared connections in the spinal cord in SCI patients. Modification of our PAS protocol demonstrated feasibility of long-term PAS in SCI individuals. In a series of experiments on healthy subjects, different parameters of the PAS protocol were tested with the objective of increasing PAS effectiveness. The most effective PAS protocols were translated to clinical research. Long-term PAS demonstrated a therapeutic effect that was accompanied with functional improvement. Long-term PAS outperformed long-term PNS, which is widely used in conventional rehabilitation in SCI. The feasibility, effectiveness, and safety of this method favours long-term PAS as a promising motor rehabilitation in SCI patients.
  • Eloranta, Katja (Helsingin yliopisto, 2021)
    Hepatoblastoma (HB) is the most common pediatric liver malignancy with an annual incidence of 1.8 cases per million children. Most cases are sporadic, although certain genetic disorders such as Beckwith-Wiedemann syndrome and familial adenomatous polyposis are associated with increased risk of HB. Developmental pathways, such as WNT/β-catenin and Hedgehog signaling, are often aberrantly activated in HB cells. The overall mutation burden in HB is generally low, but the CTNNB1 gene encoding β-catenin is altered in over 60% of cases. Surgical resection of tumor is a mainstay of HB treatment, generally supported by pre- and post-operative chemotherapy. The survival rate of HB is over 80% if tumor is confined to liver but decreases dramatically when there is extrahepatic involvement, so new treatment options are needed. In this thesis we examined two key factors, GATA4 and neuropilin-2, in the pathobiology of HB. Additionally, we conducted in vitro experiments investigating the potential of chloroquine as a novel HB therapy. Transcription factor GATA4 is crucial for early liver development. We observed that most HB patient samples and cell lines express high levels of GATA4. Moreover, GATA4 expression was associated with mesenchymal-like and motile phenotype in HB cells. Neuropilins (NRP) are multifunctional receptors involved both in physiological and pathological processes. We noted high NRP1 and NRP2 expression in HB patient samples and six HB cell models. Utilizing siRNA transfection, we observed decreased viability and motility in NRP2 knockdown cells compared to cells with intact gene expression suggesting that NRP2 promotes aggressive behavior in HB cells. Chloroquine is a traditional anti-malarial which has demonstrated potential in cancer management. We observed a drastic decrease in HB spheroid viability after chloroquine treatment. Chloroquine treatment also modified the metabolic profile of HB cells with a remarkable decrease in NAD+ and aspartate concentrations. Additionally, we noticed significant decrease in PARP1/2 expression suggesting that chloroquine may have effect on DNA repair system in HB cells. In summary, this thesis shed new light on the molecular mechanisms of HB pathogenesis. In the future, these findings may be utilized in development of novel treatment approaches and diagnostics to improve survival and life quality of HB patients.
  • Hautala, Johanna (Helsingin yliopisto, 2021)
    A congenital heart defect (CHD) is the most common congenital structural anomaly, and it is observed in 1% of live births. In Finland, approximately 550 children per year are born with a CHD. The defect is critical in 10%, requiring immediate care soon after birth. Due to developed care, most CHD patients survive adulthood, but a critical CHD is still one of the most important reasons for perinatal mortality. Information on CHD risk factors is still scarce. By uniting data from quality, nationwide registers, this study assessed the total and live birth prevalence of the selected CHDs and the effect of the implementation of national fetal anomaly screening on prenatal detection rates nationally and regionally in an 11-year cohort. In addition, the monthly variation of these CHDs was analyzed, and possible analogous variations n in viral or bacterial infections were evaluated. Prenatal anomaly and chromosomal screenings have become a pivotal part of health care. In Finland, voluntary, nationwide, free-of-charge ultrasound screenings have been provided by the municipalities for every pregnant woman since January 2010. Fetal heart screening is based on a four-chamber view and outflow tract views. This study selected the univentricular heart (UVH, in which one ventricle is taking care of circulation) as an index anomaly for the four-chamber view and transposition of the great arteries (a TGA, in which the aorta arises from the right ventricle and the pulmonary trunk from the left ventricle) for the outflow tract views. The data of all live births, stillbirths and terminated pregnancies, and maternal characteristics were gathered with ICD codes in 2004–2014. The cases with a congenital diaphragmatic hernia and a CHD in 2002–2011 were collected for Study III. The population-based cohort included data from the National Institute for Health and Welfare’s; Finnish Register of Congenital Malformations, Medical Birth Register, The Register of Induced Abortions, Hospital Discharge Register, National Infectious Diseases Register, causes of death from Statistics Finland, and the National Register of Pediatric Cardiac Surgery maintained by Children’s Hospital at Helsinki University Hospital. Nationwide prenatal detection rates increased after implementing a systematic screening program: in UVH from 50% to 83% and in TGA from 12% to 41%. However, significant regional differences were found; especially in TGA, detection rates varied between 13% and 65% and remained still non-optimal. Significant monthly variation in early pregnancy was observed in a UVH and a TGA, but no analogous variation with studied bacterial and viral infections was found. Multiple pregnancies, obesity, and pregestational diabetes were more common among the CHD population than all parturients. None of the studied risk factors affected prenatal detection. Some extra-cardiac malformations, such as a congenital diaphragmatic hernia, were associated with the development of hypoplastic left heart syndrome (HLHS), warranting the close follow-up of heart development in fetuses with a diaphragmatic hernia. Neither the screening program nor the prenatal diagnosis affected simple TGA mortality. None of the prenatally diagnosed TGA infants died, and total mortality was 9%. Most of the deceased died in maternity hospitals without a prenatal diagnosis, and postoperative mortality was only 1%. Older maternal age and lower gestational age at birth were associated with higher mortality. Maternal prepregnancy obesity and perioperative risk factors were associated with a higher need for health care resources during the first year of life. In conclusion, the implementation of a systematic fetal screening program increased prenatal detection rates of UVH and TGA. However, for a TGA, there were significant regional differences, and the prenatal detection rates of TGA were still not optimal. These results suggest establishing the previously recommended prospective fetal screening register to assure equal, adequate, and quality screening nationwide.
  • Koppatz, Hanna (Helsingin yliopisto, 2021)
    Background: Laparoscopic cholecystectomy (LCC) is one of the most common surgical procedures. Surgery involves small, but noteworthy complication risks. Malignant changes in the pathology of the gallbladder are also rare. When gallbladder surgery is so common, this aspect of surgery can have an impact on a large portion of the population. Material and Methods: The Study I was a randomized controlled trial. We investigated whether 3D laparoscopic cholecystectomy would be faster and safer than a conventional laparoscopy in day surgery patients. The primary outcome was operation time. The Study II was a retrospective cohort study in which patients had been diagnosed with severe biliary injury (BDI). We studied the success of surgical treatment as well as the quality-of-life (QOL) of patients. The Studies III and IV addressed gallbladder cancer (GBC) in a retrospective study designs. Results: The Study I included 105 and 104 patients randomized to 3D and 2D LCC groups. The 3D system did not reduce the LCC operation time (3D vs 2D; 49.0 vs 48.0 min, p=0.703). The 3D technique did not either affect complications. The Study II included 52 patients with BDI and 53 patients without complications as controls. No difference in long-term (median 90 months) QOL was observed between groups. Three patients (5.8%) died from BDI. “Primary patency” was 71%. At one and five years, the “Actuarial primary patency rate” was 58% and 53%, respectively. Patency was achieved at 83% if the reconstructive surgery was primarily performed by a liver surgeon. In the Study III, GBC was rarely found (n = 10/2034; 0.5%) in the gallbladder, which was removed for benign reasons and no cancer was found in the macroscopically normal gallbladder. In the Study IV, we found 294 patients with GBC and revealed a low and slightly declining incidence of GBC in southern Finland. The proportion of patients who underwent curative surgery was 19%, and the five-year survival after curative-intent surgery was 57%. The five-year overall survival was 12% but without surgical or oncologic treatment 1.3%. Conclusions: Cholecystectomy is a common and safe procedure when the correct surgical technique and the possibility of anatomical variations are considered. The use of a 3D laparoscopy system does not improve the safety or efficacy. If a severe bile duct injury occurs, biliary reconstruction is recommended to be performed by a hepatobiliary surgeon. In a gallbladder sample removed for a benign reason, the use of selective histopathologic examination could save a substantial amount of health care resources. The prognosis of GBC is poor. Increasing the proportion of patients undergoing curative-intent resection and adjuvant therapy, as well as the use of neoadjuvant therapy, is likely to improve the prognosis of patients with GBC.
  • Selinheimo, Sanna (Helsingin yliopisto, 2021)
    Several biological and behavioural risk factors for respiratory health have been established. However, individuals may perceive and interpret somatic symptoms and sensations differently. Psychiatric disorders, psychological dispositions, situational factors and prior experiences can influence the significance that individuals attach to bodily sensations and their regulation. Thus, as subjectively perceived respiratory complaints may not always align with objective clinical measurements, these factors may be linked with worse respiratory outcomes in populations of patients, samples of healthy individuals and moreover with persistent physical symptom (PPS) reports. In some cases, PPS appear despite good care and are associated with environmental factors such as indoor air, regardless of indoor environment quality. PPS challenge the health care system as they do not respond to standard medical treatment and cannot be explained by conventional medical models. This thesis examines the association between psychological dispositional characteristics and subjectively and objectively assessed respiratory outcomes, and the discrepancy between subjective and objective health measures in cross-sectional (Study I) and follow-up designs (Study II) at the population level. The aim was to investigate the effects of these factors on health-related quality of life (HRQoL) among cases with PPS associated with the indoor environment (Study III) and the effectiveness of psychosocial treatment for these complaints (Study IV). This thesis used two complementary datasets. First, we examined how sense of coherence, alexithymia and health anxiety were associated with respiratory health complaints without objectively assessed problems in lung function or incidence of respiratory diseases in a population-based data Health 2000 survey and its 11-year follow-up study Health 2011 (Studies I–II). Health 2000 is a nationally representative survey of over 30-year-old Finnish adults conducted by the Finnish Institute for Health and Welfare. Its data include comprehensive clinical health examinations, interviews covering respiratory symptoms and common psychiatric disorders, and validated questionnaires on psychological dispositions. The data of this study included the participants with complete information on lung function and respiratory symptoms (N=4544). The follow-up analyses in the Health 2011 sample were restricted to participants who were still working aged (<65 years) during the follow-up assessment in 2011 and had been included in the baseline study (N=2310). The second part of the thesis examined a randomized controlled trial (RCT) that compared treatment as usual (TAU) enhanced with cognitive behavioural therapy (CBT) or psychoeducation for non-specific symptoms associated with the indoor environment (Studies III–IV). The RCT included data from the participants recruited from occupational health service units. The inclusion criteria for participants were i) recurrent and persistent multiorgan symptoms and disability that they attributed to workplace indoor air, ii) symptoms not adequately explained by medical or indoor environment exposure-related reasons and iii) onset of symptoms with disability for a maximum of three years before the study. Candidates also had to be occupationally active. After baseline clinical examinations, the participants were randomized into TAU or TAU enhanced with psychoeducation or CBT. The primary outcome was HRQoL, and the secondary outcomes included measures of symptom severity and dispositional factors, which were followed up for 12 months after randomization. The findings of this study show that over a quarter of the Finnish population report respiratory health complaints without objective signs of impaired lung function. Sense of coherence, alexithymia and high illness worry associated significantly with perceived complaints. Individuals reporting respiratory health complaints without any objective signs of respiratory pathology reported 1.25 (95% CI 1.15–1.35, p<0.0001) times more health care visits than those with normal lung function and no respiratory symptoms. The difference between these groups attenuated by almost 43% following adjustment for psychological factors. Further, high illness worry and alexithymic characteristics predicted the incidence of respiratory health complaints and respiratory diseases during the eleven-year follow-up. Individuals with PPS associated with the indoor environment had significantly poorer HRQoL than comparisons derived from the general population or subgroups of those with asthma, anxiety and depressive disorder, or a chronic condition that caused work disability. Of the dispositional factors, high neuroticism showed a significantly negative, and high sense of coherence a significantly positive correlation with good HRQoL. The analyses revealed no robust effect of psychosocial interventions on HRQoL but in some cases, they prevented the deterioration of HRQoL because of PPS associated with the indoor environment. The findings from population-based Studies I and II including both objective and subjective health outcomes agree with previous studies conducted among populations of patients and in selected samples of healthy individuals and suggest that dispositional factors play a prominent part in respiratory outcomes at the population level. Our findings from Study III underline the need for improved treatment among patients with PPS associated with the indoor environment and further, that dispositional factors should be acknowledged in the treatment process. However, the results from Study IV suggest that improving the effectiveness of treatment for these health complaints requires information on the therapeutical change mechanisms related to the condition and a special focus on the acceptance of treatments. It is also important to promote further research of socio-cultural factors to explain the disease initiation and treatment process and to understand the emergence of the health complaints associated with environmental factors.
  • Pasanen, Annukka (Helsingin yliopisto, 2021)
    The ultimate goal of personalized medicine is to offer optimal treatment to each patient while avoiding under- and overtreatment. At present, clinicopathological risk algorithms determine whether lymph node dissection or postoperative radiotherapy/chemotherapy are indicated for a patient with endometrial carcinoma (EC). As traditional risk assessment methods suffer from limited accuracy, researchers are trying to find molecular biomarkers that could help guide the treatment of EC. In 2013, a landmark study by the Cancer Genome Atlas research group (TCGA) identified four prognostic molecular subgroups of EC. The study cohort of this thesis consisted of unselected EC patients who were surgically treated at Helsinki University Hospital between 2007 and 2012. Tumor samples from 842 patients were included in a tissue microarray. The aim of this thesis was to investigate clinicopathological and prognostic significance of L1 cell adhesion molecule (L1CAM) and MLH1 methylation status in EC. In addition, we explored the differences between the two largest molecular subgroups of EC with regard to the prognostic impact of individual risk factors and the expression of an immunotherapy target molecule, programmed death ligand 1 (PD-L1). I: Tumoral L1CAM positivity was associated with more frequent distant relapses and independently predicted poor prognosis in endometrioid EC but not in non-endometrioid EC. L1CAM may enhance the accuracy of post-operative risk stratification algorithms guiding adjuvant treatment of EC. II: Integrating preoperatively assessed tumoral L1CAM with the conventional risk assessment models did not facilitate stratification of patients to lymphadenectomy. III: In multiplex immunohistochemistry, PD-L1 expression was more frequent in intratumoral immune cells (27.7%) than in carcinoma cells (8.6%). Within the molecular subgroups the relative frequency of PD-L1 positivity was higher in POLE-mutated and mismatch repair deficient (MMRd) tumors than in the other molecular groups (p53 abnormal and no specific molecular profile). Further, we found differences in PD-L1 expression across histological subgroups. IV: MMR deficiency correlated with several clinicopathological risk factors. Methylated phenotype was associated with older age and larger tumor size. Methylated MMRd phenotype predicted poor disease-specific survival compared with MMR proficient EC, but the difference with non-methylated MMRd EC was not significant. V: Our study confirmed the survival differences between molecular subgroups originally proposed by TCGA: POLE-mutated EC had an excellent prognosis, followed by NSMP, MMRd, and the more aggressive p53 abnormal EC. In multivariable analysis adjusting for conventional risk factors, NSMP and MMRd molecular subgroups did not present significant survival differences. Interaction analysis confirmed differences between NSMP and MMRd with regard to prognostic impact of individual risk factors. This thesis identifies several molecular factors that may facilitate treatment planning of EC. Given their distinct prognosis and pathogenetic mechanisms, molecular EC subgroups should probably be regarded as disease entities of their own. This way optimal risk stratification algorithms can be determined leading to personalized and cancer type-specific treatment of patients with EC.
  • Kuusela, Sara (Helsingin yliopisto, 2021)
    Background: Type 2 diabetes is characterized by hyperglycemia and insulin resistance, and apart from genetic susceptibility to the disease, its major risk factor is obesity. Diabetic kidney disease is a common complication of diabetes and the leading cause of end-stage renal disease. The complex pathological mechanisms of diabetic kidney disease are not fully understood; however, both glomerular and tubular changes are involved. Tankyrases (TNKS1 and TNKS2) are ADP-ribose transferases that post-translationally modify themselves and their binding partners with ADP-ribose (termed PARylation). TNKS1/2 activity is linked to diverse cellular processes, including Wnt-signaling and cellular energy metabolism. TNKS1/2 and their PARylation activity have not been studied in the context of kidney function and injury. PARylation has been implicated in the pathological mechanism of diabetic kidney disease; however, the involvement of TNKS1/2-mediated PARylation in diabetic kidney disease is not fully understood. In the studies of this thesis, we aimed to define whether TNKS1/2 and their PARylation activity are involved in the pathophysiological processes associated with diabetes and diabetic kidney disease and whether TNKS1/2 could serve as treatment targets in these diseases. Also, we investigated the role of TNKS1/2 and their PARylation activity in kidney injury associated with the loss of CD2AP. Results: Study I of this thesis work showed that the inhibition of TNKS1/2 PARylation activity with a TNKS1/2-specific inhibitor reduces body weight gain, restrains fat accumulation, and alleviates dyslipidemia of diabetic db/db mice. We found that the inhibition of TNKS1/2 increases adiponectin, downregulates proteins associated with lipolysis, and induces browning of white adipose tissue. Furthermore, inhibition of TNKS1/2 stimulated the oxidation of lipids in muscle and reduced liver steatosis. A central finding was that TNKS1/2 inhibition leads to reduced TNKS1/2-mediated PARylation and the consequent increase of the expression of PGC-1α, the key transcriptional regulator of mitochondrial biogenesis and energy metabolism, in white adipose tissue and muscle in the db/db mice. In Study II, concentrating on the potential of TNKS1/2 inhibition to ameliorate diabetic kidney disease in the db/db mice, we found that TNKS1/2 PARylate PGC-1α and that TNKS1/2 inhibition stabilizes PGC-1α in the kidney. We showed that TNKS1/2 inhibition improves mitochondrial function and cristae morphology in the proximal tubule epithelial cells in diabetic db/db mice's kidneys. Furthermore, we found that TNKS1/2 inhibition ameliorates inflammation and fibrosis in the kidneys of db/db mice. In Study III, we showed that CD2AP, an adaptor protein essential for podocyte function, interacts with TNKS1 and TNKS2 and is a negative regulator of TNKS1/2 PARylation activity. We found that TNKS1/2-mediated total PARylation in podocytes is increased in the absence of CD2AP. We also found that active β-catenin accumulates in podocytes in the absence of CD2AP, which leads to upregulation of podocyte injury-associated molecules LEF1 and fibronectin. Inhibition of TNKS1/2 reduced total PARylation and β-catenin activation in cultured podocytes lacking CD2AP. However, knock-down of CD2AP and simultaneous inhibition of TNKS1/2 PARylation activity in zebrafish worsened kidney injury and increased mortality. Conclusion: These thesis studies showed that TNKS1/2 inhibition reduces weight gain and renal inflammation, and fibrosis in the diabetic db/db mice and suggests that TNKS1/2 are novel molecular targets for treating diabetes and diabetic kidney disease. Additionally, we found that TNKS1/2 inhibition in the absence of CD2AP induces injury in cultured podocytes and disables kidney function in zebrafish.
  • Savolainen, Marika (Helsingin yliopisto, 2021)
    Moyamoya disease (MMD) and moyamoya syndrome (MMS), referred to as the moyamoya angiopathy (MMA), is a chronic progressive steno-occlusive angiopathy at the distal portions of internal carotid arteries (ICAs) and their proximal branches, with typical collateral artery formation, called the moyamoya vessels. MMS has similar vascular abnormalities as in MMD, but is associated with other conditions such as Down’s syndrome or neurofibromatosis I (NFI). MMA is more common in East Asia (e.g. Korea and Japan) than in Western countries. Majority of the MMA studies are done in the Asian populations. Typically the clinical presentation age of MMA has a bimodal distribution, and peaks both before the age of 14 and later, at the ages 21-59 years. Women are more affected than men, with a ratio varying from 1:1-3:2. Typical presenting symptoms are ischemic or hemorrhagic strokes, transient ischemic attacks, headaches, and epileptic seizures. In East Asian countries familial form of MMD is more common than in the Western countries and a RNF213 gene has been identified as an important susceptibility gene in East Asian populations. The underlying mechanisms of MMD are still unknown, despite extensive studies. Intimal hyperplasia, medial layer thinness, and the waving of internal elastic lamina are the representative histo-pathological features found in MMD-affected vessels. Revascularization surgery is commonly used to treat MMA, although it does not cure the disease, but it can reduce the risk of an ischemic stroke. Antiplatelet therapy is also commonly used to treat the ischemic form of MMA. For this thesis we collected an MMA database at the Helsinki University Hospital (HUH), including 61 Caucasian patients of Finnish origin treated in the HUH Neurology and Neurosurgery departments between January 1987 and December 2014. The aim of the first study (I) was to investigate the prevalence of MMA in Finland, and the type of the disease, i.e. the clinical manifestations, and treatments used. The incidence of MMA in the HUH district was 0.14 per 100 000. The prevalence in the HUH district was 2.38 per 100 000 in year 2014. There was a female predominance found (ratio 4.5:1). At the time of the diagnosis, 10 patients were children. The most common clinical manifestations were ischemic stroke (51%), hemorrhagic stroke (13%), and headaches (11%). Twenty-six patients underwent revascularization surgery. Seventy percent of the patients were on antithrombotic medication. In the second (II) study our aim was to study the long term prognosis of MMA in the Finnish patient population including all 61 patients of the HUH-MMA registry. The mean follow-up period was 9.5 years. Patient-years summed up to 581. Two-thirds of the patients had no new vascular events during the follow-up period. Eight patients had an ischemic and five patients had a hemorrhagic stroke during the follow-up. The average annual rate of a recurrent stroke from the first event for all the study subjects was 3.5%. Two patients died during the follow-up period. Cause of death was intracerebral hemorrhage in both cases. Patients reported significantly poorer physical and psychological health aspects of quality of life when compared to the general Finnish population. In the third (III) study we performed a follow-up brain MRI and MRA to detect potential radiological changes over time. The mean follow-up time was 64 months between these two MRI/MRA imaging time points and we found new ischemic or hemorrhagic lesions only in one patient. All unilateral cases remained unilateral in the radiological follow-up study including 32 of our registry patients. Ivy sign was observed in 22%, cerebral microbleeds in 6% and white matter lesions in 9% of the patients. The phenotype of MMD in Finland confirms occurrence of the Western phenotype of the disease, with a disease course rather benign in the Finnish patient population, despite the less common, hemorrhagic form, which has a higher mortality.

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