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  • Kiuru, Martti (Helsingin yliopisto, 2002)
  • Korhonen, Petri (Helsingin yliopisto, 2002)
  • Palikhe, Anil (Helsingin yliopisto, 2008)
    Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).
  • Paakkanen, Riitta (Helsingin yliopisto, 2013)
    Atherosclerosis is a chronic, inflammatory, lifestyle-disease that is also influenced by genetic factors. Coronary atherosclerosis manifests as silent, chronic and acute forms. The acute forms, acute coronary syndromes (ACS) are an important cause of death due to coronary atherosclerosis. The major histocompatibility complex (MHC) region is a collection of genes on chromosome 6 that are associated with immune response. The genes in this region are usually inherited as a strongly linked genetic combination, a haplotype. Certain alleles and haplotypes of the MHC region have been linked with different forms of coronary atherosclerosis. The Thesis was set to study the role of selected MHC genes/alleles in ACS. First aim of the Thesis was to develop a novel quantitative polymerase chain reaction (qPCR) method for complement component C4 analyses. A qPCR method with a novel concentration range approach and SYBR® Green dye was developed (I). It was validated by applying it to over 1600 patient samples with available C4 protein data and by analysing 129 samples that were also assessed with other methods for C4 gene analysis. The results indicated that the developed method can be used to reliably assess C4 copy number variation. The second aim was to confirm the role of selected MHC genes and haplotypes in different clinical forms of ACS. Based on previous studies, four MHC markers that had been reported to be associated with coronary atherosclerosis were selected (HLA-DRB1*01, HLA-B*35, deficiency of complement component C4A or C4B). A haplotype with DRB1*01, with neither C4 deficiencies nor B*35 was more frequently detected in patients suffering from ACS than in healthy controls (11 vs. 5%; III). None of the markers alone was associated with a significantly increased risk of ACS (II-IV). However, the power was too low to exclude a weak association of C4 deficiency and ACS (II, IV). If only men were studied, both DRB1*01 alone and the DRB1*01-haplotype were significantly associated with ACS. The third aim was to evaluate the possible inflammatory mechanisms through which the association of MHC genes/haplotypes and ACS could be mediated. The DRB1*01-haplotype was associated with higher hsCRP levels (III). C4 deficiency segregated patients that benefitted from macrolide treatment in secondary prevention of recurrent ACS (II). C4 deficiency was also associated with elevated heat shock protein 60 IgA autoantibody levels, which, in turn, was shown to be associated with ACS and recurrent cardiovascular end points (IV). C4 deficiency was also increased in patients suffering from recurrent infections (I). The genetic polymorphism in MHC may be linked with ACS by affecting the inflammatory responses. These data are observational and thus do not indicate causality. However, these data might help to elucidate the complex interplay of inflammatory reactions in ACS and in directing patient care.
  • Helenius, Katja (Helsingin yliopisto, 2011)
    All protein-encoding genes in eukaryotes are transcribed into messenger RNA (mRNA) by RNA Polymerase II (RNAP II), whose activity therefore needs to be tightly controlled. An important and only partially understood level of regulation is the multiple phosphorylations of RNAP II large subunit C-terminal domain (CTD). Sequential phosphorylations regulate transcription initiation and elongation, and recruit factors involved in co-transcriptional processing of mRNA. Based largely on studies in yeast models and in vitro, the kinase activity responsible for the phosphorylation of the serine-5 (Ser5) residues of RNAP II CTD has been attributed to the Mat1/Cdk7/CycH trimer as part of Transcription Factor IIH. However, due to the lack of good mammalian genetic models, the roles of both RNAP II Ser5 phosphorylation as well as TFIIH kinase in transcription have provided ambiguous results and the in vivo kinase of Ser5 has remained elusive. The primary objective of this study was to elucidate the role of mammalian TFIIH, and specifically the Mat1 subunit in CTD phosphorylation and general RNAP II-mediated transcription. The approach utilized the Cre-LoxP system to conditionally delete murine Mat1 in cardiomyocytes and hepatocytes in vivo and and in cell culture models. The results identify the TFIIH kinase as the major mammalian Ser5 kinase and demonstrate its requirement for general transcription, noted by the use of nascent mRNA labeling. Also a role for Mat1 in regulating general mRNA turnover was identified, providing a possible rationale for earlier negative findings. A secondary objective was to identify potential gene- and tissue-specific roles of Mat1 and the TFIIH kinase through the use of tissue-specific Mat1 deletion. Mat1 was found to be required for the transcriptional function of PGC-1 in cardiomyocytes. Transriptional activation of lipogenic SREBP1 target genes following Mat1 deletion in hepatocytes revealed a repressive role for Mat1apparently mediated via co-repressor DMAP1 and the DNA methyltransferase Dnmt1. Finally, Mat1 and Cdk7 were also identified as a negative regulators of adipocyte differentiation through the inhibitory phosphorylation of Peroxisome proliferator-activated receptor (PPAR) γ. Together, these results demonstrate gene- and tissue-specific roles for the Mat1 subunit of TFIIH and open up new therapeutic possibilities in the treatment of diseases such as type II diabetes, hepatosteatosis and obesity.
  • Seppänen, Johanna (Helsingin yliopisto, 2008)
    A population-based early detection program for breast cancer has been in progress in Finland since 1987. According to regulations during the study period 1987-2001, free of charge mammography screening was offered every second year to women aged 50-59 years. Recently, the screening service was decided to be extended to age group 50-69. However, the scope of the program is still frequently discussed in public and information about potential impacts of mass-screening practice changes on future breast cancer burden is required. The aim of this doctoral thesis is to present methodologies for taking into account the mass-screening invitation information in breast cancer burden predictions, and to present alternative breast cancer incidence and mortality predictions up to 2012 based on scenarios of the future screening policy. The focus of this work is not on assessing the absolute efficacy but the effectiveness of mass-screening, and, by utilizing the data on invitations, on showing the estimated impacts of changes in an existing screening program on the short-term predictions. The breast cancer mortality predictions are calculated using a model that combines incidence, cause-specific and other cause survival on individual level. The screening invitation data are incorporated into modeling of breast cancer incidence and survival by dividing the program into separate components (first and subsequent rounds and years within them, breaks, and post screening period) and defining a variable that gives the component of the screening program. The incidence is modeled using a Poisson regression approach and the breast cancer survival by applying a parametric mixture cure model, where the patient population is allowed to be a combination of cured and uncured patients. The patients risk to die from other causes than breast cancer is allowed to differ from that of a corresponding general population group and to depend on age and follow-up time. As a result, the effects of separate components of the screening program on incidence, proportion of cured and the survival of the uncured are quantified. According to the predictions, the impacts of policy changes, like extending the program from age group 50-59 to 50-69, are clearly visible on incidence while the effects on mortality in age group 40-74 are minor. Extending the screening service would increase the incidence of localized breast cancers but decrease the rates of non-localized breast cancer. There were no major differences between mortality predictions yielded by alternative future scenarios of the screening policy: Any policy change would have at the most a 3.0% reduction on overall breast cancer mortality compared to continuing the current practice in the near future.
  • Alestalo, Paula Kaarina (Helsingin yliopisto, 2015)
    ABSTRACT The general objective of this thesis was to study management and leadership in the Public Dental Service (PDS) in Finland during the major Dental Care Reform in 2003–2011. The specific aims were to study 1) how dentists became leaders in the PDS and 2) what characterised their leadership, 3) distribution of leadership positions between women and men, 4) chief dentists’ position in the municipal hierarchy, as seen from their own superiors’ and subordinates’ points of view and 5) chief dentists’ attitudes to the Dental Care Reform and the changes it caused in the work environment. Four questionnaire surveys based on four data sets were carried out in 2003. The target groups were the chief dentists of the municipal PDS units (health centres) (n=265), the chief physicians (n=233), the line managers (superiors) of the chief dentists, PDS dentists who were subordinates to the chief dentists (n=365), and the chairpersons of Municipal Boards of Social Affairs and Health (MBSH) (n=233). In 2011, the target group was the chief dentists alone (n=161). Factor analysis, linear regression analysis, parametric and non-parametric tests were used in the analysis of the materials. The results showed that only fewer than a fifth (17%) of chief dentists were full-time leaders in 2011 and they worked in the largest health centres. The rest also provided patient care, to varying degrees. In 2003 and 2011, nearly two thirds (62%) of the chief dentists identified themselves as leaders instead of seeing themselves only as dentists among other dentists in the PDS, though fewer than a third (31%) of them had applied for their posts. More precisely, just 21% of female and 43% of male chief dentists (p<0.001) had applied for the chief dentist posts they held. Chief dentists felt they were better people-oriented leaders than goal-oriented managers (p<0.001). Nevertheless, 49% p<0.001) of their job satisfaction was explained by the fact that they were motivated to lead, their position as superiors to their subordinates was good, they had enough (3-4 on a scale 1-4) decision making power, they were good goal-oriented managers (good in sum variables ≥3 on a scale 1-4 from very bad to very good), and had received enough (3 on a scale 1-3) leadership education. In addition, if they had a good position as subordinates to their superiors, chief physicians and MBSH chairpersons (p<0.001), and when they had applied for their posts, they felt contented (p<0.001). In 2003, their subordinates, the PDS dentists, appreciated their people-oriented female superiors highest. Chief dentists themselves as subordinates felt they received little feedback and support from their superiors (chief physicians), and their own subordinates, the PDS dentists, felt the same (p<0.001). Chief dentists’ superiors (chief physicians and MBSH chairpersons) evaluated the chief dentists as good managers and leaders, whereas the chief dentists’ subordinates evaluated their superiors in less positive terms. In 2003, more than half (59%) of the chief dentists were very positive (4 on a scale 1-4) towards the recently introduced reforms of the Health Insurance Act (HIA) promoting adults’ treatment in the private sector by increasing reimbursement for dental care. Fewer than half (43%) were very positive towards the reform of the Primary Health Care Act (PHCA) improving adults’ access to care in the public sector (p<0.001). In 2011, only 20% totally agreed (4 on a scale 1-4) that the implementation of the Dental Care Reform had succeeded. The respondents’ general opinion was that the timetable for the Reform was too tight and not enough resources were allocated. In general, the chief dentists considered their position as isolated and rather weak in the municipal hierarchy. Effective and rewarding leadership in the PDS needs more attention in the future.
  • Koskenmies, Sari (Helsingin yliopisto, 2004)
  • Aalto, Hannele (Helsingin yliopisto, 2007)
    Sepsis is associated with a systemic inflammatory response. It is characterised by an early proinflammatory response and followed by a state of immunosuppression. In order to improve the outcome of patients with infection and sepsis, novel therapies that influence the systemic inflammatory response are being developed and utilised. Thus, an accurate and early diagnosis of infection and evaluation of immune state are crucial. In this thesis, various markers of systemic inflammation were studied with respect to enhancing the diagnostics of infection and of predicting outcome in patients with suspected community-acquired infection. A total of 1092 acutely ill patients admitted to a university hospital medical emergency department were evaluated, and 531 patients with a suspicion of community-acquired infection were included for the analysis. Markers of systemic inflammation were determined from a blood sample obtained simultaneously with a blood culture sample on admission to hospital. Levels of phagocyte CD11b/CD18 and CD14 expression were measured by whole blood flow cytometry. Concentrations of soluble CD14, interleukin (IL)-8, and soluble IL-2 receptor α (sIL-2Rα) were determined by ELISA, those of sIL-2R, IL-6, and IL-8 by a chemiluminescent immunoassay, that of procalcitonin by immunoluminometric assay, and that of C-reactive protein by immunoturbidimetric assay. Clinical data were collected retrospectively from the medical records. No marker of systemic inflammation, neither CRP, PCT, IL-6, IL-8, nor sIL-2R predicted bacteraemia better than did the clinical signs of infection, i.e., the presence of infectious focus or fever or both. IL-6 and PCT had the highest positive likelihood ratios to identify patients with hidden community-acquired infection. However, the use of a single marker failed to detect all patients with infection. A combination of markers including a fast-responding reactant (CD11b expression), a later-peaking reactant (CRP), and a reactant originating from inflamed tissues (IL-8) detected all patients with infection. The majority of patients (86.5%) with possible but not verified infection showed levels exceeding at least one cut-off limit of combination, supporting the view that infection was the cause of their acute illness. The 28-day mortality of patients with community-acquired infection was low (3.4%). On admission to hospital, the low expression of cell-associated lipopolysaccharide receptor CD14 (mCD14) was predictive for 28-day mortality. In the patients with severe forms of community-acquired infection, namely pneumonia and sepsis, high levels of soluble CD14 alone did not predict mortality, but a high sCD14 level measured simultaneously with a low mCD14 raised the possibility of poor prognosis. In conclusion, to further enhance the diagnostics of hidden community-acquired infection, a combination of inflammatory markers is useful; 28-day mortality is associated with low levels of mCD14 expression at an early phase of the disease.
  • Parviainen, Ville (Helsingin yliopisto, 2014)
    The field of biological sciences has expanded enormously within the last few decades. Developments in techniques and instrumentation have allowed biologist to explore biological mechanisms in an unprecedented detail. One of the most evolved disciplines is the field of proteomics. In general, proteins function in many different biological roles. They serve as structural molecules, in signaling routes mediating information in the cell, in intra- and extracellular transport and trafficking as well as in numerous other cellular functions. The area of protein research entails the study of all things relating to proteins and their functions. These include cellular protein composition, expression changes, protein structure, post-translational modifications and protein-protein interactions. Mass spectrometry (MS) has become one of the key technologies in proteomic research. The relative ease of sample handling and automated MS machinery has made proteomic analysis relatively straightforward. Mass spectrometers work by measuring the weight of intact proteins or protein-derived peptides. Proteomic MS identification is usually done by fragmenting the proteins or peptides in the mass spectrometer and using the resulting mass spectral information in identification of peptide sequence. There are two main strategies of peptide sequence identification: database dependent and de novo identification. Database dependent algorithms utilize known sequence information stored in databases to decipher the peptide amino acid sequence of the MS-observed spectra and use that information to predict the protein from which the peptide is derived from. On the other hand de novo methods try to construct the peptide sequence solely based on the fragmentation patterns of the peptide. The completeness of sequence databases of many species and the speed and efficiency of the search engines have made the database dependent search as the main method in peptide and protein identification. The modern high resolution mass spectrometers along with ultra-performance liquid chromatography have enabled the detection of thousands of protein in one single MS run. This, together with advances in MS-based protein quantification has extended the use of mass spectrometers in discovery type biomarker search. Mass spectrometers are able to produce a large amount of data on numerous proteins that can be used to detect and quantify differences in patient and control samples. This in turn can be used as starting point for more focused validation studies on the acquired data and ultimately lead to useful clinical biomarkers. The focus of this study was to utilize and learn mass spectrometric methodologies and to analyze different proteomic processes in sample types. We analyzed the protein-protein interactions in Baker´s yeast PSA1 protein in various points of batch cultivation using database dependent and de novo protein identification methods. We showed that the interactome of PSA1 is very dynamic depending on the phase of the cultivation. We also showed the limitations and benefits of de novo identification and the combined use of both search strategies in improving the confidence of the identifications. In another study using affinity purification and mass spectrometry we identified Fibrillin-2 as the binding partner of lung cancer associated Gremlin-1 protein. This finding elucidates functions and mechanisms of Gremlin-1 and Fibrillin-2 in malignant tissues. In two mass spectrometry-based protein quantification studies we characterized the protein concentration changes in human plasma during liver transplantation surgery as well as the effect of excess sialic acid production in HEK293 model cell line. In the liver transplantation plasma project we identified protein concentration changes in liver in response to the trauma caused by the surgery using label-based iTRAQ method. We showed consumption and secretion of several coagulation related proteins within the liver suggesting activation of coagulation cascade in the very early phases of the craft reperfusion. In the study of excess sialic acid production we first verified the amounts of sialic acid using mass spectrometry-based multiple reaction monitoring method. We were able to induce the production of sialic acid to almost 70-fold compared to control cells. We also monitored the protein abundance changes in sialic acid producing cells using label free proteins quantification method identifying 105 changed proteins. We analyzed those proteins with several functional enrichment tools revealing modifications in cellular protein transport, metabolic and signaling pathways and in remodeling of cellular adherens junctions. Such large scale MS-analyses using ontology-based tools can significantly aid in deciphering the effect of perturbations to complex systems but also reveal novel functional targets for biomarker discovery. The results obtained from targeted interaction experiments as well as large scale quantification studies can be used as basis for more rigorous investigations on the various subjects in search for potential biomarkers for clinical use. The techniques and methods used in the studies also demonstrate the many uses of mass spectrometric techniques in several fields of proteomic and biological research.
  • Leskinen, Markus (Helsingin yliopisto, 2003)
  • Mäyränpää, Mikko (Helsingin yliopisto, 2007)
    More than 40% of all deaths in Finland are caused by atherosclerosis. The complications of atherosclerosis are due to either detachment of the luminal endothelium (erosion) or rupture of the fibrous cap of an atherosclerotic plaque (rupture). As a result, a thrombus is formed at the site of the intimal lesion. Indeed, erosions cause roughly 40% of sudden atherothrombotic deaths and 25% of all atherothrombotic deaths. Erosions are overrepresented in young subjects, diabetics, smokers and women. This dissertation focuses on endothelial erosion. Endothelial erosions were studied in the context of arterial grafting and vascular inflammation. Special attention was given to the role of intimal mast cells and the methodological viewpoints of reliable identification of endothelial erosions. Mast cells are inflammatory cells mostly known for their ability to cause allergic symptoms. In addition to occurring in skin and mucosal surfaces, mast cells are abundant in arterial intima and adventitia. In this study, mast cells were found to associate with endothelial erosions in non-lesional and atherosclerotic human coronary arteries. Thus, mast cells may participate in atherogenesis at the initial phases of the disease process already. We also showed that the mast cell proteases tryptase, chymase, and cathepsin G are all capable of cleaving molecules essential for endothelial cell-to-cell and cell-to-extracellular matrix interactions, such as VE-cadherin and fibronectin. Symptom-causing carotid plaques were found to contain more inflammatory cells, especially mast cells, than non-symptom-causing plaques. Furthermore, the atherogenic serum lipid profile and the degree of carotid stenosis turned out to correlate with the density of carotid plaque mast cells. Apoptotic and proliferating cells were more abundant in non-symptom causing plaques (active renewal of endothelial cells), but erosions were larger in symptom-causing plaques (capacity of endothelial regeneration exceeded). The process of identifying endothelial erosions with immunostainings has been ambiguous, since both endothelial cells and platelets express largely the same antigens. This may have caused inaccurate interpretations of the presence of endothelial erosion. In the last substudy of this thesis we developed a double immunostaining method for simultaneous identification of endothelial cells and platelets. This method enables more reliable identification of endothelial erosions.
  • Hannula, Katariina (Helsingin yliopisto, 2001)
  • Mäkitalo, Laura (Helsingin yliopisto, 2010)
    Matrix metalloproteinases (MMPs) represent a family of 23 metalloendopeptidases, collectively capable of degrading all components of the extracellular matrix. MMPs have been implicated in several inflammatory processes such as arthritis, atherosclerosis, and even carcinomas. They are also involved in several beneficial activities such as epithelial repair. MMPs are inhibited by endogenous tissue inhibitors of matrix metalloproteinases (TIMP). In this study, MMPs were investigated in intestinal mucosa of inflammatory bowel diseases (IBD), chronic intestinal disorders. The main focus was to characterize mucosal inflammation in the intestine, but also cutaneous pyoderma gangrenosum (PG), to assess similarites with IBD inflammation. MMPs and TIMPs were mainly examined in colonic mucosa, in adult Crohn s disease (CD), and paediatric CD, ulcerative colitis (UC), and indeterminate colitis (IC). Ileal pouch mucosa of proctocolectomized paediatric onset IBD patients was also investigated to characterize pouch mucosa. The focus was on finding specific MMPs that could act as markers to differentiate between different IBD disorders, and MMPs that could be implied as markers for tissue injury, potentially serving as targets for MMP-inhibitors. All examinations were performed using immunohistochemistry. The results show that immunosuppressive agents decrease stromal expression of MMP-9 and -26 that could serve as specific targets for MMP-inhibitors in treating CD. In paediatric colonic inflammation, MMP-10 and TIMP-3 present as molecular markers for IBD inflammation, and MMP-7 for CD. MMP expression in the the pouch mucosa could not be classified as strictly IBD- or non-IBD-like. For the first time, this study describes the expression of MMP-3, -7, -9, -12, and TIMP-2 and -3 in pouch mucosa. The MMP profile in PG bears resemblance to both intestinal IBD inflammation and cutaneous inflammation. Based on the results, MMPs and their inhibitors emerge as promising tools in the differential diagnosis of IBD and characterization of the disease subtype, although further research is necessary. Furthermore, the expression of several MMPs in pouch has been described for the first time. While further research is warranted, the findings contribute to a better understanding of events occurring in IBD mucosa, as well as pyoderma gangrenosum.