Faculty of Medicine

 

Recent Submissions

  • Lantto, Juulia (Helsingin yliopisto, 2021)
    Changes in oxygenation and placental function affect foetal cardiac function and circulation. Congenital heart defects alter normal circulation of blood flow, which may further alter cardiac development and function. In distress, the foetus can adapt to the situation by altering energy consumption, enhancing cardiac function, and redirecting blood to critically vital organs. The main objective of this thesis was to investigate the responses of foetal cardiovascular function to hypoxaemia and in situations when the normal circulation is altered either by pharmacologically increasing the placental vascular resistance or by mechanically occluding the ventricular outflow tracts. The specific aims were to study the factors affecting the redistribution of blood flow to critical organs, the responses of the ventricles and the role of different foetal shunts during changes in blood flow. Chronically instrumented late gestation foetal sheep were studied. Foetal hypoxaemia was induced by increasing placental vascular resistance with angiotensin II (ATII) infusion (I) or by maternal hypo-oxygenation (IV). In addition, blood circulation was altered by mechanically occluding the foetal ascending aorta (AaO, II) or the main pulmonary artery (PaO, III). In each study, foetal cardiac function and central and peripheral haemodynamics were assessed with spectral and pulsed-wave Doppler ultrasonography concomitantly with invasively monitored foetal blood pressures and arterial blood samples. Additionally, tissue Doppler imaging (TDI) was used in studies I-III to study myocardial systolic and diastolic function. ATII infusion led to foetal hypoxaemia and metabolic acidaemia (I). The earliest signs of cardiac dysfunction were seen on TDI parameters describing diastolic function, and these changes were seen earlier in the left ventricle (LV). AaO led to LV systolic and diastolic dysfunction, while the right ventricle (RV) was able to improve its systolic function; however, RV diastolic dysfunction developed (II). There were signs that the AoI could not provide adequate blood flow to the cerebral circulation (II). During PaO, the FO blood flow slightly increased, but it could not fully compensate for the drop in RV cardiac output and pulmonary return (III). In addition, PaO led to RV systolic and diastolic dysfunction, and LV systolic dysfunction developed, while LV diastolic function was preserved. During foetal hypoxaemia without placental compromise, the peripheral chemoreflex was seen to function even during prolonged hypoxaemia and metabolic acidaemia (IV). AoI was able to increase blood flow towards the brain, however, the perfusion pressure towards the brain decreased. The findings of the current study show that the foetal special circulatory areas, such as FO and AoI, may have limited capacity to support adequate redistribution of highly oxygenated blood towards cerebral circulation in late-term pregnancy. In addition, the cardiac outputs did not increase during hypoxaemia and metabolic acidaemia. This is of great importance as foetuses with congenital heart defects have altered circulation and cardiac function, and foetuses with placental insufficiency have diminished reserves to enhance their oxygenation and cardiac function. These foetuses could be at great risk of foetal compromise in late-term gestation.
  • Awad, Shady (Helsingin yliopisto, 2021)
    Chronic myeloid leukemia (CML) is an adulthood leukemia that accounts for 15% of all leukemia patients. The hallmark of the disease is the BCR-ABL1 fusion gene. The development of Tyrosine kinase inhibitors targeted therapy has led to dramatic improvement in CML management with the life expectancy of CML patients approaching normal rates. Nevertheless, certain challenges in CML management remain unmet. CML progression from the chronic phase (CP) to the blast phase (BP) occurs in 5% of CML patients and is associated with dismal survival. Additionally, a minority of CML patients express p190-BCR-ABL1 isoform, which is associated with inferior treatment responses. Characterization of the mechanisms underlying the pathogenesis of high-risk CML is warranted to enable optimization of the therapeutic approaches for these patients. We aimed to investigate the role of mutations at different phases of CML, and to identify genetic biomarkers that allow better risk assessment and optimization of CML management. Furthermore, we integrated genetic and drug sensitivity profiling to identify effective drugs in a personalized medicine approach. In study I, we applied comprehensive sequencing techniques to a cohort of 59 CML patients, including 19 BP and 40 CP patients. In comparison to CP, BP patients demonstrated higher mutational load and were associated with recurrent mutations involving ABL1, ASXL1, and RUNX1 genes. Fusion genes were also identified as recurrent genetic abnormalities in BP patients. Cancer-associated gene mutations correlated with inferior treatment outcomes in CP patients. Finally, we presented two BP patients who were managed using a personalized approach and achieved good responses, highlighting the potential power of this strategy in BP management. In study II, RUNX1 was selected as an example driver mutation in CML progression. RUNX1 mutations were identified in 35% of patients. We also identified mutagenic AID/RAG axis activity which was specific to RUNX1-mutated patients. RUNX1-mutated BP patients demonstrated the overexpression of lymphoid transcription factors as well as aberrant expression of lymphoid antigens (CD19/CD7). We identified targeted therapies with enhanced sensitivities in RUNX1-mutated blasts. We further used CRISPR/Cas9 gene editing to model RUNX1 mutations in cell lines. Finally, we demonstrated the efficiency of CD19 CAR T-cell immunotherapy in targeting RUNX1-mutated blasts. In study III, we performed genomic and phosphorylation profiling for four p190-CML patients and validated findings in two progenitor cell lines (Ba/f3 and HPC-LSK). We demonstrated novel evidence for the p190-specific activation of IFN/JAK1/STAT1 pathway in CML. Other important signaling pathways included Src and PAK1 signaling. We also identified novel p190-specific drug vulnerabilities. Finally, we validated these findings in a cohort of Ph+ ALL patients. In conclusion, our study provides novel insights into the molecular pathogenesis of high-risk CML patients, including BP-CML and p190-CML patients. We identified potential biomarkers, which can be involved in the risk-assessment of CML. Integration of genetic and drug screening data enabled the identification of promising drug candidates. Taken together, we highlighted the potential value of the personalized medicine approach for optimizing the management of high-risk CML patients.
  • Ala-Houhala, Mari (Helsingin yliopisto, 2021)
    Invasive fungal diseases (IFD) cause significant morbidity and mortality in immunocompromised and critically ill patients. Candida is the most frequent species that causes IFDs. However, other invasive mycoses such as aspergillosis and infections caused by other rarer fungal pathogens, have been reported to emerge. Diagnosis of IFDs is challenging. Culture and histopathology are the foundation of the diagnosis. However, more accurate and rapid diagnostic methods are needed. The purpose of this study was to assess the clinical use of panfungal polymerase chain reaction (PCR) in diagnosing IFD. The study also aimed to provide recent epidemiological data for candidemia in the hospital district of Helsinki and Uusimaa and to analyse the risk factors for 30-day mortality in candidemia. The risk factors for persistent and characteristics of recurrent candidemia were also analysed. Study I was a retrospective cohort study that analysed the clinical use of panfugal PCR to diagnose IFD. We focused on specimens taken from normally sterile tissues and fluids. We compared results of panfungal PCR test to the results of culture and histopathology in 307 specimens. The likelihood of an IFD was evaluated with the criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG). Studies II-IV were retrospective, cohort studies. All patients with candidemia were identified from the microbiological database between 2007 and 2016. Study II included 350 patients with a positive blood culture results for Candida species. The study period was divided into two 5-year periods to analyse the changes in the epidemiology of candidemia. The main outcome mesure was 30-day overall mortality after the diagnosis of candidemia. In study III, we compared the patients with persistent candidemia (PC) with non-persistent cases. PC was defined as an isolation of the same Candida species from blood culture ≥5 days. In study IV, we compared patients with late recurrent (LR) candidemia and patients with a single candidemia episode to analyse the characteristics of LR candidemia. LR candidemia was defined as having at least two episodes of candidemia ≥30 days apart. Panfungal PCR was positive in 48 specimens, and 23 patients of these had a proven or probable IFD. The sensitivity and specificity of panfungal PCR in diagnosing IFD was 61% and 92%, respectively; the negative predictive value and positive predictive value were 93% and 54%, respectively. C. albicans was the leading cause of candidemia and the distribution of Candida species showed no significant change during the study period. The overall 30-day mortality was 31%. McCabe score 3, ICU stay at the onset of candidemia and age >65 years were independent risk factors for 30-day mortality. An association between 30-day mortality and early start of effective antifungal treatment was not observed, although an effective antifungal treatment was a protective factor against mortality. PC was observed in 75 (21%) patients. Metastatic infection foci, presence of central venous catheter (CVC) and ineffective empirical antifungal therapy were independent risk factors for PC. LR candidemia was an uncommon event and diagnosed in 6% of all patients with candidemia. LR candidemia was associated with a history of intravenous drug use (IDU) and underlying gastrointestinal diseases. Diagnosis of IFDs remains challenging. Our results show that panfungal PCR aids in the diagnosis of IFDs; however, panfungal PCR should be combined with other diagnostic methods. A significant shift to non-albicans Candida species causing candidemia was not observed in our hospital district during the study period. An early start of effective antifungal agent was not a protective factor against 30-day mortality. Effective antifungal treatment is beneficial in candidemia, but the early initiation of the medication seems not to be as crucial as it is in bacterial septic shock. Removal of CVC as early as possible, and search and treatment for metastatic infection foci are key elements for preventing PC.
  • Hackman, Greetta (Helsingin yliopisto, 2021)
    Prostate cancer is the second most common cancer worldwide and the most common cancer in Finland among men. While the prognosis of low-risk localised prostate cancer is excellent, a substantial proportion of prostate cancer patients experience disease progression after first-line treatment. This doctoral dissertation includes four studies that focused on active treatment options for prostate cancer patients with adverse pathologic features or risk factors associated with increased disease recurrence and/or mortality. Epidermal growth factor receptor (EGFR) overexpression is associated with poor prognosis in prostate cancer and resistance to radiotherapy in several solid tumours. In the first two non-randomised trials, the objective was to evaluate the safety and efficacy of 250 mg once daily gefitinib, an orally active EGFR inhibitor, in prostate cancer patients. In the first (phase I/II) trial, 42 patients with nonmetastatic prostate cancer received gefitinib in combination with radical radiotherapy as the first-line treatment. In the second (phase II) trial, 30 patients with biochemical recurrence following radical treatment received gefitinib monotherapy. The third study was a retrospective patient series of 46 patients with previously untreated metastatic prostate cancer—a diagnosis that continues to have poor overall survival. This study evaluated the safety and efficacy of multimodal treatment, including androgen deprivation and radical radiotherapy. In addition, the patients received various individually planned treatments. The fourth study was a multicentre trial that randomised 250 radical prostatectomy-treated patients into an adjuvant radiation (126 patients) or observation (124 patients) group. All patients had positive surgical margins or extracapsular extension, both of which have been associated with increased prostate cancer progression; however, it was unclear whether these patients benefit from adjuvant radiation after surgery. While most of the adverse events in the first study were mild to moderate, the toxicity of gefitinib in combination with radiation was unacceptable, considering that most patients had low-risk prostate cancer with a favourable prognosis even without any active treatments. In studies II–IV, the toxicity was acceptable. The efficacy of gefitinib in prostate cancer patients was modest, both in combination with radical radiotherapy and as a monotherapy. The multimodal treatment approach in metastatic prostate cancer was promising but requires further confirmation in randomised trials. Adjuvant radiotherapy following radical prostatectomy resulted in significant improvement in patients’ biochemical recurrence-free survival when compared to surgery alone. However, salvage radiation upon biochemical recurrence following surgery appeared equally effective in terms of overall survival. Prostate cancer patients with adverse pathologic features or risk factors form a heterogeneous group of patients with different prognoses. To balance the subjective experience of treatment toxicity and the treatment’s expected efficacy on survival, the patient must be adequately informed about the toxicity profiles of the treatments available as well as the risk for later disease progression. The aims of future research include more accurate risk-profiling for each prostate cancer patient, a better understanding of individual disease characteristics, and, thus, the identification of optimal treatments.
  • Kaartokallio, Tea (Helsingin yliopisto, 2021)
    Pre-eclampsia is a complex vascular pregnancy disorder characterised by systemic endothelial dysfunction and often impaired placental development. The course of the disease is unpredictable and severe forms of the disease may threaten the lives of both the mother and her child. Indeed, pre-eclampsia is a major cause for maternal and perinatal mortality, especially in countries with limited access to maternal and neonatal health care. The risk of pre-eclampsia involves a genetic component conveyed by both the maternal and fetal genomes. Based on the largest genome-wide association study on maternal pre-eclampsia, the risk of the disease is influenced by many of the genetic loci previously implicated in hypertension. However, the hypertension risk variants do not fully explain the genetic risk of pre-eclampsia and variants involved in other physiological processes, such as placental development, are also likely to play a role. As pre-eclampsia reduces reproductive success, any predisposing large-impact variants are assumed to be pruned out to low population frequencies by negative evolutionary selection. Deleterious low-frequency variation, on the other hand, is enriched in population isolates, where evolutionary selection has not had enough time to effectively prune out the harmful variation. Due to the consequent boost in statistical power to detect genetic associations, studies in population isolates, such as the Finnish population, offer a feasible option for exploring especially low-frequency pathogenic variation. The aim of this thesis was to identify genetic risk factors of pre-eclampsia in the Finnish population isolate. The thesis includes two studies on a protective candidate gene HMOX1 that is crucial for vascular and placental function and has previously been linked to cardiometabolic diseases. In the other two studies, we performed genome- and exome-wide scans in families and sporadic cases of pre-eclampsia, respectively. The main sources for the samples in the thesis were the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) case-control cohort and the pre-eclampsia family cohort predominantly originating from a north-eastern population isolate. Initial findings of the screening studies were also evaluated in samples from two large population-based studies, Sequencing Initiative Suomi (SISu) and FinnGen. In the HMOX1 studies, we found that a functional promoter microsatellite was associated with both maternal and fetal pre-eclampsia. The long maternal repeat was associated with the late-onset, less severe phenotype whereas the fetal association was observed especially for the early-onset and severe forms of the disease. In accordance with these findings, we confirmed the previously reported impact of the microsatellite length on the heme oxygenase 1 (HO-1) protein levels, but here for the first time during pregnancy. The long maternal repeat was associated with reduced gestational serum HO-1 while the long fetal repeat showed an association with decreased HO-1 levels in placenta. These results suggest that HMOX1 is associated with pre-eclampsia through both maternal and fetal genomes, likely acting through separate mechanisms. In the exome-wide screen, we searched for pre-eclampsia-enriched low-frequency variants based on whole-exome sequencing data from a hundred pre-eclamptic women and aimed to validate the identified candidates by testing their association with pre-eclampsia in a larger set of case-control samples (1344 cases and 6817 controls). Four variants were nominally associated with pre-eclampsia, but none of them reached statistical significance after correcting for multiple testing. We conclude that larger sample sizes will likely be needed to reveal large-impact risk variants of sporadic pre-eclampsia, even in a population isolate like Finland. In the family study of pre-eclampsia, we performed linkage analysis in 15 extended families, both for the maternal and fetal phenotypes, using genome-wide imputed SNP markers and parametric linkage analysis. We identified 15 maternal and 27 fetal loci that segregated with the disease. In order to evaluate if there was an overlap between the risk loci in the families and the general population, we tested whether the suggestive case-control association signals from the maternal meta-analysis of ~90 000 samples and fetal analysis of ~1700 samples would be enriched in the linkage regions. Evidence of such enrichment was, however, not observed in our study, implying that the risk loci identified might be largely specific to the families. Finally, we searched for linkage region candidate variants in 21 exome sequenced pre-eclamptic women from 6 families and genotyped the identified variants in the whole family cohort. Four previously unknown or rare missense variants in the linkage region genes COL6A3, DGKB, EEPD1, and PITPNM1 were found to segregate with pre-eclampsia either fully or partially. As each of the candidates was found in only one of the families, additional support from independent genetic or functional studies will be required to establish their connection with pre-eclampsia. In conclusion, the studies of the thesis shed light on genetic susceptibility of pre-eclampsia by identifying novel risk variants and loci for both the maternal and fetal phenotypes. We also demonstrate the benefits of utilising the family and population isolate based designs, especially in the search for rare risk variants of complex reproductive diseases such as pre-eclampsia.
  • Seppälä, Miia (Helsingin yliopisto, 2021)
    Tongue squamous cell carcinoma (TSCC) is one of the most aggressive cancers in the oral cavity or oropharynx. Its 5-year survival rate is approximately 70%. TSCC has a poor prognosis due to early metastasis via lymphatic vessels. This thesis describes the immunoexpression of CIP, IDO, Podoplanin, vWf, and CDH3 in primary tumor, tumor recurrence, and lymph-node metastasis in the mobile tongue and base of TSCC. We also determined the level of mucosal inflammation, lymphatic vessel density, and diameter and their predictive role in TSCC. We characterized the clinical background, operation, follow-up treatment, and outcomes. We performed immunohistochemistry on whole-section slides from primary tumors, recurrent tumors, and lymph nodes. CIP2A was similarly detected in TSCC and tongue hyperplasia, whereas local inflammation was stronger in cancer. CIP2A expression was increased in metastasized cancer compared to non-metastasized cancer. IDO had stronger expression in tongue hyperplasia than in TSCC. However, IDO expression was associated with poor survival in the subgroups with primary tumor stage T2 to T4 and in the sub-group with strong inflammation at the tumor invasive front. Strong CDH3 expression was associated with better disease-specific and overall survival in the group of tumor size T3. In TSCC patients who had recurrence during follow up, strong CDH3 expression in the primary tumor was associated with poor disease-specific and overall survival. CDH3 expression in lymph nodes without metastasis was negative in all cases. Lymph nodes with extranodal extension CDH3 expression in lymph-node metastasis were positive in all cases. CDH3 expression in lymph-node metastasis was not associated with patient disease-specific survival or overall survival. The high relative density of lymphatic vessels (≥ 80 %) was associated with poor prognosis in TSCC. The relative density of lymphatic vessels was associated with poor prognosis in the group of primary tumor size T1 to T2 and in the group of non-metastatic cancer. In conclusion, CIP2A, IDO, and CDH3 do not appear to be specific biomarkers for TSCC. CIP2A may have some use as it was associated with progression of TSCC. IDO may have some use as it was associated with progression of primary tumor stage T2 and T3 in TSCC. CDH3 may have some use as it was associated with patient survival for tumor recurrence in TSCC. The density and diameter of lymphatic vessels were not associated with TSCC survival. On the other hand, high relative density of lymphatic vessels was associated with tumor invasiveness and poor survival. Thus, the relative density of lymphatic vessels could have a role as a prognostic marker.
  • Karjalainen, Essi (Helsingin yliopisto, 2021)
    Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown aetiology. UC is associated with increased risk of colorectal cancer (CRC). The risk of CRC has declined over recent decades, but the risk is still approximately 2-fold. CRC is one of the indications for surgery, but most UC patients undergo surgery due to medically refractory disease, suggesting that they often use immunomodulative medication and corticosteroids prior to surgery. The most common operation performed is proctocolectomy with ileal pouch-anal anastomosis (PC-IPAA), avoiding a permanent ileostomy. Controversy exists about whether or not a diverting ileostomy should be constructed. The most common long-term complication of PC-IPAA is pouchitis. Episodes of pouchitis are frequently successfully treated with antibiotics. However, a chronic antibiotic-refractory pouchitis can be difficult to manage. Aims: The dissertation comprises four original studies. The aims of these studies were to determine the incidence and prognosis of UC-associated CRC (Study I), to assess the effect of preoperative anti-tumour necrosis factor (TNF) therapy and corticosteroids on postoperative complications and pouch failure (Study II), to determine the effect of covering ileostomy on postoperative morbidity after IPAA (Study III), and to investigate the efficacy and safety of faecal microbiota transplantation (FMT) in the treatment of chronic pouchitis (Study IV). Patients and methods: All studies were carried out at Helsinki University Hospital, Finland. Studies I, II, and III were retrospective. Study I included all 71 patients with UC-associated CRC who were operated on at Helsinki University Hospital between 1991 and 2018. Moreover, 108 patients with dysplasia in the surgical specimen were analysed. Study III included all 510 consecutive patients who underwent PC-IPAA or proctectomy with IPAA between January 2005 and June 2016. A diverting ileostomy was constructed in 119 of these patients. Study II was a subgroup of Study III that excluded patients who underwent proctectomy or were using tacrolimus or vedolizumab prior to surgery. In total, 445 patients were included. Study IV was a randomized, double-blinded, placebo-controlled trial. We randomly allocated 26 patients in a 1:1 ratio to either donor FMT or autologous transplant (placebo). The recruitment was implemented between December 2017 and August 2018. Results: In Study I, CRC was diagnosed preoperatively in 47 patients (66.2%). Altogether 34 patients (47.9%) had synchronous CRC or dysplasia. The incidence of CRC among patients undergoing surgery has not changed during the 28-year study period (P = 0.113). The overall survival was 71.8%, while the 5-year CRC-specific survival was 81.5%. In Study II, anti-TNF therapy was not associated with postoperative complications. Corticosteroids with a dose equivalent to prednisolone 20 mg or more increased the incidence of anastomotic leak (12.6% vs. 2.5%, P = 0.002) and wound dehiscence (4.2% vs. 0%, P = 0.019), but not pouch failure (2.1% vs. 0%, P = 0.141). Patients with a lower dose of corticosteroids had more pouch failures than patients without corticosteroid treatment (4.4% vs. 0%, P = 0.015). In Study III, patients with a diverting ileostomy had more postoperative complications than patients without ileostomy (55.4% vs. 30.2%, P < 0.0001). Although clinical anastomotic leak was more common in patients without an ileostomy (6.6% vs. 1.7%, P = 0.04), the re-laparotomy rate due to an early complication did not differ between the groups (P = 0.58). Re-admission rate was higher among patients with an ileostomy (42.0% vs. 13.0%, P < 0.0001). Of patients, 43.4% had problems with an ileostomy after discharge. There was no difference in pouch failure rate between the groups (1.7% vs. 2.8%, P = 0.74). In Study IV, relapse-free survival was similar between the groups (log rank P = 0.183; HR 1.90, 95% CI 0.73 to 4.98, P = 0.190). Patients reported no major adverse effects. Conclusions: The incidence of UC-associated CRC has remained constant among patients undergoing surgery. The prognosis of patients with UC-associated CRC was no worse than the prognosis of all CRC patients in the Finnish population. The most important finding in Study I was that one-third of the CRCs were not diagnosed until surgery. Moreover, synchronous lesions were common. These findings are alarming and should be considered before the endoscopic management of UC-associated dysplasia. Anti-TNF agents seem to be safe prior to surgery. By contrast, corticosteroids were associated with a higher incidence of anastomotic leak, wound dehishence, and pouch failure. An ileostomy is associated with a considerable number of complications without any reduction in pouch failure rate. Based on our results, a single-stage PC-IPAA is safe in low-risk UC patients. FMT was not effective in the treatment of chronic pouchitis with the FMT treatment protocol used in our trial. The safety profile of FMT was good with no major adverse effects.
  • Woldegebriel, Rosa (Helsingin yliopisto, 2021)
    Rare diseases are a heterogenous group of disorders, which together affect a large number of people. These diseases typically have a genetic cause, which affects various cellular processes and pathways. One of the key processes in the cell is RNA metabolism, which is crucial for gene expression. Defects in RNA processing steps contribute to a variety of human diseases ranging from motor neuron diseases to cancer. Most rare diseases currently have no cure. In this dissertation, biallelic variants in Minichromosome maintenance complex 3 associated protein (MCM3AP) gene were found to underlie a neurological syndrome: early-onset peripheral neuropathy with/without intellectual disability. Through international collaboration, patients from multiple families and the disease variants in MCM3AP were identified. Germinal center associated nuclear protein (GANP), encoded by MCM3AP, is a large scaffold protein in the Transcription and Export 2 (TREX-2) complex, which is responsible for the transport of mRNAs from nucleus to the cytoplasm. The effects of different disease-causing variants on GANP abundance on the nuclear envelope were determined, which led to establishment of a genotype/phenotype correlation regarding the severity of the motor phenotype. This research showed that GANP regulates gene expression in a cell type specific manner. In patient fibroblasts, GANP was found to dysregulate genes depending on intron content. In addition, stem cell-based technology and genome editing was utilized to allow modeling of GANP defects in human cultured motor neurons. We found that GANP is a major regulator of gene expression in developing motor neurons, with GANP defects altering the expression of genes related to synaptic functions and resulting in compensatory induction of protein synthesis and mitochondrial pathways. Understanding of the causes and mechanisms of rare diseases is needed for improving diagnostics, therapeutic development and personalized treatments. In this dissertation, the foundation for understanding the mechanisms of MCM3AP-linked neurological syndrome has been established through identification of the causative gene and description of the disease phenotype. The different cellular models including patient-specific motor neurons used in this study have revealed molecular pathways that may be targets for treatment in preclinical trials.
  • Koskela, Mikael (Helsingin yliopisto, 2021)
    Henoch-Schönlein purpura (HSP) is a vasculitis occurring predominantly in children. Symptoms of HSP typically manifest in the skin, joints, gastrointestinal tract, and kidneys; the severity of kidney symptoms principally determines the long-term outcome of HSP. The pathophysiology and optimal treatment of HSP nephritis (HSN) is still unclear. The International Study of Kidney Disease in Children (ISKDC) classification is a classic and widely used histological scoring system for HSN. It is based primarily on the percentage of glomeruli with crescents. HSN has common pathophysiologic features with IgA nephropathy (IgAN). The Oxford classification for histologic classification for IgAN appeared in 2009. However, its feasibility in HSN is still unclear. This thesis aimed to evaluate the prognostic capability and clinical utility of diagnostic and follow-up kidney biopsies in HSN, to describe the long-term outcome of HSN patients treated in Finland with methylprednisolone (MP) pulses and cyclosporine A (CyA), and to perform a genome-wide association study (GWAS) in HSP patients. Histologic findings from diagnostic kidney biopsies in 53 pediatric HSN patients were retrospectively scored with the ISKDC classification and a new semiquantitative classification (SQC). SQC takes into account 14 histologic variables and divides them into activity and chronicity scores. HSN patients were categorized into those with favorable and unfavorable outcome according to their latest laboratory results; active kidney disease or reduced kidney function denoted unfavorable outcome. The ability of the ISKDC classification and SQC to predict unfavorable outcome was evaluated comparatively with two methods: receiver operating characteristics and logistic regression analyses. According to results from both analyses, SQC seemed better in predicting patient outcome than the ISKDC classification. Our results thus suggest that it is possible to develop a more sensitive histologic classification for HSN than the current ISKDC classification. Serial kidney biopsies were evaluated from 26 HSN patients. Median time between diagnostic and follow-up biopsy was 2.1 years. In addition to the ISKDC classification and SQC, biopsies underwent evaluation with the Oxford classification. Eleven patients had no proteinuria and fifteen patients had proteinuria at the follow-up biopsy; these patients formed groups I and II, respectively. Analysis also involved expression of pro-fibrotic and inflammatory molecules in diagnostic biopsy samples. SQC activity scores decreased in 21 (81%) patients and SQC chronicity scores increased in 22 (85%) patients. The active and chronic parameters of the Oxford classification showed similar results. These changes in the SQC and Oxford classification occurred similarly in groups I and II. All five patients with unfavorable outcome had proteinuria at the follow-up biopsy (group II). Expression of pro-fibrotic and inflammatory molecules showed no clinically significant difference between groups I and II. Our results suggest that in predicting the outcome of HSN, follow-up kidney biopsies provide limited additive information to ongoing clinical symptoms. Long-term follow-up after severe HSN is nonetheless needed as also patients with favorable outcome developed progressive chronic lesions in the follow-up biopsy. The outcome of 62 pediatric HSN patients treated with MP pulses and CyA was studied after a mean follow-up of 10.8 years. Forty-two were initially treated with MP pulses and 20 with CyA. One patient developed kidney failure and one patient had reduced kidney function; both received MP pulses as an initial treatment. These two patients account for 3% of the whole cohort. Additionally, 18 (29%) patients had persisting urinary abnormalities, which justifies long-term follow-up after severe HSN. GWAS was performed for 46 HSP samples against a reference population comprising 18,757 Finnish bone marrow and blood donor samples. Forty-two (91%) HSP patients had undergone kidney biopsy. Analysis also involved imputation of human leukocyte antigen (HLA) alleles. GWAS revealed several polymorphisms from the HLA region in chromosome 6 that were associated with HSP. Three HLA class II alleles (DQA1*01:01, DQB1*05:01, and DRB1*01:01) also occurred significantly more frequently in HSP than in the reference population; a haplotype containing these three alleles occurred in 44% of the HSP patients and in 15% of the reference population. Our results suggest that haplotype DQA1*01:01/DQB1*05:01/DRB1*01:01 is associated with HSP susceptibility in Finnish HSN patients.
  • Lundell, Richard (Helsingin yliopisto, 2021)
    In the Arctic diving occurs in cold water throughout the year. At a depth of 20 meters sea/fresh water (msw/mfw) temperatures are 4°C even in the warmest summer. Cold is not only a discomfort factor for divers. It also impairs physical and cognitive performance, which in turn may jeopardize diving safety, and is one of the major risk factors for decompression illness (DCI). In our study we looked for factors that influence diving safety in Arctic water temperatures and searched for possible ways to reduce the risks. Based on the results from our three studies we aimed to suggest improvements for cold water diving procedures. First, we studied special features of DCI in the Finnish diving population, and different factors associated with DCI. Second, we investigated different thermal protection properties by comparing argon and air used as drysuit inflation gas. Argon is a widely-used passive method to reduce heat loss in cold water diving. Third, we studied the autonomic nervous system (ANS) responses to cold water diving with heart rate variability (HRV) measures. With results from these studies we aimed to give recommendations to make diving in Arctic conditions safer. We carried out a retrospective study to evaluate DCI treated in Finland over a time period of 20 years (1999-2018). The study included by estimation over 95% of all hyperbaric oxygen- treated DCI patients (n = 571) during the years studied. We divided cases into technical divers (n = 200) and non-technical divers (n = 371) and examined differences between the groups. Technical diving was defined as the usage of mixed breathing gases, closed circuit rebreather diving or planned decompression diving. The mean annual number of treated DCI cases in Finland was 29 (range 16– 38). The number of divers treated seemed to show a shift towards technical diving. Technical dives were deeper and longer and were mainly performed in cold water or in an overhead environment. Technical divers were more likely to utilize first aid 100% oxygen (FAO2) and sought medical attention earlier than non-technical divers. The symptom profiles were similar in both groups. Recompression was performed using US Navy Treatment Table Six (USN TT6) in the majority of the cases and resulted in good final outcomes. Eighty two percent of the patients were asymptomatic on completion of all recompression treatment(s). To study the difference in thermal insulation properties of argon compared to air we measured the rectal temperature and eight standardized skin temperatures during tests for navy drysuit diving equipment development in Arctic water conditions. Four divers completed 14 dives, each lasting 45 minutes: seven dives used air insulation and seven used argon insulation. From the measured temperatures, changes in the calculated mean body temperature (MBT) were assessed. There was a significant reduction in the area weighted skin temperature over time (0-45min) during air dives (ΔTskin = -4.16°, SE = 0.445, p<0.001). During argon dives the reduction was significantly smaller compared to the air dives (difference between groups = 2.26°C, SE = 0.358, P<0.001). There were no significant changes in rectal temperatures, nor was a significant difference seen between groups in rectal temperatures. We investigated the human diving responses in cold water temperatures by retrospectively analyzing repeated 5-minute HRV measures and mean body temperature measurements from dives at regular intervals using naval diving equipment measurement tests in 0°C water. The human body reacts to cold through ANS-mediated thermoregulatory mechanisms. Diving also induces ANS responses as a result of the diving reflex. Three divers performed seven dives without engaging in physical activity (dive time 81–91 min), and two divers performed four dives with physical activity after 10 min of diving (0–10 min HRV recordings were included in the study). Based on changes in the root mean square of successive RR interval differences (RMSSD) an increase in parasympathetic nervous system (PNS) activity could be seen at the beginning of the dive. First from the measure at rest to the measure 0 to 5 min showed a significant increase of 14.67 ms (SE = 6.09, p = 0.02). After this PNS activity decreased: the measure from 0 to 5 min to the measure at 5 to 10 min had a significant decrease of 12.92 ms (SE = 6.09, p = 0.04). The remaining measures showed an increase in PNS activity over time: the measure from 5 to 10 min until the measure at 75 to 80 min (n = 7), showed a significant increase of 97.86 ms (SE = 2.28, p < 0.001). As a conclusion there has been a shift towards technical diving in the Finnish diving population, and hence a more demanding types of diving. The number of diagnosed DCI cases in Finland has been quite constant over the last 20 years (1999 to 2018), estimated from a mean incidence of 29 primary decompression treatments annually. Fortunately, the majority of divers recovered well and could continue diving after successful treatment. The use of FAO2 was relatively uncommon in the treatment of DCI symptoms. Compared to air, argon seemed to have better thermal insulation properties when used as drysuit inflation gas, suggesting it would be beneficial when diving in Arctic conditions. Using argon could make diving safer and reduce the risks of fatal diving accidents. The results of the HRV measures suggested a rapid decrease in parasympathetic activity (PNS) after an initial PNS increase at the beginning of the dive. This decrease in PNS activity has not been described in previous studies. To avoid concurrent sympathetic (SNS) and PNS activity at the beginning of dives, which in turn could increase the risk of malign arrhythmia, we suggest a short adaptation phase before physical activity. Moreover, we suggest it is prudent to pay special attention to cardiovascular risk factors during fit-to-dive evaluations for cold water divers.
  • Westberg, Anna (Helsingin yliopisto, 2021)
    The prevalence of obesity is increasing globally both in the general population and in women of a reproductive age. Maternal obesity is a well-known risk factor for acute adverse pregnancy outcomes. Furthermore, accumulating evidence suggests that maternal obesity may impact offspring health later in life. The finding is supported by the Developmental Origins of Health and Disease (DOHaD) paradigm, which suggests that adverse environmental influences during early development, such as maternal obesity, may have an unfavorable impact on later health through the mechanisms of developmental programming. Previous observational studies have found associations of maternal obesity and a number of offspring long-term health outcomes, including obesity, diabetes and asthma. While most preceding research in the field have focused on offspring health outcomes in childhood and adolescence, the associations of maternal obesity and offspring health in adulthood are less studied. The aim of this thesis was to examine the associations between maternal body mass index (BMI) in pregnancy and offspring health in late adulthood. The main offspring health outcomes in the thesis are physical and psychosocial functioning (study I), asthma (study II), glucose metabolism (study III) and physical activity (study IV). Study IV also examine associations between a change in participants’ weight status from childhood to late adulthood and physical activity in older age. This thesis is a part of the Helsinki Birth Cohort Study (HBCS). HBCS includes 13,345 individuals who were born in Helsinki between 1934 and 1944. In 2001- 2004, a subset of 2003 cohort members participated in a baseline clinical examination and in 2011-2013, 1094 participants attended a follow-up clinical examination. The 4 articles of the thesis produced several findings. Higher maternal BMI predicted a poorer physical and psychosocial functioning (study I) and an increased risk of asthma (study II) in men in late adulthood, whereas no similar associations were observed in women. In participants without diabetes, maternal BMI was associated with a favorable glucose metabolism in non-obese men (study III). Overweight in childhood, especially in women, and overweight in adulthood were associated with lower levels of physical activity in older age. In participants with childhood overweight, reaching normal weight by adulthood was associated with higher physical activity levels in older age. There was a slightly U-shaped association of maternal BMI and physical activity levels in older aged women, whereas there was no association on men (study IV). This thesis provides further insight into maternal obesity as a risk factor for offspring health later in life. Findings from this thesis support the evidence that maternal BMI may affect offspring later health in a sex-dependent way and suggest that the associations can still be observed in the offspring in late adulthood. Strategies to reduce overweight and obesity in young women should be implemented in order to improve the long-term health of their offspring.
  • Casado, Julia (Helsingin yliopisto, 2021)
    Cancer continues to be a major clinical and societal challenge. Globally, the cancer burden rises every year with a new record of 18.1 million new cases and 9.6 million cancer deaths, as reported by the World Health Organization. Despite the increased financial efforts of western countries to cure this disease, it is expected that in the year 2040, over one-third of the population will be diagnosed with cancer. The gap in translating basic research into clinical benefit requires cross-disciplinary approaches to harness large data from the complex molecular systems and cellular organization within the tumor. The main obstacles in current cancer care are late detection and therapy resistance. While high-throughput and single-cell methodologies have become an advisable tool to analyze molecular profiles, their use for clinical decision-making is still missing. This thesis aims to propose efficient methodologies to connect molecular and cellular profiling research to cancer therapy outcomes. In the first and second publications, we made available software to rapidly analyze large mass cytometry data with high resolution and interaction-assisted interpretation steps for the analysis. These methods allow the rapid profiling of tumor cell populations and their association with therapy response. As part of the third project, we developed new image analysis methods to identify therapy response predictors from highly multiplexed images. We found that spatial organization within the tumor microenvironment was highly associated with DNA damage genome scarring. In the fourth study, we designed a new method to identify epigenetically reversible drug resistance mechanisms in tumor cells. The application of novel methodologies contributed to a better understanding of the roles of genomic and proteomic features in the tumor-immune microenvironment in response to modern anti-cancer therapies.
  • Mattila, Simo (Helsingin yliopisto, 2021)
    A variety of surgical techniques have been described to treat trapeziometacarpal (TMC) osteoarthritis (OA). The simple removal of the trapezium and partial trapeziectomy are traditional techniques. As an alternative treatment, the poly-L/D-lactide 96/4 implant RegJoint™, has shown positive results in small joint arthroplasty of the rheumatic hand and foot. The purpose of this study was to investigate the clinical results and biocompatibility of the RegJoint™ implant in partial and total trapeziectomy for TMC OA. The retrospective patient cohort for Studies I-II comprised 23 patients (23 hands) operated on for isolated TMC OA by partial trapeziectomy and interposition of the RegJoint™ implant. Subjective Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH), Patient Evaluation Measure (PEM), pain Visual Analog Score (VAS), objective strength and range of motion, and radiographic evaluation were done preoperatively and at follow-up visits up to 3 years postoperatively. Overall, the results showed an unacceptable rate of complications related to clinically manifested foreign body reactions resulting in three revision procedures. The findings were unacceptable due to the high incidence of tissue reactions and revision surgery. The implant should not be used as an interpositional implant in partial trapeziectomy for TMC OA. In retrospective Study III, 34 patients (38 hands) were operated on for TMC OA by total trapeziectomy and interposition of the RegJoint™ implant. Subjective (QuickDASH, PEM, pain VAS), objective (strength and range of motion) and radiographic evaluation were done preoperatively and at follow-up visits up to 33 months postoperatively. The results showed better biocompatibility of the implant than in partial trapeziectomy with only three of 34 patients having signs of a clinically manifested foreign-body reaction. One patient was scheduled for revision surgery. Osteolysis was present in most cases. Strength and subjective outcomes improved and pain decreased during the follow-up. The clinical results were more favorable than in partial trapeziectomy. However, the high incidence of osteolysis casts doubt on the safety of the implant and there is a small risk of clinically manifested foreign-body reactions. The RegJoint™ implant cannot be recommended for use in the treatment of TMC OA in total trapeziectomy. Revision surgery is sometimes required after primary surgery for TMC OA. In Study IV, a single-center retrospective cohort of 1142 TMC arthroplasties performed on 930 patients was evaluated for risk factors for revision and possible factors affecting the final outcome of revision. There were 121 male and 809 female patients (mean age 61 years). Fifty patients (65 revision surgeries) were identified and invited for a follow-up visit. The revision rate was 5%. Patients ≤ 55 years had a revision rate of 9% and age ≤ 55 was identified as a risk factor for revision. Surgery on both thumbs at some point during the follow-up was a negative risk factor for revision. No difference was identified in revision risk between primary surgical techniques, and no difference in final outcome was found between patients having had one versus several revision procedures. The outcome measures QuickDASH and pain VAS were in the same range as previously reported for TMC OA revision surgery. The majority of patients exhibited residual symptoms after revision surgery. However, most patients felt subjectively that they had benefited from the treatment.
  • Paananen, Riku (Helsingin yliopisto, 2021)
    The ocular surface is covered by the tear film that provides a smooth optical surface required for clear vision, offers lubrication necessary for eye movement, and maintains a suitable microenvironment for the ocular surface cells. The tear film can be divided into two separate layers: the aqueous layer, which makes up the bulk of the tear film, and a thin tear film lipid layer (TFLL), which covers the aqueous layer. The TFLL is considered to stabilize the tear film by slowing down evaporation of water from the aqueous tear film. The loss of this stabilizing function of the TFLL is believed to play a central role in the development of most cases of dry eye disease. However, the structure of the TFLL remains poorly characterized, and currently no consensus exists on the molecular level organization within the TFLL. Due to this lack of understanding of TFLL organization on a molecular level, the basis underlying the stabilizing mechanism has remained unclear. In this thesis, the aim was to gain insight into the molecular level structure of the TFLL by dissecting how different lipids in the TFLL organize at the aqueous interface. Further, the effects of interfacial organization of different lipid classes on the evaporation resistance was studied to understand how these structural characteristics are related to the evaporation resistance of the TFLL. The lipids studied in this work cover most of the lipid classes that are found in the TFLL, namely wax esters (WEs), cholesteryl esters (CEs), Type II diesters (DiE), and O-acyl-ω-hydroxy fatty acids (OAHFAs). For each lipid class, model compounds representing the most abundant lipids in the TFLL were used. The organization and evaporation resistance of each lipid class was studied experimentally using a Langmuir trough model system, and this work was complemented by molecular dynamics simulations. This thesis project identified OAHFAs and WEs as the most important lipid classes related to TFLL evaporation resistance. The evaporation resistance of both OAHFAs and WEs was related to the formation of solid monomolecular structures at the aqueous interface. WEs formed solid crystals of monomolecular thickness at the aqueous surface, which spread to cover the surface at a narrow temperature range below the melting point of the WE facilitated by coexistence with a disordered WE monolayer. Long-chained OAHFAs formed a solid monolayer phase on the aqueous interface that provided up to 5 s/cm of evaporation resistance, making OAHFAs even more effective in resisting evaporation than WEs. However, such condensed monolayers inhibited the spreading of nonpolar CE multilayers, which challenges the role of polar lipids in spreading and stabilizing the TFLL. Instead, these results suggest that polar TFLL lipids, mainly OAHFAs, form a condensed monolayer that is directly responsible for the evaporation resistance of the TFLL.
  • Helin, Noora (Helsingin yliopisto, 2021)
    Background: Small children often experience gastrointestinal (GI) symptoms such as regurgitation or abdominal pain. These are usually benign, functional symptoms. In infants, spilling or vomiting can also be suspected to be a symptom of food allergy. In children of developed countries, the most common organic disease in the upper GI tract is gastroesophageal reflux disease (GERD), which refers to troublesome symptoms caused by reflux and related complications such as esophagitis. Upper endoscopy has traditionally been used to verify or exclude esophagitis in patients with symptoms suggestive of GERD, or to exclude other diseases such as eosinophilic esophagitis. Distinguishing children with functional symptoms from those in whom symptoms are associated with disease is often challenging, although guidelines to facilitate differential diagnosis are available. Good availability of endoscopy and lack of data on the long-term prognosis of common upper GI symptoms may have increased the number of upper endoscopies in young children. However, clinical experience suggests that upper endoscopies performed on young patients with non-acute, non-specific symptoms seldom provide clinically relevant data.   Aim: This study aimed to evaluate the diagnostic role of upper endoscopy in children younger than 7 years presenting with non-acute, non-specific GI symptoms. The aim was also to estimate school-age outcomes of symptoms suggestive of GERD or cow’s milk protein allergy with GI symptoms (GI-CMPA) in early childhood.  Patients and methods: The first substudy included children who had undergone primary upper endoscopy at Helsinki University Children’s Hospital in 2006-2016. At an age younger than 7 years, these children had presented with non-specific, non-acute symptoms (n=1850). Patients with acute symptoms, a known disease, or a foreign body for which endoscopy was performed, and congenital malformations of the head, neck and respiratory tract or GI tract, as well as patients with antibody-positive suspected celiac disease were excluded from the study. The remaining patients’ records (n=666) were retrospectively searched for data on the symptoms leading to upper endoscopy, the use of anti-acid medication at the time of endoscopy, the endoscopy results, and the impact of the results on the follow-up or treatment plans.  In the second substudy, children who had undergone primary upper endoscopy due to suspicion of GERD (n=254) were further investigated by evaluating the effect of predisposing conditions to GER on endoscopy findings. Also, the current well-being and anti-acid medication use of children in whom the primary upper endoscopy had resulted in normal findings (n=199) was assessed using the Patient Data Repository and Prescription Service. Of these patients, those whose native language is Finnish (n=175) were invited to an electronic follow-up survey on current symptoms, medications, and GI health-related quality of life (QoL).  The third substudy investigated children who had in early childhood undergone a double-blind, placebo-controlled food challenge due to suspicion of GI-CMPA (n=57). The suspicion of GI-CMPA had not been confirmed in the majority (68%) of these patients. Mothers of both challenge-positive and challenge-negative children were invited to respond to an electronic follow-up questionnaire on children's current GI symptoms, diet, and QoL.    Results: In the majority (81%) of the 666 children who had undergone primary upper endoscopy to investigate non-acute, non-specific symptoms, the endoscopy results were completely normal. Especially in infants, the findings were minor. The most common symptoms leading to endoscopy were related to gastroesophageal reflux, and the most common cause of endoscopy was suspicion of GERD. None in the cohort had erosive esophagitis. The most common histological finding was mild to moderate histological esophagitis (9%). The number of histological findings increased significantly with age. The use of acid blocker medication at the time of endoscopy did not significantly affect the histological findings. There were no unsuspected celiac disease cases, and the number of histologically confirmed eosinophilic esophagitis in this group was low (0.3%). In children diagnosed with inflammatory bowel disease using concomitant colonoscopy, upper endoscopy findings (most often mild gastritis) did not require active treatment. Regardless of the original cause of the primary upper endoscopy, the results affected treatment or follow-up plans in less than 10% of the patients. In children with suspicion of GERD, the upper endoscopy findings were normal in 83%. Vomiting was the most common symptom associated with suspicion of GERD, but none of the symptoms leading to the endoscopy predicted positive findings. Some of the children (39%) had also undergone 24-hour pH- monitoring, but increased esophageal acid reflux did not predict positive endoscopy findings. Thirty-one patients had undergone more than one upper endoscopy. In these patients, the histological degree of changes in the esophageal biopsies remained unchanged or became less severe. Based on medical records, after a median of eight years of follow-up, the otherwise healthy children had seldom reported reflux-related complaints. However, 4% of children with initially normal primary upper endoscopy findings were currently using long-term acid blocker medication, with most of them having underlying conditions predisposing them to GER. Of the 175 Finnish-speaking families who were invited to the follow-up survey, 51 parents (29%) completed the questionnaire on their child’s current reflux symptoms and use of anti-acid medications. Current daily or weekly occurring reflux-related complaints were parentally reported by 24% of survey responders. The use of anti-acid medications was uncommon, but many parents described their child to use a restricted diet. The GI health-related QoL was reported as good by both parents and children older than 8 years of age who responded to the survey. Of the children who had previously undergone a double-blind, placebo-controlled food challenge for symptoms suggestive of GI-CMPA, all had a normal diet after an average of five follow-up years, and mothers reported their children's quality of life as good. After the food challenge, two challenge-negative children with on-going GI symptoms had also undergone an upper endoscopy with normal results.   Conclusions: Based on this study, the diagnostic role of upper endoscopy is minimal in young children presenting with non-specific, non-acute symptoms. There were only a few positive findings in the endoscopy, most non-specific, and the findings rarely led to changes in the child's treatment or follow-up plan. In children who had undergone more than one upper endoscopy, the histological changes either remained unchanged or became less severe. The suspicion of symptom-based diseases in early childhood was not associated with long-term morbidity at school age. Also, the early childhood symptoms leading to suspicion of GERD were not associated with long-term use of acid blocker medications at school age, especially if the child did not have underlying diseases predisposing to GERD. Moreover, no subsequent dietary restrictions occurred in children investigated due to an early childhood suspicion of GI-CMPA.  It is essential to educate clinicians referring children to upper endoscopy about the rarity of diagnostic findings in association with non-acute, non-specific GI symptoms in young children. Also, centers performing pediatric upper endoscopies should ensure that their endoscopy indications are in line with the scientifically approved data, reserving the endoscopy for those who most benefit from it.
  • Zafar, Sadia (Helsingin yliopisto, 2021)
    Dendritic cells (DCs) are the sentinels of the immune system and are specialized in initiating adaptive immune responses by presenting foreign antigens to T cells. Thus, dendritic cells are critical regulators of immune responses and accordingly have been a focus of cancer immunotherapy research. DC therapy is considered as a promising approach in cancer immunotherapy. However, DC-based vaccines have shown limited efficacy in clinical trials. Oncolytic adenovirus replicates and lyses only cancer cells. Virus-mediated lysis of cancer cells also induces danger signals and exposes tumor epitopes that promote immune system activation against cancer. This study investigated the oncolytic adenovirus 3 coding for CD40 Ligand: Ad3-hTERT-CMV-CD40L (also known as TILT-234) as an enhancer of DC therapy. In the first study, human cancer patient data suggested that intravenous adenovirus administration is able to transduce distant tumors and virally-produced CD40L can activate DCs in situ. Studies with mice suggested that the virus possesses potent antitumor activity. In the second study, treatment with Ad3-hTERT-CMV-CD40L and DCs showed 100% survival of humanized mice and resulted in greater antitumor efficacy than either approach as monotherapy. The third study focused on the treatment of prostate cancer. In this study, treatment with companion therapy (i.e. Ad3-hTERT-CMV-CD40L and DC therapy) was shown to induce greater antitumor immune responses in vivo and in established prostate cancer histocultures. In the fourth study, we focused on the interaction of a chimeric adenovirus Ad5/3 with human lymphocytes and erythrocytes. This study showed that the binding of Ad5/3 with human lymphocytes and erythrocytes occurs in a reversible manner, which enables the virus to transduce different tumors and to retain oncolytic potency both in vitro and in vivo, with or without neutralizing antibodies. In summary, the first three studies demonstrated the ability of Ad3-hTERT-CMV-CD40L to modulate the tumor microenvironment and that local delivery of CD40L is safe and efficient regarding DC therapy. In conclusion, Ad3-hTERT-CMV-CD40L was shown to be a potential enabler of DC therapy. The fourth study revealed the ability of a chimeric Ad5/3 adenovirus to transduce non-injected tumors through blood, even in the presence of neutralizing antibodies.
  • Suojärvi, Nora (Helsingin yliopisto, 2021)
    The diagnosis and treatment of distal radius fractures (DRFs) are currently guided by the interpretation of findings in conventional radiographs. However, measurement of radiographic parameters has not been standardized. Cone-beam computed tomography (CBCT) is an alternative imaging technology that is increasingly applied in primary wrist imaging. The purpose of this thesis was to study the potential of CBCT in wrist imaging. Conventional radiographs and CBCT were compared with respect to the reliability of the measurements used in evaluation of DRFs. The potential of CBCT in diagnosing ligamentous and chondral lesions of the wrist was also assessed. CBCT arthrography and magnetic resonance arthrography findings were compared to wrist arthroscopic findings in a group of patients with wrist pain. To improve DRF analysis, a 3D image processing tool based on segmentation of CBCT images and mathematical modelling was developed. The first step was to analyse the osseous anatomy of the normal distal radius and to define the radiographic measurements in the 3D modality. Second, the reliability of computer-aided analysis in performing the measurements in normal radii was compared to physicians’ manual measurements on reformatted 2D wrist radiographs. CBCT images provided better reliability than radiographs for evaluation of intra-articular incongruency. Determining the angular parameters revealed variation in both imaging modalities. CBCT was similar to magnetic resonance imaging in assessment of ligament or triangular fibrocartilage complex tears and chondral lesions of the wrist. The location where the longitudinal axis of the distal radius is determined affects the measurements of the angular parameters used in fracture analysis. The volar tilt angle varies along the distal articular surface and the measurements of ulnar variance and radial inclination vary according to the ulnar reference point. The intraobserver reliability analysis of the new computer-aided analysis showed excellent reliability for the measurements, whereas physicians’ assessment of reformatted 2D radiographs demonstrated variable reliability, particularly for angular measurements. CBCT is a valuable imaging option in wrist imaging. For accurate DRF analysis, the reference points for the radiographic measurements should be standardized. Computer-aided 3D analysis of the radiographic measurements of the intact distal radius is very reliable.
  • Voutilainen, Silja (Helsingin yliopisto, 2021)
    Subclinical fibrosis and inflammation are common findings in pediatric hepatic diseases and in liver grafts after liver transplantation (LT). The mechanisms of these histopathological changes are unclear. Biomarkers that would precede these changes and non-invasive ways to assess fibrosis are needed. The aim of this thesis was to investigate the molecular and cellular markers of subclinical graft fibrosis and non-invasive methods to determine the stage of fibrosis and varices. Study I included 99 pediatric patients with chronic liver disease who underwent liver biopsy sampling for histology and transient elastography (TE) study for liver stiffness (LS) between January 2012 to March 2015. In addition, data on spleen size, platelet count, and aspartate aminotransferase to platelet ratio index (APRI) were collected. Esophagogastroduodenoscopy (EGD) was performed for 61 patients to assess varices. LS had the best accuracy in the prediction of liver fibrosis stage, compared to APRI, spleen size, and platelets to spleen size score. For moderate fibrosis (≥F2) and cirrhosis (F4) the area under receiver curve (AUROC) values for LS were 0.831 (95%CI: 0.745-0.981, p<0.001) and 0.919 (95%CI: 0.861-0.977, p<0.001, respectively). APRI performed little better in the prediction of varices with AUROC 0.832 [95%CI: 0.730-0.934, p<0.001), compared to LS with AUROC 0.818 (95%CI: 0.706-0.930, p<0.001). Patients of studies II-IV consisted of pediatric LT patients who underwent LT in Finland between 1987-2007 and were available for the study in 2009-2011 (time point referred here as cross-section/first follow-up) (n=54). The median time from LT was 11 years. All patients underwent liver biopsy sampling for histological evaluation and blood sampling for liver biochemistry. In Study II, serum concentrations of matrix metalloproteinase-8 (MMP-8), MMP-9, and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were analyzed from 52 patients and 94 healthy controls. Also, hepatic gene expression of MMPs and TIMPS genes was measured from 29 patients and eight control liver samples. The findings were correlated with histological findings. Patients with graft fibrosis had significantly increased hepatic gene expression of MMP-2, MMP-9, MMP-14, TIMP-1, and TIMP-2, compared to those without fibrosis (Mann-Whitney U test p-values ranging from 0.001 to 0.031). However, serum concentrations of MMP-8, MMP-9 TIMP-1 were unrelated to the presence of graft fibrosis. In Study III, hepatic gene expression of 40 fibrosis-related genes was measured from 29 patients and eight healthy controls. Gene expression levels were correlated with histological findings. Compared to patients with normal histology, patients with fibrosis and no inflammation had higher hepatic gene expression of genes related to fibrosis: transforming growth factor-3 (TGF-3), connective tissue growth factor (CTGF), platelet-derived growth factor subunit- (PDGF-), PDGF-, integrin-subunit-1, -smooth muscle cell actin (-SMA), and type I collagen and lower gene expression of decorin and vascular endothelial growth factor A (VEGF A) (Mann-Whitney U test p-values ranging from 0.005 to 0.049). However, compared to patients with normal histology, patients with fibrosis and inflammation had much fewer differences in the gene expression of the fibrosis-related factors than patients with solely fibrosis. Study IV included 51 patients who had sufficient liver biopsy for immunohistochemical staining of six biomarkers; -SMA, type I collagen, decorin, vimentin, p-selectin glycoprotein-1 (PSGL-1), and CD34. Immunohistochemical staining was also performed on 29 patients' biopsy samples taken at the LT operation available from the biobank. Before November 2019, at the final assessment, data on patient (n=48, three deceased) liver status was evaluated. Second liver biopsies (taken after the first follow-up) were available from 24 patients for histological evaluation. The immunohistochemically assessed expression of the six biomarkers at the time of the operation was unrelated to the presence of graft fibrosis at the first follow-up. The expression -SMA, type I collagen, decorin and vimentin correlated with simultaneous graft fibrosis (r=0.693, p<0.001; r=0.612, p<0.001; r=0.464, p=0.003; and r=0.562, p<0.001, respectively). Increased portal expression of -SMA, decorin, and vimentin in liver grafts without fibrosis at the first follow-up were observed in patients who later developed fibrosis (second follow-up biopsies) (Fisher's exact test p-values: 0.014, 0.024, and 0.024 respectively). Moderate fibrosis (≥F2) and increased expression of -SMA, type I collagen, decorin, and vimentin at the first follow-up associated with suboptimal liver status at the final assessment (Fishers exact test p=0.002, p=0.003, p=0.003, p=0.003, p=0.042, p=0.014 and p=0.004 respectively). Our findings implicate that the hepatic gene expression of MMPs and TIMPs is altered in patients with fibrosis after LT. Still, the serum concentration of MMPs and TIMPs was unrelated to graft fibrosis or their gene expression. The genes related to fibrosis were mostly associated with graft fibrosis without inflammation after LT. The immunohistochemically assessed expression of -SMA, decorin, and vimentin might precede the development of later fibrosis and suboptimal liver status, but their expression at the time of LT seems to be unrelated to later graft fibrosis. LS and APRI could be used in some cases to rule out moderate fibrosis and varices and thus avoid invasive liver biopsy sampling and EGS.
  • Maukonen, Mirkka (Helsingin yliopisto, 2021)
    Chronotype refers to preferences in timing the daily activities; accordingly, individuals can be divided from extreme morning to extreme evening types. The evening type has been associated with unhealthier behavior and higher morbidity and mortality risk than the morning type. Furthermore, twin studies have suggested genetic underpinnings behind chronotype trait. However, chronotype associations of dietary habits, obesity and genetics have not been thoroughly examined. The aim of this thesis was to study the associations between chronotype, dietary habits (overall diet quality, energy and macronutrient intake timing) and obesity (weight, body mass index [BMI], waist circumference, body fat percentage) and the interrelationships between these factors. This thesis additionally aimed to clarify the genetic basis of chronotype (clock gene analysis, genome-wide association study [GWAS] of chronotype, developing a genetic risk score [GRS] for chronotype). The study population included participants from the population-based National FINRISK 2007 (n=9958) and 2012 (n=9905) studies and the following sub-studies of FINRISK 2007: DIetary Lifestyle and Genetic determinants of Obesity and Metabolic syndrome (DILGOM) 2007 (n=5024), DILGOM 2014 (n=3735, follow-up) and National FINDIET 2007 (n=2054) conducted at the Finnish Institute for Health and Welfare. Chronotype was assessed with a shortened morningness–eveningness questionnaire. Overall diet was assessed with a validated food frequency questionnaire and measured with the Baltic Sea Diet Score (BSDS), which illustrates adherence to the healthy Nordic diet. Energy and macronutrient intake timing was assessed with 48-hour dietary recalls and 3-day food records. Anthropometric measures were based on measured and self-reported values. Statistical analyses were conducted with analysis of covariance and with linear and logistic regression. The evening type was associated with lower adherence to the healthy diet and with lower energy and macronutrient intake (except for sucrose [E%]) in the morning (by 10:00 a.m.) and higher energy, sucrose (E%), fat (E%) and saturated fat (E%) intakes in the evening (after 8:00 p.m.). Differences between morning and evening types in energy and macronutrient intake timing were even more pronounced at the weekend. Those with a higher tendency towards eveningness more likely had a lower baseline BMI in men but not in women. A higher percentage of evening-typed women had at least a 5% increase in weight and BMI than did morning-typed women during a seven-year follow-up period. These associations, however, attenuated after excluding participants with depression. When interrelationships between chronotype, dietary habits and obesity were examined, no evidence was found that the BSDS would mediate the association between chronotype and obesity or that chronotype would modify the association between the BSDS and obesity. Instead, higher evening energy intake was associated with a higher obesity risk independent of chronotype. Clock gene analysis revealed a novel association between chronotype and the NR1D2 gene. No genome-wide significant associations were found, but the genetic risk score based on 313 single nucleotide polymorphisms (SNPs) that have previously been associated with chronotype predicted the chronotypes in the present study population. In conclusion, despite unhealthier dietary habits (lower adherence to the BSDS, later energy intake timing) of evening chronotypes, evening types were not significantly more prone to obesity nor did chronotype play a role in the association between healthy diet/energy intake timing and obesity. Furthermore, a novel clock gene association was found with the NR1D2 clock gene, which has previously been demonstrated to have a role in carbohydrate and lipid metabolism. A GRS based on GWAS studies of chronotype may be a useful tool for capturing the genetic aspect of chronotype in different populations.
  • Kelppe, Jetta (Helsingin yliopisto, 2021)
    Ameloblastoma is a benign albeit locally aggressive odontogenic tumor originating from remnants of the dental lamina, primarily affecting the mandible, and potentially mutilating if is left untreated. Ameloblastomas are classified as ameloblastoma (conventional), unicystic ameloblastoma, and peripheral ameloblastoma. Annual incidence is estimated to be 0.5/1 million population. In the Helsinki University Hospital (HUS) district, approximately five ameloblastoma patients are treated each year. Etiology has yet to be elucidated, although new genetic findings have emerged relating to the mitogen-activated protein kinase (MAPK) pathway. We surveyed the Q-pati system to identify all ameloblastoma patients (n = 64) treated at the Head and Neck Surgery Unit of HUS. A total of 30 to 36 patient records and the formalin-fixed paraffin-embedded tumor tissue samples were suitable for use from the Department of Pathology at HUS (HUSLAB) from 1985 through 2016. All patient reports were studied, and the parameters were collected using clinical data, Q-pati records, and imaging reports. A total of 26 ameloblastoma patients’ radiological findings were re-evaluated and studied. All tissue samples were revised microscopically, and representative paraffin blocks were chosen for immunohistochemistry with tested dilutions and protocol methods including positive and negative controls. BRAF, MMP-7, MMP-8, MMP-9, E-cadherin, and beta-catenin were of interest. For statistics, we used R studio, seeking correlations between parameters using the Fisher’s exact test, z-test, t-test, χ², and logistic regression to determine statistical significance. We considered p < 0.05 significant. Our results mostly coincide with previous knowledge with minor deviations and some notable differences to consider in future studies. Specifically, maxillary tumors occurred mostly in older, male patients. BRAF-positive tumors seemed to recur more often than BRAF-negative tumors in the mandible area. In addition, all maxillary tumors were BRAF-negative. Maxillary tumors are likely to recur easily, presumably along complex anatomical structures. Unlike previous studies, ameloblastoma cells did not express MMP-7, MMP-8, or MMP-9. MMP-9 positivity, however, was observed in inflammatory cells, macrophages, and osteoclasts. Beta-catenin expression appeared on the cell membranes. E-cadherin expression varied, although maxillary tumors presented with a weak E-cadherin expression. Radiologic re-evaluation revealed that ameloblastomas eradicate cortical bone already during the early stages of tumor growth. Ultimately, we found that CT and MRI imaging remain essential in differential diagnostics, serving to protect the patient from radical surgery. In conclusion, maxillary tumors might be reasonable to study separately from mandibular tumors because of their different protein properties. Our investigations among this Finnish ameloblastoma patient cohort have expanded our knowledge of a rare odontogenic tumor and further substantiated previous findings.

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