Lääketieteellinen tiedekunta


Recent Submissions

  • Salo, Veijo (Helsingin yliopisto, 2020)
    Cells store excess energy mostly as neutral lipids (NLs), inside fat-specialized organelles called lipid droplets (LDs). LDs are metabolic hubs involved in many cellular processes, such as lipid metabolism and endoplasmic reticulum (ER) homeostasis. There is increasing evidence linking LDs and human pathologies, including e.g. cardiovascular and fatty liver disease. Thus, detailed understanding of LD biology should be beneficial for tackling these disease states. LDs consist of a core of NLs surrounded by a phospholipid monolayer. Their biogenesis begins in the ER, with accumulation of NL lenses within the ER bilayer. These lenses subsequently grow and bud into the cytosol. All this likely occurs in specialized ER subdomains, but a detailed understanding of the earliest steps of LD biogenesis is still lacking. After formation, LDs retain an intimate relationship with the ER, through ER-LD contacts. Seipin is an oligomeric ER transmembrane protein implicated in LD assembly and adipocyte development. Mutations in seipin give rise to three disease states: a severe form of congenital lipodystrophy (BSCL2), a motor neuron disease called seipinopathy and a fatal neurodegenerative disease called Celia’s encephalopathy. The molecular function of seipin is unclear. In this thesis, using advanced cell biological techniques, we sought to expand the understanding of the function of seipin and how mutations in seipin lead to human disease. In the first study, we focused on the seipinopathy-linked seipin mutant, N88S-seipin, which is known to form misfolded aggregates. We found that overexpression of N88S-seipin leads to ER stress, decreased NL storage and voluntary swimming in zebrafish larvae. Upon increasing NL stores, these defects were alleviated, and the mutant protein was translocated from the ER to regions flanking LDs. Increasing cellular LDs also alleviated ER stress induced by tunicamycin. We propose that increasing LDs may aid cells in coping with misfolded proteins. We next focused on deciphering the function of seipin in LD formation and ER-LD contacts. Endogenously tagged seipin localized stably at ER-LD contact sites, but a BSCL2-linked mutant did not. Knockout (KO) of seipin led to the formation of numerous tiny LDs, which failed to grow and recruit protein and lipid cargo from the ER. Furthermore, whilst all LDs in control cells were connected to the ER, seipin KO lead to a subset of LDs completely detaching from the ER. The remaining LDs of seipin KO cells also had morphologically aberrant and dysfunctional ER-LD contacts. As similar defects were also evident in BSCL2 patient fibroblasts, we propose that seipin is crucial for the formation of ER-LD contacts and cargo delivery. Finally, we found that seipin can determine the site where LDs start to form, as relocalization of seipin to a subdomain of the ER, the nuclear envelope, was sufficient to relocalize LD biogenesis to that site. We found that seipin-mediated ER-LD contact sites display a uniform neck-like architecture, suggesting that the seipin oligomer may structurally restrain this site. Seipin was also required for ER-LD contact maintenance, as acute removal of seipin from ER-LD contacts lead to strikingly heterogonous LD growth. This LD growth heterogeneity arises via a biophysical ripening process, with NLs partitioning from smaller to larger LDs via LD-ER contacts. These data suggest seipin-mediated ER-LD contacts function as a valve facilitating NL flux from the ER to LDs, thus allowing controlled LD growth. Overall, this thesis provides insight into the function of seipin and mechanisms of LD formation. Our data suggest that the ER-LD nexus could be considered as a joint system, with continuous bidirectional trafficking of cargo occurring via ER-LD contacts mediated by seipin. Future work will be required to investigate the molecular intricacies of the lipid flux occurring at ER-LD contacts. A deeper understanding of LD biology will aid in the development of new therapeutics for a number of pathologies, including seipin-related disorders and common metabolic disturbances.
  • Wang, Yilin (Helsingin yliopisto, 2020)
    Microbial infections share many symptoms in common, rendering diagnosis difficult solely on clinical grounds. Thus, rapid, cost-effective and reliable tests are necessary for the diagnosis of infectious diseases. While the traditional diagnosis is mostly confined to detection of one pathogen at a time, a multiplex array could be a feasible alternative to improve the efficiency in the detection of infections. The Luminex xMAP-based high-throughput platform can provide simultaneous analysis of multiple analytes from the same sample by utilising differentially dyed microspheres. In this thesis, I developed xMAP-based Suspension Immuno Assays (SIAs) for the determination of IgG antibodies, IgM antibodies, as well as the avidity of IgG against the human cytomegalovirus (HCMV), Toxoplasma gondii (T. gondii) or human parvovirus B19 (B19V). Moreover, I also developed xMAP-based multiplex DNA assays for 13 human polyomaviruses (HPyVs). Primary infections by HCMV, T. gondii and B19V during pregnancy can result in severe consequences to the foetus. The serological status of the mother is critically important in counselling and recognition of infections. Hereby, I developed and evaluated SIAs for IgG, IgM and IgG-avidity against these three important pathogens. Diagnostic performances of the new assays were assessed with more than 1000 well-characterised serum samples. All the newly developed assays exhibited excellent performance compared to corresponding high-quality reference methods. The positive and negative percent agreements of the antibody-SIAs in comparison with high-standard reference assays were 92-100% and 95-100%, respectively. Kappa efficiencies between SIAs and corresponding reference assays were 0.94-1. Intra-assay and inter-assay coefficients of variations ranged between 2-12% and 1-19%, respectively. Among clinical samples from individuals with primary infection, the IgM- and IgG-SIAs served as highly sensitive screening means for detection of acute infections and immune status; and IgG-avidity-SIAs as a highly specific confirmatory approach for separation of primary infections from long-term B-cell immunity. On the other hand, during the past 12 years, a dozen viruses have joined the known family members of HPyVs. Serological studies have shown that HPyV infections occur at young age and most of the viruses circulate widely in the general population. Although HPyV infections are generally asymptomatic, severe complications can arise due to virus reactivations in immunocompromised or elderly individuals. HPyVs can persist lifelong after primary infection; however, their tissue specificities, persistence sites and transmission routes are still unclear. Also, the clinical manifestations of HPyVs with regard to immune suppression are largely unidentified. To this end, I developed xMAP-based multiplex DNA assays for all 13 HPyVs known before 2017, by using primer pairs and probes targeting the respective HPyV major capsid protein VP1 genes. The xMAP-based multiplex assays allowed for simultaneous detection of all the HPyVs with detection limits of 1-100 copies/µl. At high copies (105 copies/µl) each of the 13 target sequences were identified correctly with no cross-reactions. With this novel and specific assay, the extent to which the lymphoid system plays a role in the HPyV infection and persistence was assessed. The frequency of occurrence of HPyV viral genomes was explored in 78 lymphoid tissues from children and adults with tonsillar diseases. HPyV-DNA was found in 17.9% (14/78) of tonsils: JC polyomavirus (JCPyV, n=1), WU polyomavirus (WUPyV, n=3), Merkel cell polyomavirus (MCPyV, n=1), human polyomavirus 6 (HPyV6, n=6), trichodysplasia spinulosa polyomavirus (TSPyV, n=3). The observed frequent occurrence of HPyVs in human tonsils suggests the lymphoid tissue plays an important role as a potential transmission route and a location of persistence for these viruses. However, whether or not the undetected HPyVs share the same infection route requires more investigation with different sample types. Furthermore, to determine the occurrences in skin and clinical associations of HPyVs, I studied their genoprevalences in biopsies of premalignant [squamous cell carcinoma in situ (SCCis) or actinic keratosis (AK)] lesional vs. benign skin from 126 liver transplant recipients (LiTRs); as well as in healthy skin of 80 immunocompetent adults. Multiplex screening was followed by singleplex qPCRs of positive samples, for reference and quantification of the viral DNAs. In total, five dermal HPyVs – MCPyV, HPyV6, human polyomavirus 7, TSPyV, and Lyon IARC polyomavirus (LIPyV) – were found in 26.2% (58/221) skin biopsies. The prevalences and quantities of MCPyV in premalignant vs. benign skin of LiTRs were similar to those in healthy skin of controls. TSPyV was found in a single skin lesion at very low copies. The other three HPyVs occurred exclusively in benign skin. Overall, in 10 out of 12 SCCis/AK patients the viral DNA findings in skin were alike. Thereby, the occurrences of HPyVs in the skin of LiTRs and controls speak against a role for any of HPyVs in SCC development. The work presented in this thesis shows that the xMAP-based serological approaches exhibit excellent diagnostic performances compared to corresponding conventional methods. Moreover, the developed xMAP-based multiplex PCR for 13 HPyVs could be applied successfully in a variety of clinical materials. Altogether, the newly developed systems provide a powerful tool for medical diagnosis and research.
  • Porkka, Noora (Helsingin yliopisto, 2020)
    Lynch syndrome is the most prevalent cancer predisposition syndrome that causes significantly increased lifetime risk of cancer in multiple organs such as colorectum, endometrium and ovarium. The predisposition is caused by germline mutations in DNA mismatch repair (MRR) genes MLH1, MSH2, MSH6, and PMS2. Lynch-like syndrome colorectal tumors, like Lynch syndrome tumors, are MMR-deficient. Nevertheless, Lynch-like syndrome tumors do not bear germline mutations in MMR genes nor methylation of the promoter regions of MMR genes that would explain the deficiency. Instead, MMR deficiency in majority of Lynch-like syndrome tumors is caused by two somatic mutations in the MMR genes. Dysfunctional MMR protein complex enables accumulation of mutations in the genome (mutator phenotype) and, eventually, microsatellite instability and cancer. The reasons behind organ selectivity in MMR-deficient tumors are unknown, and whether breast cancer is part of the Lynch syndrome spectrum is under debate. The germline mutations in MMR genes are well studied, but molecular characteristics of Lynch syndrome tumors remain to be studied further. Lynch-like syndrome tumors remain less well characterized: besides the double somatic MMR mutations as a cause of MMR deficiency, their molecular and clinicopathological features as well as their incidence in the population remain poorly known. Currently, the only possibility to diagnose Lynch-like syndrome is by ruling out the possibility of germline mutations in MMR genes (Lynch syndrome) or methylation of the promoter regions of MMR genes. Synchronous ovarian and endometrial carcinomas are common in Lynch syndrome; whether they are of same origin (metastatic cancer) or two independently developed primary cancers remains to be resolved. We aimed to characterize the epigenetic and somatic mutation profiles in Lynch syndrome representing colorectal, ovarian, endometrial and breast carcinomas, and to identify new unique features that could be used in evaluating cancer risk, diagnosis and targeted treatment. We used targeted high-throughput sequencing of 578 known cancer genes to investigate the somatic mutation profiles, and methylation-specific multiplex ligation-dependent probe amplification to study the epigenetic profiles of the tumors. Non-synonymous somatic mutations were detected from sequencing data of the paired tumor and normal tissues to determine mutation signatures and identify potential driver genes. The data was also compared statistically between tumors of different origin, epigenetic status, and between breast carcinomas from Lynch syndrome mutation carriers and their known non-carrier family members. We observed that Lynch- and Lynch-like syndrome tumors have unique somatic mutation and methylation profiles. We were able to link the methylator phenotype to high somatic mutation rates, and Lynch-like colorectal tumors to hypermethylated CpG island methylator phenotype (CIMP), which are novel findings. Our discovery of high mutation burden in genes associated with epigenetic regulation provides a new link between genetic and epigenetic factors in tumorigenesis. Genetic and epigenetic characterization of synchronous ovarian and endometrial carcinomas indicated shared origin, in analogy to sporadic cases. Molecular characteristics and especially mutational signatures of breast tumors of Lynch syndrome mutation carriers indicated that breast carcinoma is likely to be part of the Lynch syndrome tumor spectrum. These findings bear potential clinical relevance since the molecular tumor profiles may be used in diagnosis and may guide tailored management of the patients. Many of the mutated genes are part of signaling routes to which targeted molecules either exist or can be developed.
  • Korhonen, Suvi (Helsingin yliopisto, 2020)
    Chlamydia trachomatis is a common cause of sexually transmitted infections (STI), which can lead to pelvic inflammatory disease (PID) and reproductive complications. C. trachomatis genotypes D–K cause urogenital infections and genotypes L1–L3 cause lymphogranuloma venereum (LGV). LGV infection outbreaks were reported in 2003 in Europe after decades among men who have sex with men (MSM). A new variant of C. trachomatis (nvCT) was identified in Sweden in 2006 with a large deletion in the plasmid leading to failing detection by two nucleic acid amplification tests (NAAT). Other microbes causing genitourinary tract infections, such as the human papillomavirus (HPV), herpes simplex virus (HSV), Mycoplasma genitalium and human herpesvirus 6 (HHV-6) may also contribute to the epidemiology of C. trachomatis. C. trachomatis promotes its survival inside the host cell through secreted effector proteins. The transcriptional expression of the genes encoding these effectors has mainly been studied using C. trachomatis reference strains propagated in cultured cells and not with currently circulating strains. In this thesis, the C. trachomatis genotype distribution and the occurrence of the Swedish nvCT in Finland was studied with real-time polymerase chain reaction (PCR) in urogenital specimens. The three most prevalent C. trachomatis genotypes were E, F, and G. The percentage of infections due to genotypes F and G increased slightly in 1987–2008, but otherwise the proportion of genotypes has remained quite stable in Finland during the last ten years. The Swedish nvCT was rare in Finland, as only two specimens (0.4%) containing the variant plasmid were detected. The prevalence of the LGV types in extragenital specimens in Finland was investigated with real-time PCR. Among the C. trachomatis positive samples, nine samples (8%) contained LGV types and one sample (1%) contained both non-LGV and LGV types. The LGV types were detected mainly in rectal swabs. Altogether nine LGV positive patients were identified and they were all MSM, and all except one were human immunodeficiency virus (HIV) positive. LGV infections among MSM also occur in Finland, which should be taken into account when considering the management of rectal C. trachomatis infections. The prevalence of HSV, HHV-6, HPV and M. genitalium in urogenital samples of C. trachomatis NAAT positive and negative young women in Finland was analysed with real-time PCR. The prevalence of HPV was significantly higher among the C. trachomatis positives than the negatives (66% vs. 25%). The prevalence of HSV, HHV-6, and M. genitalium was not significantly different between the C. trachomatis positives and negatives or between those who cleared the C. trachomatis infection and those who did not. The high prevalence of HPV among the C. trachomatis positives suggests greater sexual activity and increased risk for sexually transmitted pathogens. Low-passage-number C. trachomatis clinical isolates originating from cervical swabs and reference strains were used to study the expression of C. trachomatis protease (cpaf), phosphoprotein (tarp) and cytotoxin (tox) genes in a cell line during the chlamydial developmental cycle with real-time reverse transcriptase PCR (RT-PCR). Dissimilarities were observed in the gene expression patterns of cpaf and tox between the clinical isolates and the reference strains. All the strains had a similar tarp expression profile. Most clinical isolates showed slower growth kinetics compared to the reference strains. The use of low-passage-number C. trachomatis clinical isolates instead of reference strains in the gene expression studies might better reflect the situation during human infection.
  • Jousi, Milla (Helsingin yliopisto, 2020)
    Background Clinical decisions in prehospital critical care are often based on limited information about the patient’s medical history and the events preceding the acute illness. In addition to physical examination, point-of-care (POC) laboratory diagnostics can provide information for decision-making. Unfortunately, obtaining blood samples for POC analysis from critically ill patients can sometimes be difficult, thus diminishing the usefulness of POC analyses in prehospital critical care. Intraosseous (IO) access is used as an optional vascular route for fluid and medication administration for emergency patients when difficulties with venous access are encountered. Using IO access as a source of POC laboratory samples is interesting; however, evidence regarding the feasibility and agreement of these samples with arterial and venous samples is scarce. We therefore designed a series of studies to obtain more information on the POC analysis of IO samples. Materials and methods First, we performed a systematic literature review of the studies addressing the POC analyses of IO samples (Study I). We included all publications using POC or conventional laboratory methods to compare IO samples with venous or arterial samples for the parameters relevant to emergency care. Second, we performed an observational study within 31 healthy volunteers (Study II). We evaluated the feasibility of IO analyses with an i-STAT® POC device and the agreement of IO values with arterial and venous values. The agreement was evaluated using the Bland–Altman method. In addition, we examined the necessity to draw waste blood before taking the actual IO sample for POC analysis. Third, we studied the usability of IO POC values under unstable haemodynamic conditions with an experimental porcine (n = 23) resuscitation model (Study III). The model simulated a real-life course of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Four repeated samples were simultaneously drawn from the IO access, artery and central vein during CA and CPR, and were instantly analysed with an i-STAT® POC device. The laboratory values were plotted as a function of time to demonstrate their development during CA and CPR. The arterial, venous and IO samples which were taken during CPR were compared with the arterial baseline samples to observe how they resemble the pre-arrest state. Last, in an observational study, we analysed the agreement between IO and arterial samples obtained from 35 critically ill prehospital patients, using the Bland–Altman method (Study IV). Moreover, we administered a questionnaire about acceptable biases in clinical practice to 16 experienced emergency physicians and compared our results with their responses. Results The 27 reviewed studies had heterogeneous populations: healthy volunteers, haemodynamically stable and unstable animals, adult and paediatric haematologic patients, and emergency patients. Only three of these studies followed the recommended guidelines for method comparison studies. The sample sizes were relatively small (n = 14–20) and the populations were heterogeneous in these three studies precluding the combining of results for meta-analysis. However, in IO samples, potassium values were generally higher than in arterial or venous samples. In the observational studies, we found that the POC analyses of IO samples were often feasible. Higher failure rates were associated with higher age. Agreement of IO values with arterial values appeared acceptable for base excess, pH, standard bicarbonate, lactate, glucose, ionised calcium and sodium within healthy volunteers and critically ill emergency patients. Potassium values from IO samples were systematically higher than arterial and venous values (biases 1.8–2.2 mmol/l). Agreement within haemoglobin and haematocrit measurements showed very large variety (95% limits of agreement in the bias of haemoglobin up to 95 g/l). However, the sample sizes were too small to unequivocally prove the agreement. Using the resuscitation model, we discovered that IO, arterial and venous values changed differently from one another during CA and CPR. Acidaemia was detectable in IO samples during untreated ventricular fibrillation, but in arterial samples the acidaemia was evident only after the initiation of CPR. The average potassium values during CPR from IO, arterial and venous samples were 4.4, 3.3, and 2.8 mmol/l higher than the pre-arrest arterial values, respectively. Conclusions When obtaining vascular access is challenging, IO access can be used for emergency POC analyses; however, the results of IO POC analyses should be interpreted with care. Waste blood does not need to be taken before the sample. POC analyses of IO samples may fail for older patients. In general, potassium values from IO samples are usually higher than those from arterial and venous samples, haemoglobin and haematocrit measurements from IO access are not reliable, and partial pressure measurements of oxygen and carbon dioxide from IO samples represent venous rather than arterial values.
  • Niinikoski, Laura (Helsingin yliopisto, 2020)
    Background: The aim of the thesis is to assess different surgical and biopsy techniques, which can help to treat breast cancer patients with less invasive methods. Material and methods: The thesis comprises four original retrospective studies. All study patients were treated at the Breast Surgery Unit of Helsinki University Hospital 2010-2016. Study I evaluated the feasibility of Breast Lesion Excision System (BLES) procedure in the treatment of breast lesions (n=80) with a needle biopsy –based suspicion of an intraductal papilloma. In studies II and III 1800 breast cancer patients underwent breast conserving surgery (BCS). Study II compared reoperations due to insufficient surgical margins and local recurrences (LR) after conventional or oncoplastic BCS and study III after BCS using radioguided occult lesion localization (ROLL) or radioactive seed localization (RSL). In study IV patients (n=507) with primary invasive breast cancer and axillary nodal metastasis preoperatively diagnosed by ultrasound (US)-guided needle biopsy underwent breast surgery and axillary lymph node dissection. The aim was to determine which factors were related to high nodal tumour burden in clinically node-positive breast cancer patients and to construct a predictive tool for evaluating the patient-specific risk of having >2 axillary lymph node metastases (LNM). Results: In study I, the BLES biopsy diagnosis was a benign intraductal papilloma or other benign in 61 lesions, 10 lesions were upgraded to high-risk tumours and five to malignancy. Four procedures failed, no major complications occurred. In study II, 1189 patients underwent conventional BCS and 611 oncoplastic BCS. Patients with oncoplastic BCS were younger, they had more often multifocal, larger and more aggressive tumours. There was no difference, however, in reoperation rates or in local recurrence-free survival (LRFS) between the groups during the follow-up. In study III, 318 patients underwent ROLL and 426 RSL. The reoperation rate or the 5-year LRFS was not different between these groups. In study IV, 153 patients had 1-2 LNM and 354 >2 LNMs. Patient’s age, larger size and lymphovascular invasion of the primary tumour, extracapsular extension of the LNMs, and morphology of the lymph nodes in US were the factors, which remained statistically significantly associated with >2 LNMs. These factors were included in the predictive model. Conclusions: The BLES procedure is an acceptable method for the management of small benign and high-risk breast lesions. Thus, a great amount of diagnostic surgical biopsies can be avoided. Oncoplastic BCS is as safe as conventional BCS, though it is used for larger, multifocal and more aggressive tumours. It enables BCS for patients who otherwise were candidates for mastectomy. Reoperation rates and LRFS were comparable for ROLL and RSL in patients with impalpable breast cancer treated with BCS. The 4th study provides a well performing patient-specific prediction model for evaluating the nodal tumour burden in clinically node-positive breast cancer patients. The prediction model could be helpful in the decision-making on the optimal axillary treatment in these patients.
  • Saku, Sami (Helsingin yliopisto, 2020)
    Total joint arthroplasty (TJA) is the gold-standard treatment for severe hip and knee osteoarthritis. In recent decades, the hospital length of stay (LOS) has reduced substantially largely due to the widespread implementation of fast track protocols. Although the results are now better than ever and most patients have joint replacement without complications, some patients experience one or several deviations from the fast track protocol. In healthcare systems similar to those in Finland, knowledge on these deviations is sparse. This doctoral thesis sought to elucidate some of the deviations in TJA in a Finnish healthcare system. The study population consisted of TJA (i.e. total hip arthroplasty [THA] and total knee arthroplasty [TKA]) patients that underwent surgery at Helsinki University Hospital between 2014 and 2017. The study aimed to identify the reasons and risk factors for delayed discharge and 90-day readmissions after primary TKA. The study also aimed to assess early postoperative emergencies by evaluating the use of an Emergency Response Team (ERT) in the arthroplasty ward. Lastly, the study aimed to evaluate a novel phone consultation service for TJA patients and thereby elucidate common post-discharge concerns. The median LOS after TKA was 3 days. The main reasons for delayed discharge were related to functional recovery and pain. Risk factors for a discharge after the third postoperative day were older age, higher American Society of Anaesthesiologists (ASA) score, shorter preoperative walking distance, general anaesthesia, longer duration of surgery, longer time spent in Post-Anaesthesia Care Unit, and surgery later in the week. The 90-day readmission rate was 8.0% after primary TKA. The most common reasons for readmission were surgical site infection and knee pain. Independent predictors of readmission were psychiatric disease, asthma, a preoperative valgus malalignment, and a preoperative knee flexion deficit. The rate of ERT calls was approximately 7 per 1000 admissions. The most common criteria that triggered the ERT call were decreased level of consciousness, hypotension, and low oxygen saturation. Half of the patients could be treated at the ward after ERT intervention, and the other half was moved to the Intensive Care Unit. Common causes of the emergency included drug-related side effects, pneumonia, and pulmonary embolism. Concerns regarding prescribed medication, wound problems, and mobilization triggered most of the phone consultation service calls. The answering nurse alone resolved two thirds of all calls. Thirteen percent of the patients received instructions to visit the Emergency Department (ED) and half of them had a condition requiring treatment. Only two patients (0.7%) that should have been directed to the ED did not receive such instructions. This study identified several new risk factors for deviations in TJA. Due to the single-payer healthcare system, the possible confounding effect of insurance status did not confound the results. Despite differences in healthcare systems, both LOS and the readmission rate were similar to those previously reported. Considering the present study, a phone consultation service seems to reduce the amount of unnecessary ED visits. Employing an ERT service likely reduces the amount of ICU admissions after TJA surgery.
  • Grönvall, Maiju (Helsingin yliopisto, 2020)
    Postpartum hemorrhage (PPH) complicates approximately 4% of all deliveries. In recent years, abnormally invasive placenta (AIP) has emerged as an increasing cause of PPH. Women with a history of cesarean section and presenting with placenta previa (PP) are at high risk for AIP. Antenatal detection of PP and AIP diminishes the risk of PPH and other complications. It is inconclusive whether PP type (major or minor) makes a significant difference in obstetric outcomes. Although there are several known risk factors, PPH still occurs often in primiparous women without any detectable risk factors. First-line treatment of PPH includes the use of uterotonics and tranexamic acid, laceration repair, and removal of placental tissue. If bleeding continues, the following treatment options have traditionally been surgical procedures: ligation of the uterine arteries, uterine compression sutures and hysterectomy. These procedures require advanced surgical skills. Avoiding hysterectomy, and thus infertility, is also one main goal, especially among primiparous women. Readily available, simple, fertility-sparing treatment modalities are needed. Over the last two decades, the Bakri balloon (Cook® Medical Incorporated, Bloomington, IN, USA) tamponade (BBT) and interventional radiology procedures (IRP) have been used increasingly in the treatment of PPH. The Bakri balloon is exclusively designed for obstetric use. It can be inserted into the uterus or the vagina depending on the bleeding site. IRPs include pelvic arterial embolization (PAE) and the use of prophylactic balloon occlusion in the pelvic arteries. IRPs can be used both in acute massive PPH and also prophylactically in women who are at high risk for PPH due to anticipated AIP. There were only a few publications on BBT before our study commenced (2012). In our hospital, PAE had been used for 10 years without evaluating the effectiveness and safety of our own process. The psychological and physical outcomes of complicated deliveries and IRPs were also controversial and unclear. Our treatment protocol arranged follow-up only for women with major PP. The aims of the studies were to determine the effectiveness and safety of BBT and PAE. The long-term psychological and physical sequelae of IRPs were also explored. The accuracy of our screening and treatment protocol for PP was evaluated, and obstetric outcomes between major and minor PP were compared. The study data were collected retrospectively from Helsinki University Hospital registers (years 2001 ¬̶ 2014). A structured questionnaire concerning long-term psychological and physical outcomes was sent to women who underwent IRPs. The success rate for BBT was 86% and for PAE 89%. None of the complications was related to BBT. Only one major PAE-related complication occurred (rupture of the iliac artery). The results of the inquiry showed a high incidence of adverse psychological outcomes. Many women consider counseling to be inadequate after a stressful delivery. Due to our mid-pregnancy screening protocol, almost one-fifth of the women with major or minor PP at term had no follow-up after mid-pregnancy. The first bleeding episode occurred at 37 gestational weeks or later in 21.9% of these women and massive PPH (>2500 ml) in 15.6% of these women. Two women had undiagnosed AIP leading to hysterectomy. These complications could possibly have been avoided if the diagnosis had been established earlier. It was also remarkable that the incidence of AIP was equal in women with major and minor PP. Additionally two out of seven women with BBT failure and three out of five with PAE failure had AIP as a cause of PPH. In conclusion, BBT and PAE are safe and effective treatment modalities. The procedures had a significant role in the PPH treatment protocol. The availability of these procedures is mandatory due to the unpredictable nature of PPH and the increasing incidence of AIP. Our screening and follow-up protocols for PP are revised based on our results. A systematic counseling protocol for women who had PPH and a complicated delivery should be implemented in every delivery unit.
  • Haavisto, Anna-Kaisa (Helsingin yliopisto, 2020)
    Eye injuries cause inconvenience at least and permanent disability at worst. Yet most of the injuries are preventable. Therefore, it is essential to have updated information on the circumstances leading to eye injuries. By reporting on the causes and contexts, we can promote proper eye protection and safe behaviour to reduce the number of accidents. In this thesis, the focus of analysis was leisure-time eye injuries; injuries in children and those caused by toy guns, sports and wooden projectiles in Southern Finland. Patients were gathered from all new eye trauma patients (n = 1151) taken into care at the Helsinki University Eye Hospital during a one-year period in 2011-2012. The follow-up time was three months and patients injured by toy guns were examined also five years after the eye injury. Children comprised 18 % (n = 202/1151) of all patients. Eye injury was most likely at the age of 13-16, and the leading causes were a hit of a sporting equipment (15 %), contact with the human body (12 %) and superficial foreign body (11 %). The main diagnosis was mild ocular or periorbital trauma (50 %). Six open globe traumas were caused by fireworks, tools, ski pole and a gun. Permanent disability was estimated for 9 % (n = 19) of children. Toy guns caused 1 % (n = 15/1151) of all eye injuries, consisting of 12 airsoft guns, 2 peashooters and 1 paintball gun. The main diagnosis was contusion (87 %). At the five-year follow-up, 47 % (n = 7) had subjective impairment, and 53 % had (n = 8) abnormal clinical findings. Sports caused 13 % (n = 149/1151) of all eye injuries. Floorball, football and tennis were the main sports to come up in the study. Floorball eye injuries decreased from 45 to 32 % of all sports-eye injuries from the season 2002-2003. The main diagnosis was contusion (77 %). Regarding participants, rink bandy had the highest risk. Permanent disability was diagnosed in 11 % of patients and was more common (p = 0.033) in ice hockey than in other sports in the number of injuries. Wooden projectiles caused 6 % (n = 67/1151) of all eye injuries. Males aged 51-67 were at the highest risk. The most common activity during the accidents was playing (27 %), gardening (18 %) and forest work (16 %). In relation to time spent in the activity, the risk of eye injury was the highest in gardening, forest work and woodwork. Permanent disability was diagnosed for 10 % due to various activities. Children should be guided safe play with sticks, and fireworks and tools should be avoided among children. The sale of toy guns should be more restricted and put under the Firearms Act to increase awareness of the risk. The use of eye protection in floorball is recommended for all age groups, and in ice hockey, the use of visors should be emphasised. In gardening, forest work and woodwork, the use of protective eyewear should be enhanced.
  • Sarviaho, Riika (Helsingin yliopisto, 2020)
    Neurologic and neuropsychiatric conditions are a major welfare issue, affecting millions of people worldwide. Neurologic diseases and behavioural problems are also diagnosed in dogs. These disorders are known to have a strong genetic component, but the largely complex genetic background behind brain disorders such as epilepsy and anxiety remains elusive. In this thesis work the genetic background of a novel epilepsy syndrome and three anxiety- related phenotypes were studied in dogs. The studies provided a causative variant for the canine epilepsy in a novel epilepsy gene and identified three loci for canine fear and anxiety, revealing parallels to the genetic background of human neuropsychiatric disorders. A novel epilepsy characterised by myoclonic seizures and a juvenile age of onset was described in Rhodesian Ridgeback dogs. Genetic analyses revealed a four base pair deletion mutation in a DIRAS1 gene, which encodes a poorly characterized neuronal GTPase protein with a suggested role in cholinergic neurotransmission. DIRAS1 is a novel candidate gene for human myoclonic epilepsies and the genetic test developed based on the results is now actively used in veterinary diagnostics and dog breeding. Anxiety and fearfulness were studied in two breeds, German Shepherds and Great Danes, using a behavioural survey and behavioural testing. Genome-wide association analyses of a cohort of over 300 fearful and non- fearful German Shepherd dogs with behavioural scores for noise sensitivity and fear revealed a noise sensitivity locus on chromosome 20 and fear locus on chromosome 7. The noise sensitivity locus included several relevant candidate genes, such as the oxytocin receptor gene OXTR that has been linked to anxiety and social behaviour in humans and dogs, and the glutamate receptor gene GRM7, that has been linked both to anxiety and age-related hearing. The fear locus was largely syntenic to a human locus 18p11.2 that has been linked to human schizophrenia and bipolar disorder, suggesting shared genetic factors behind canine fearfulness and human psychiatric disorders. A similar approach in Great Danes studying fear towards strangers, with the level of socialisation as a cofactor, revealed a novel locus on chromosome 11 including candidate genes such as a mitogen-activated protein-coding gene MAPK9, that has been linked to anxiety and behaviour. In summary, this study has described a novel epilepsy in dogs and revealed new candidate genes and loci for canine epilepsy and anxiety. The results bring new insight into the molecular background of canine brain disorders, establish a novel large animal model of human psychiatric conditions and provide new diagnostic tools for veterinary medicine and canine breeding programs.
  • Verho, Anastasiya (Helsingin yliopisto, 2020)
    Non-communicable diseases, especially cardiovascular disease (CVD), account for the majority of deaths worldwide. It is well known that population health is vulnerable to political and economic influences. Russia has faced significant political and economic changes over the past few decades. The country became world-famous for its high CVD morbidity and mortality rates after the collapse of the USSR and the shift towards a market economy (transitional period). While CVD risk factors in adults have been widely investigated by international scholars, scientific evidence regarding changes in CVD risk factors among Russian youth in recent decades appears to be rather scarce. Long ago it was established that CVD risk factors, both biological and behavioural, develop early in life and are shaped by environmental influences. Once formed, CVD risk factors – especially health behaviours – are difficult to change. Epidemiological studies have shown that the development of CVD risk factors among children and adolescents follows the same trends as among adults. This thesis investigates changes in cardiovascular risk factors among Russian youth during the transition in the country between 1995 and 2013. No previous study has investigated changes in CVD risk factors in relation to adolescents’ social environment in Russia. The study period coincides with the highest CVD mortality rates in the country and significant economic changes. Biological and behavioural CVD risk factors were studied using an internationally comparable methodology among all 15-year-old students, from all schools in the Pitkäranta district in the Republic of Karelia in Russia, between 1995 and 2013. Cross-sectional surveys were conducted in 1995, 2004 and 2013. The total sample consisted of 1072 students: 368 in 1995 (response rate of 95%), 340 in 2004 (response rate of 85%) and 364 students in 2013 (response rate of 98%). In 1995 and 2004, to study changes in biological risk factors for CVD, the blood pressure, height and weight of subjects were obtained for calculation of body mass index, together with venous blood samples for the determination of lipid levels. Participants filled in a questionnaire with pre-coded answers to study smoking, drinking of alcohol and nutrition behaviour, together with nutrition literacy in all years of the survey. In 2013, data was collected using only the questionnaire. Biological risk factors (blood pressure, total cholesterol, high-density lipoprotein cholesterol, weight, height, BMI) were within a normal range initially and did not show negative changes until 2004 for both genders. Significant changes were observed in behavioural risk factors. They mostly worsened during the study period, especially among girls. Thus, the prevalence of daily smoking among boys was high and did not change during the study period. Among girls, daily smoking had more than doubled by 2004 from 7% to 15%. The proportion of non-smoking girls, believing that quitting smoking is easy for young people, increased by 2004. Also, the proportion of girls smoking at home with parents being aware of it increased compared with 1995. Both genders reported smoking at school during breaks. Especially among boys the prevalence was high but did not change between the survey years. Among girls, smoking at school during breaks increased by 2004. Even though the prevalence of smoking remained high among boys and increased among girls during the study period, the prestige of smoking, as a means of improving image, decreased among participants of both genders, except for smoking boys. Among both genders, the odds of smoking were higher among participants if the best friend smoked compared with the presence of smokers among family members. In both survey years, adolescents reported that it was possible to purchase tobacco and alcohol themselves. The self- purchasing of alcohol increased among both genders by 2004. The proportion of youth that had never tried alcohol decreased among both genders. Early initiation of alcohol-drinking increased among boys and in 2004 they consumed alcohol more frequently compared with 1995. Weekly alcohol consumption increased among both genders by 2004. Gender differences in alcohol-drinking behaviour have mostly diminished. Beer consumption among girls almost tripled and the prevalence of drunken behaviour increased among girls by 2004. The consumption of vegetables increased by 2013 but was still insufficient among both genders. Fruit and berry consumption among girls increased by 2004, with a consequent decrease almost to previous levels by 2013. The purchasing of food high in saturated fat during breaks at school and after school increased among both genders by 2004 and decreased again by 2013. The consumption of sugary drinks and snacks among boys increased from 2004. Among girls it increased steadily from 1995. Milk-drinking decreased among both genders. Nutritional literacy was rather low and further deteriorated among both genders. The country’s transition had an effect on behavioural CVD risk factors among Russian adolescents. With regard to some positive changes, negative changes in smoking, alcohol use and some dietary aspects prevailed. It is important to be aware of potential environmental influences on youth CVD risk factors, especially in countries with fluctuating economic situations. A regular country-wide surveillance system should be established to monitor changes in CVD risk factors with regard to environmental influences. Public health interventions and control over the implementation of health policies are needed to improve the CVD risk factor profile of Russian adolescents.
  • Katainen, Riku (Helsingin yliopisto, 2020)
    The research in cancer genetics aims to detect genetic causes for the excessive growth of cells, which may subsequently form a tumor and further develop into cancer. The Human Genome Project succeeded in mapping the majority of the human DNA sequence, which enabled modern sequencing technologies to emerge, namely next-generation sequencing (NGS). The new era of disease genetics research shifted DNA analyses from laboratory to computer screens. Since then, the massive growth of sequencing data has been facilitating the detection of novel disease-causing mutations and thus improving the screening and medical treatments of cancer. However, the exponential growth of sequencing data brought new challenges for computing. The sheer size of the data is not only expensive to store and maintain, but also highly demanding to process and analyze. Moreover, not only has the amount of sequencing data increased, but new kinds of functional genomics data, which are instrumental in figuring out the consequences of detected mutations, have also emerged. To this end, continuous software development has become essential to enable the utilization of all produced research data, new and old. This thesis describes a software for the analysis and visualization of NGS data (publication I) that allows the integration of genomic data from various sources. The software, BasePlayer, was designed for the need of efficient and user-friendly methods that could be used to analyze and visualize massive variant, and various other types of genomic data. To this end, we developed a multi-purpose tool for the analysis of genomic data, such as DNA, RNA, ChIP-seq, and DNase. The capabilities of BasePlayer in the detection of putatively causative variants and data visualization have already been used in over twenty scientific publications. The applicability of the software is demonstrated in this thesis with two distinct analysis cases - publications II and III. The second study considered somatic mutations in colorectal cancer (CRC) genomes. We were able to identify distinct mutation patterns at the CTCF/Cohesin binding sites (CBSs) by analyzing whole-genome sequencing (WGS) data with BasePlayer. The sites were observed to be frequently mutated in CRC, especially in samples with a specific mutational signature. However, the source for the mutation accumulation remained unclear. On the contrary, a subset of samples with an ultra-mutator phenotype, caused by defective polymerase epsilon (POLE) gene, exhibited an inverse pattern at CBSs. We detected the same signal in other, predominantly gastrointestinal, cancers as well. However, we were not able to measure changes in gene expressions at mutated sites, so the role of the CBS mutations in tumorigenesis remained and still remains to be elucidated. The third study considered esophageal squamous cell carcinoma (ESCC), and the objective was to detect predisposing mutations using the Finnish Cancer Registry (FCR) data. We performed clustering analysis for the FCR data, with additional information obtained from the Population Information System of Finland. We detected an enrichment of ESCC in the Karelia region and were able to collect and sequence 30 formalin-fixed paraffin-embedded (FFPE) samples from the region. We reported several candidate genes, out of which EP300 and DNAH9 were considered the most interesting. The study not only reported putative genes predisposing to ESCC but also worked as a proof of concept for the feasibility of conducting genetic research utilizing both clustering of the FCR data and FFPE exome sequencing in such studies.
  • Pietiläinen, Olli (Helsingin yliopisto, 2020)
    Socioeconomic position has been consistently found to be connected to health, with those in lower socioeconomic positions having worse health. Inequalities are observed in mortality, individual illnesses, self-rated health, and functioning. While socioeconomic inequalities have been studied extensively over the last decades, there are still gaps in our knowledge. Previous studies have indicated a widening of socioeconomic differences in physical health in late middle-age, but our understanding on the causes of this widening is lacking. Some ageing employees are confronted with work disability and disability retirement, while most eventually transition to mandatory retirement. We also lack knowledge on how different retirement pathways affect the trajectories of physical health in different socioconomic positions. Work ability may also be affected differently in socioeconomic positions after occurrence of serious ill-health. Studies comparing a wide range of causes of inequalities in work disability are scarce. Studying all these different aspects of health and work disability across socioeconomic positions provides a fuller understanding of health and ill-health in different socioeconomic positions, and may provide justification and targets for interventions aiming to reduce the inequalities. The aim of this study was to examine occupational class inequalities in physical health and work disability, assess the major explanations of these inequalities, and examine occupational class inequalities in consequences of ill-health on work disability. The evidence of this study provides material for designing focused interventions to tackle socioeconomic inequalities in physical health and work disability. This study was conducted among the Helsinki Health Study cohort of employees of City of Helsinki, Finland, using both questionnaire survey data and register based data of City of Helsinki employees. The baseline survey data were collected in 2000-2002 (N=8960, response rate 67%), and the follow-up surveys in 2007 (N=7332, response rate 83%) and 2012 (N=6816, response rate 79%). In one sub-study the baseline questionnaire was linked to the retirement register from Finnish Centre for Pensions. In another sub-study the personnel register of all City of Helsinki employees from 1990 to 2013 (N=170510) were linked to national retirement and hospital discharge registers. Information on occupational class was based on job title, and categorized to professionals, semi-professionals, routine non-manual workers and manual workers. Physical health was measured by the physical component summary of the Short Form 36 (SF-36) questionnaire, summarizing different aspects of physical health. The data were analysed using Bayesian hierarchical linear random effects models, mixed effects growth curve models, Cox proportional hazards regression models, and competing risks regression models. Overall the findings of this study indicate the existence of clear occupational class inequalities in physical health among late middle-aged employees, and inequalities are also observed in how health changes over age. The health inequalities also manifest as inequalities in subsequent work disability and as inequalities in consequences of ill-health on work disability. The occupational class differences are likely to be related to differences in accumulation of exposures, particularly physical exposures related to work, and possibly to opportunities to deal with the exposures. .
  • Forsström, Saara (Helsingin yliopisto, 2020)
    Mitochondria are best known for ATP production in oxidative phosphorylation (OXPHOS), dependent on nuclear factors and the organellar genome of the mitochondria, mtDNA. Mitochondrial disorders are categorically OXPHOS diseases, presenting with extreme variability of tissue-specific manifestation that cannot be explained by genetic causes or impaired ATP production. Comprehensive understanding of the pathophysiology is key for better diagnostics and therapy targets. AdPEO is a mitochondrial disease characterized by mtDNA deletions and mitochondrial deficiency in muscles and brain. Studies on a transgenic mouse model of AdPEO, Deletor, have revealed similar pathology to human patients, and induction of a local stress response in muscle that involves expression of a metabolic hormone, FGF21. In physiological challenges, FGF21 is secreted by the liver to enhance systemic energy-metabolism. Therefore, chronic exposure to FGF21 in mitochondrial disease exposes the body to non-homeostatic regulation of metabolism. In this thesis, we have studied the stress responses upon mitochondrial myopathy with emphasis on the actions of FGF21. We utilized parallel analysis of patient samples and established mouse models, and generated a Deletor-FGF21 knockout model. In the first part, we discovered novel rearrangements of whole-cellular metabolism in post-mitotic muscle with mtDNA maintenance defect, including induction of one-carbon metabolism and glucose-driven glutathione synthesis. We demonstrated that the transcriptional and metabolic stress programs progressed sequentially along the primary disease pathology, and that FGF21 expression was indisputable for initiation of the glucose-driven metabolic programs in the muscle. Systemically, FGF21 expectedly impaired maintenance of adiposity and altered tissue-level glucose preferences. Additionally, we discovered FGF21-dependent mitochondrial and metabolic pathology in CA2-region of hippocampus in Deletor. The second part highlighted that the mitochondrial stress responses dependent on the primary disease mechanisms, not on the fundamental OXPHOS-deficiency. Our clinical analyses showed that the circulating mitochondrial disease biomarkers FGF21 and GDF15 were specific for primary or secondary mitochondrial translation defects, not for isolated OXPHOS mutations. In summary, this work has revealed clinically relevant local and systemic metabolic rearrangements in response to mtDNA maintenance and expression defects, and demonstrated tissue-specific regulation of pathophysiology by FGF21.
  • Sabell, Tuija (Helsingin yliopisto, 2020)
    Cardiogenic shock (CS) is a medical emergency in which cardiac dysfunction causes a state of shock resulting in end-organ hypoperfusion. The most common cause of CS is acute coronary syndrome (ACS), ST-segment elevation myocardial infarction (STEMI) being the leading aetiology. Other causes of CS may include exacerbation of chronic heart failure, valvular dysfunction, myocarditis, and stress-induced cardiomyopathy. Despite progress in revascularization and development of mechanical circulatory support-devices, short-term mortality is still high at 40%, which calls for further advances in CS management and in risk stratification. The electrocardiogram (ECG) plays a major role at the first instance of CS management, as it provides essential information about cardiac ischaemia, rhythm, and conduction. After initial evaluation, emergent coronary angiography is the next step in CS management with the possibility of immediate revascularization with percutaneous coronary intervention (PCI). The aim of this study was to examine electrocardiographic and angiographic features in CS. The patient data in this thesis are primarily included in a multinational, prospective, observational cohort study called the CardShock study, which investigated 219 CS patients with diverse CS aetiologies. Study I evaluated baseline ECG ST-segment patterns in patients with differing CS aetiologies. ST-segment elevation was associated with ACS, and in patients with ST-segment elevation, CS was often the first manifestation of coronary artery disease. One-third of patients with ST-segment depression did not have ACS, but ST-segment depression was associated with a high burden of previous comorbidities. ST-segment elevation was associated with 90-day mortality in patients with mixed CS aetiologies. In the subgroup of ACS patients, no difference in revascularization or mortality rates emerged between the studied ST-segment patterns. Study II examined ventricular conduction disturbances in patients with ACS-related CS. In this population, ventricular conduction disturbances occurred more often in older patients with a higher burden of comorbidities. The temporal evolution of ventricular conduction blocks from baseline to day three was high, because one-third of the blocks were transient. All ventricular conduction disturbances were associated with poor prognosis, and the reversal of the block during the first three days was not associated with better one-year survival. Study III examined the prognostic value of the SYNTAX scores in STEMI-related CS patients. The SYNTAX score is a tool created for assessment of the complexity of coronary artery disease. In this study, SYNTAX score was calculated before PCI (baseline SYNTAX score) and after PCI (residual SYNTAX score). The baseline SYNTAX score was associated with mortality, but its additive value in risk prediction beyond clinical assessment and risk scores was marginal. Residual SYNTAX score did not associate with outcome in STEMI-related CS. Study IV examined angiographic features and their prognostic value in ACS-related CS. Multivessel disease and unsuccessful revascularization of the infarct-related artery were associated with poor prognosis. In addition, assessment of procedural PCI complications showed that arrhythmic complications were the most common, but they did not associate with worse outcome. In conclusion, electrocardiography is an important tool for differentiating the aetiologies of CS and it can be useful in risk assessment. ST-segment elevation and ventricular conduction blocks are markers of high mortality risk. In addition, some angiographic features may prove useful in prognosis assessment. Multivessel disease carries a high mortality risk, whereas successful revascularization of the infarct-related artery is associated with better outcome.
  • Jokelainen, Jarno (Helsingin yliopisto, 2020)
    Endoscopic retrograde cholangiopancreatography (ERCP) is a relatively common procedure utilizing fluoroscopy in the imaging of bile and pancreatic ducts and is used for diagnostic and therapeutic purposes. ERCP usually requires moderate to deep sedation to be successful due to considerable discomfort and pain for the patient. During the last two decades propofol, has become the sedative drug of choice for ERCP. There are several ways to administer propofol sedation. Traditionally, the anesthesiologist administers propofol, but today there are other ways to organize the procedural sedation, such as patient controlled sedation (PCS) and patient-maintained sedation (PMS). All these techniques have their advantages and disadvantages and no method has been found to be superior to others. Assessment and recording the level of sedation during endoscopic procedures is advocated for in guidelines for sedation. There is currently no consensus on which method of assessment should be used. Doxapram is a respiratory stimulant with both peripheral and central effects. Since respiratory depression is a common adverse effect caused by propofol sedation, the role of doxapram in ameliorating this depression is worth exploring. Routine preoperative laboratory testing (RPLT) has been increasingly been questioned and several guidelines are advising against RPLT for surgery and endoscopy in general, but studies regarding ERCP and RPLT are lacking. Four studies were performed in the gastrointestinal endoscopy unit of Helsinki University Hospital. First study (Study I) was a prospective study to investigate how PCS is adopted in clinical practice for ERCP and differences in outcomes using different sedation methods commonly in use. It included all patients with ERCP performed during a one-year period. The analysis of 1196 ERCPs on 956 patients revealed that patients using PCS consumed less propofol with a similar incidence of adverse effects when compared to the other methods. Second study (Study II) was performed to evaluate different methods of assessment of sedation used in scientific literature for ERCP sedation, Bispectral index (BiS), Richmond Agitation/Sedation Scale (RASS), a modified Ramsay Sedation Scale (mRSS) and modified Observer Assessment of Alertness and Sedation (mOAAS) in 200 patients with all scales simultaneously used. All scales were found to be reliable in assessing the level of sedation when compared to each other. However, in the clinical setting of ERCP sedation BiS may be preferable to the other methods because it does not require communication with the sedated patient. The purpose of the third study (Study III) was to find out if RPLT was useful in predicting adverse effects caused by ERCP or sedation in the patient cohort of Study I. RPLT included basic blood count, creatinine, potassium, sodium, amylase and International Normalized Ratio/thromboplastin time. Multivariate analysis showed no association with RPLTs and post-ERCP pancreatitis. The rate of other adverse effects related to ERCP was too low for statistical analysis. Respiratory depression caused by sedation was not associated with abnormal RPLTs. Cardiovascular depression caused by sedation was found to be related to thrombocytopenia and in male patients, hyponatremia. The clinical significance of the relation to cardiovascular depression remains unclear and is probably related to other health issues. The fourth study (Study IV) investigated the use of doxapram as an adjunct to BiS-guided deep propofol sedation in order to reduce respiratory apneic episodes and hypoxemia. Fifty-six patients were randomized to receive either doxapram or placebo in 1:1 ratio in a prospective double-blinded protocol and resulted with no statistically significant differences between the groups. In conclusion, no superior method for sedation or for the assessment of sedation for ERCP could be identified, but PCS and BiS remain the most clinically desirable protocols. The results suggest that the practice of RPLT should be abandoned and an individual preoperative laboratory testing should be adopted. Doxapram seems ineffective in preventing respiratory depression caused by propofol sedation.
  • Ruuth, Maija (Helsingin yliopisto, 2020)
    Atherosclerotic cardiovascular disease (ASCVD) is the leading worldwide cause of mortality and morbidity, being both an economic burden for healthcare systems and a source of great individual suffering. The low density lipoprotein cholesterol (LDL-C) concentration in plasma is a causal and modifiable risk factor for ASCVD. Atherogenesis is initiated and then driven by retention, modification, and aggregation of low density lipoprotein (LDL) particles in the arterial intima. Experimental work by our group and others has demonstrated that aggregated LDL particles can induce lipid accumulation in macrophages and this can also activate intimal cells and thereby induce inflammation in the arterial wall. Here, it was hypothesized that it is not only the plasma concentration of LDL particles that influences atherogenesis, but also their characteristics. This thesis aims at testing whether subjects with aggregation-prone LDL have an increased risk for ASCVD and/or ASCVD death. For this purpose, a method to measure LDL aggregation susceptibility was developed, and it was further studied if LDL aggregation susceptibility is modifiable. In addition, it is studied here if LDL aggregates cause a heightened inflammatory response in cells present in the artery wall. The first publication was a hypothesis-generating study, where it was discovered that LDL particles from ASCVD patients are more prone to aggregate in comparison to those from healthy individuals, and importantly, that aggregation-prone LDL predicted future ASCVD death in a group of patients with established ASCVD. It was found that the aggregation-prone LDL particles are rich in sphingolipids but have less phosphatidylcholines than their aggregation-resistant LDL counterparts. Three interventions in animal models aimed at altering the LDL composition, were observed not only to lower the susceptibility LDL particles to aggregate but also to slow the development of atherosclerosis. Similar compositional changes induced in humans by proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition or adoption of a healthy Nordic diet also lowered the LDL aggregation susceptibility. In the second publication it was demonstrated that genetic background, here ethnicity, influenced LDL aggregation susceptibility. LDL particles from South Asians were more prone to aggregate compared to those from white Caucasians, a finding that may partly explain why South Asians are at higher risk for ASCVD. The third study was a dietary intervention to investigate if different macronutrients could alter LDL particles aggregation susceptibility. Saturated fats were found to increase LDL aggregation, while unsaturated fats or simple sugars had no effect. In addition, the consumption of plant stanol esters, that are known to reduce the LDL-C concentration, was found to decrease the LDL aggregation susceptibility. This thesis propose that the aggregation susceptibility of LDL particles is a novel modifiable risk factor of ASCVD; its assessment may add predictive power to the conventional ASCVD risk estimation. The evaluation of this biomarker may facilitate the identification of those patients who would benefit most from aggressive LDL-C-lowering therapies.
  • Vesterinen, Tiina (Helsingin yliopisto, 2020)
    Pulmonary carcinoid (PC) tumors are rare neuroendocrine lung neoplasms that have in general an indolent clinical course. Based on morphology, PC tumors are categorized into typical carcinoid (TC) tumors and atypical carcinoid (AC) tumors. The primary treatment is surgery, while there is no consensus on the treatment of metastatic disease. Thus, effective and more targeted therapies are needed for the PC patients who are inoperable or not curable with surgery alone. While histologic subtype and stage are important prognostic factors, other predictive and prognostic factors or biomarkers are urgently needed for individualized treatment and patient-tailored surveillance protocols. The main aim of this thesis was to find tissue-based biomarkers in PC tumors by utilizing the Finnish hospital-integrated biobanks as a source of the study material. Moreover, the performance of these biobanks in distributing material for a research of a rare cancer was evaluated. The nationwide study cohort consisted of 224 PC tumor patients operated on between 1990 and 2013 in Finland. Eighty-one per cent (n=182) of the tumors were classified as TCs and 19% (n=42) as ACs. Hilar/mediastinal lymph node involvement at the time of primary surgery as well as metastatic disease in general was more common in AC patients than in TC patients. The follow-up time of the patients varied between <1 and 28.0 years. Of 224 patients, 14 died with evidence of disease and 33 died from unrelated causes. The 10-year disease-specific survival rate was 98% for TC patients and 81% for AC patients. Five clinicopathological factors were recognized to be associated with the risk of disease-specific mortality. These were age over 56 at diagnosis, tumor size over 2.5 cm, atypical subtype, hilar/mediastinal lymph node involvement at diagnosis, and presence of metastatic disease. Similarly, lack of somatostatin receptor (SSTR) 1 expression and presence of SSTR3 expression as well as a Ki-67 proliferation index over 2.5% were identified as risk factors for shorter DSS. In addition, lack of SSTR2 expression and presence of SSTR4 expression was associated with a risk of shorter DSS in AC patients. Metastasized PC tumors expressed significantly more SSTR4 and programmed death ligand 1 (PD-L1) than non-metastasized tumors. In addition, lack of SSTR1 or SSTR2 expression was associated with metastatic disease. Based on biobank registry data, Finnish hospital-integrated biobanks were able to identify 88% of the histologically verified PC tumor patients registered by the FCR. However, they were only able to deliver 63% of the tumor samples because samples were missing in the archives or they were too scarce. The most challenging part of the biobanks’ workflow was collecting the clinical data.
  • Rusanen, Peter (Helsingin yliopisto, 2020)
    Oral lichenoid disease (OLD) encompasses oral lichen planus (OLP) and oral lichenoid lesion (OLL), which are chronic T-cell-mediated mucocutaneus inflammatory disorders of unknown aetiology. Both OLP and OLL are classified as potentially malignant disorders. Although various antigens have been considered, it is not known what triggers the inflammatory response of T-cells. Suggested predisposing factors include stress, genetic factors, trauma, viral, fungal and bacterial infection. Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral cavity. It is a multifactorial disease with no single clearly recognizable cause. Chronic inflammation is one of the most important causes of OSCC. Chronic oral candidiasis has also been associated with oral carcinoma in several studies. It is still debatable whether microbial infections initiate cancer or is the preexisting cancer colonized by microbes secondarily. Acetaldehyde is the first metabolite of ethanol and it is carcinogenic. Acetaldehyde is also produced by microbes and poor oral hygiene increases acetaldehyde production. Recent studies of the oral microbial acetaldehyde production are mainly based on uncultured saliva samples. Saliva and mouth rinse samples are often used for general sampling but do not represent the microbes at a specific lesion or site. Toll-like receptors (TLRs) and nuclear factor-κB (NF-κB) signalling transduction pathway play important roles in the pathogenesis of several chronic inflammatory diseases. Tumour suppressor protein p53 regulates TLR expression. It was not known clearly what the optimal sampling site and method to study the microbial colonisation on mucosal lesions is and what impact specific microbial colonisation has on TLR expression. In addition, the immunohistochemical localisation of all TLRs in OLD was not established. Therefore, the aim of the first study was to investigate how the method and site of microbial sampling affect the discovery of Candida species on OSCC lesions. The objective of the second study was to develop a site-specific sampling method that would give quantitative results for samples from the oral mucosa. The aim of the third study was to explore lesion specific microbes and their ability to produce acetaldehyde in OSCC and OLD patients. Furthermore, the aim of the fourth study was to investigate the immunohistochemical staining and tissue localization of TLR1-10, p53 and NF-kB in mucosal biopsies from patients with OLD. In the first study, four different sampling methods in oral cancer patients were compared for culture of yeasts. In the second study, two site-specific sampling methods, filter paper and swab, were compared for microbiological analyses of the healthy oral mucosa. The filter paper sampling method was developed for the second study. In the third study, microbial samples from OSCC and OLD patients for microbiological analyses and acetaldehyde measurement were obtained using the filter paper sampling method. In the fourth study, oral mucosal biopsies from patients with OLD and from healthy controls were analysed for the expression of TLR1-10, NF-κB and p53 by immunohistochemistry. This work has demonstrated that after cancer treatment, the incidence of Candida albicans was found to be increased and a shift from C. albicans to other Candida species was found. The optimal sampling site for Candida in these patients was found to be the labial sulcus. Moreover, the filter paper sampling method was found to be an ideal technique for obtaining quantitative data from defined areas of the oral mucosa. Based on the filter paper sampling method, it was detected that the bacterial composition on OSCC and OLD lesions differed from that of the healthy appearing contralateral mucosa and from healthy controls. Candida colonization was higher in OSCC and OLD lesions and patients with Candida colonization produced significantly more frequently mutagenic amounts of acetaldehyde. The staining intensity of several TLRs was markedly stronger throughout the epithelium and in the basement membrane zone of OLD samples. Likewise, the staining for NF-κB and p53 were more intense in OLD samples compared to the control samples. We did not find any correlations between the microbial samples and the immunostaining of TLRs. In conclusion, this study showed that the composition of lesional microbes differs on OSCC and OLD lesions compared to the healthy appearing mucosa and to the healthy controls. Furthermore, the composition rather than the number of microbes is a significant factor that influences the production of carcinogenic level of acetaldehyde. Our results indicate that acetaldehyde and Candida colonisation may have an impact on TLR4 expression that may play a role in OSCC pathogenesis. The role of soluble TLR forms in the basement membrane zone calls for further studies.
  • Bollepalli, Sailalitha Spurthy (Helsingin yliopisto, 2020)
    Obesity and smoking are the two major preventable causes of global mortality associated with a multitude of comorbidities, inflicting greater public health and economic burden. Complex interactions between genetic and environmental factors influence susceptibility to obesity and smoking. Epigenetic modifications provide a mechanistic link between genetic and non-genetic factors causing complex diseases or traits. Epigenetic modifications also function as an additional layer of gene regulation by modifying the structure and accessibility of DNA and chromatin. The fundamental objective of this thesis is to elucidate the role of epigenetic and transcriptomic markers in obesity and smoking. Hence, this thesis focuses on identifying epigenetic and transcriptomic markers associated with weight loss and smoking behavior using different study designs and by applying computational and statistical approaches. Genome-wide transcriptome and methylome were assessed in an unbiased, hypothesis-free setting to identify weight-loss and smoking-associated signals in Study I and II, respectively. Validation of the main findings from the discovery analyses and integration of transcriptomic and methylation data were performed to assess the validity and biological significance of the identified markers. A machine learning approach was employed in Study III to develop a robust smoking status classifier based on DNA methylation profiles. The performance of the classifier was tested in three different test datasets and also in comparison with two other existing approaches. Therefore, this thesis encompasses both application and method development aspects to achieve the corresponding aims of the studies. In Study I, clinical parameters, genome-wide transcriptome, and methylome analyses were assessed longitudinally at three time points during a one-year weight loss intervention study, to understand the temporal changes in transcriptome and methylome of subcutaneous adipose tissue (SAT) in response to weight-loss. Results from the discovery analyses were validated using monozygotic (MZ) twin pairs discordant for acquired obesity, to examine whether weight loss and acquired obesity exhibit reciprocal transcriptome and methylome profiles. Gene expression and methylation profiles of the SAT at the three time points were also integrated to enhance our understanding of their interaction and thereby their contribution in weight loss. Based on the weight loss trajectory of the participants, three comparisons were performed: short-term (baseline to the fifth month), continuous (fifth to twelfth month), and long-term weight loss (baseline to twelfth month). Clinical parameters were improved with the weight loss (e.g. from baseline to fifth month, total and low-density lipoprotein cholesterol; triglycerides; and systolic blood pressure decreased and insulin sensitivity increased) and several significant transcriptome profiles were identified in response to weight loss at the three comparisons. No genome-wide significant methylation profiles were identified for the three comparisons. However, several significant correlations were observed between expression and methylation, indicating a potential regulatory role of DNA methylation in weight loss -associated transcriptome profiles. At the pathway level, short-term weight loss was implicated in lipoprotein metabolism and long-term weight loss associated with various pathways associated with multiple functions of the SAT. Furthermore, several weight loss -associated genes exhibited opposite direction of expression in acquired obesity in the validation cohort of MZ twins, validating the robustness of identified associations. In Study II, discovery analyses focused on understanding the widespread effects of smoking on SAT by simultaneous assessment of genome-wide transcriptome and methylome of SAT. Discovery analyses performed on the current (n=54) and never (n=291) smokers in the TwinsUK cohort identified 42 significantly differentially methylated signals and 42 significant differentially expressed genes (DEG) indicating a substantial impact of smoking on metabolically important SAT. Integration of these results revealed an overlap at five genes (AHRR, CYP1A1, CYP1B1, CYTL1, and F2RL3) comprising 14 CpG sites. To further characterize the widespread effects of smoking on metabolic disease risk three adiposity phenotypes (total fat mass [TFM], android-to-gynoid fat ratio [AGR] and visceral fat mass [VFM]) were assessed with regards to the identified smoking-associated methylation and expression signals. Three CpG sites in CYP1A1 showed significant associations with VFM and AGR, and an inverse association was identified between methylation levels of cg14120703 (NOTCH1) and AGR. To validate these associations, a subset of younger Finnish twins (n=69, 21 current smokers) was used as a replication cohort. The overall inverse association between cg10009577 (CYP1A1) and AGR was replicated and exhibited a similar direction for interaction effects between smoking status and AGR. However, this association did not reach the genome-wide significance level. Expression levels of F2RL3 showed a significant association with all three adiposity phenotypes. While OR51E1 expression levels were significantly associated with AGR and VFM. Our results show that smoking affects both the methylome and transcriptome of the SAT with overlapping signals. Furthermore, smoking-associated methylation and transcriptome profiles are also associated with adiposity phenotypes indicating a broader impact of smoking on human metabolic health. In Study III, I developed a methylation-based smoking status classifier using a machine learning approach to overcome the limitations of cotinine and carbon monoxide biomarkers (i.e. limited to measuring recent exposure to smoking due to their short half-lives in body fluids) and the existing DNA methylation score-based approaches and to advance the practical applicability of smoking-associated methylation signals. I considered three smoking status categories (current, former and never) and used multinomial LASSO regression coupled with internal cross-validation to build the classifier. I demonstrated the global applicability and robustness of our classifier by evaluation of its performance in three independent test datasets from different populations and also compared the performance with two existing approaches. Our classifier differs from the existing approaches by curtailing the need to compute a threshold value specific to each dataset to predict smoking status. Our classifier showed good discriminative ability in identifying current and never smokers compared to other approaches. I also performed an extensive phenotypic evaluation to understand the results of our classifier. Accurate classification of former smokers is challenging as their classification is affected by cessation time and smoking intensity prior to quitting. I provide the functionalities of our classifier including other the two methods as an R package EpiSmokEr (Epigenetic Smoking status Estimator), facilitating prediction of smoking status in future studies. In conclusion, this doctoral thesis (1) enhances our understanding of obesity and smoking by integrating methylation and transcriptome data and identifying several weight-loss and smoking-associated signals, (2) shows wide-spread impact of smoking on metabolic health risk by evaluating the associations between smoking-associated signals and adiposity measures, and (3) demonstrates the role of DNA methylation profiles as a robust biomarker to predict smoking status by developing a smoking-status classifier.

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