Browsing by Issue Date

Sort by: Order: Results:

Now showing items 41-60 of 1649
  • Orivuori, Laura (Helsingin yliopisto, 2015)
    ABSTRACT Allergies have become a major public health problem in recent decades. Approximately 60 million people in Europe, and one billion worldwide, are affected by allergies. In Europe, approximately 30% of the population suffers from allergic rhinoconjunctivitis, approximately 20% from asthma, and 15% from allergic skin conditions. The farming environment has been shown to protect against allergies. In the present thesis, we studied the development of Immunoglobulin E (IgE) sensitization, allergic diseases, gut permeability and inflammation in children from farming and non-farming environments who participated in the PASTURE project (Protection against allergy: study in rural environments). Our results showed that early-age inflammation, measured with serum high-sensitivity C- reactive protein (hs-CRP) at the age of 1 year, was associated with decreased risk of IgE-sensitization at the age of 4.5 years. Our findings suggest that poor inflammatory response at infancy may predispose to IgE-sensitization. The allergy-protective effect of breastfeeding is debated. Our findings illustrated that increased breast milk soluble immunoglobulin A (sIgA) levels were associated with decreased risk of atopic dermatitis (AD) up to the age of 2 years. When the ingested dose of sIgA during the first year of life was considered, it was associated with decreased risk of AD up to ages of 2 and 4 years. Elevated sIgA levels in breast milk were associated with environmental factors indicating increased environmental microbial load. Serum immunoglobulin A (IgA) and immunoglobulin G (IgG) against cow s milk β- lactoglobulin (BLG) and wheat gliadin are induced by the antigen exposure, but the antibody levels are also affected by the gut permeability and inflammation. Elevated serum IgA or IgG antibody levels against BLG at the age of 1 year increased the risk of sensitization to at least one of the measured allergens or food allergens at the age of 6 years. Elevated levels of IgG against gliadin at the age of 1 year increased the risk of sensitization to any, at least one inhalant, or at least one food allergen at the age of 6 years. Our findings suggest that an enhanced antibody response to food antigens reflects alterations in mucosal tolerance, such as altered increased gut permeability and/or microbiota, which is later seen as sensitization to allergens. Fecal calprotectin is a marker of intestinal inflammation. We studied the association of early-infancy fecal calprotectin levels to the later development of allergic diseases in children from farming and non-farming environments, and further evaluated the effect of gut microbes on the levels of fecal calprotectin. Infants from a farming environment had higher levels of fecal calprotectin at the age of 2 months when compared to the infants from a non-farming environment. However, farming did not explain the very high levels of calprotectin, i.e. the levels above the 90th percentile. The infants with remarkably high fecal calprotectin levels at the age of 2 months, i.e. levels above the 90th percentile, had an increased risk of developing AD and asthma/asthmatic bronchitis by the age of 6 years. Infants who had high fecal calprotectin concentrations had less Escherichia in their feces. Our findings suggest that strong intestinal inflammation at early infancy represents a risk of allergic diseases, and the colonization with Escherichia coli is an important regulator of the inflammation implicating long-term health effects mediated by early intestinal colonization. In conclusion, the findings in this thesis show that the environmental microbial load plays an important role in the development of the immune system in infants and ultimately may affect the risk of developing allergic diseases. In addition to the direct effects on the infant, the effects of the environmental microbial load are also mediated indirectly through alterations in maternal milk composition. Interestingly, we found that an early intestinal inflammation is associated with the later risk of allergic diseases and gut colonization indicating a crucial role of gut colonization in the development of allergy. Keywords: Allergy, antibody, asthma, atopic dermatitis, farming environment, fecal calprotectin, IgE-sensitization, inflammation.
  • Knaster, Peter (Helsingin yliopisto, 2015)
    Chronic pain is a long-lasting burdensome condition. Comorbid psychological symptoms are common in chronic pain patients and they tend to worsen the treatment outcome. The aim of this cross-sectional study was to assess the prevalence of psychiatric comorbidity in chronic pain patients and to assess the associations between chronic pain, anxiety, anger, and depression. The study participants in the study were one hundred consecutive chronic pain patients referred to the Meilahti Pain Clinic of Helsinki University Hospital. The subjects were interviewed with the Structured Clinical Interview for DSM-IV for Axis I disorders (SCID-I). In addition self-report questionnaires were used in the assessment. Most (75%) patients had at least one lifetime mental disorder. The prevalence of major depressive disorder (MDD) was 37% and of a specific anxiety disorder 25% over the past 12 months. The psychiatric comorbidity was associated with increased pain intensity, measured by Visual Analogue Scale (VAS). The majority (77%) of the anxiety disorders had their onset before the onset of pain, whereas only 37% of the mood disorders preceded pain onset. The Harm Avoidance (HA) scale of the Temperament and Character Inventory (TCI) of Cloninger was associated with pain-related anxiety measured with Pain Anxiety Symptom Scale-20. The pain intensity influenced the strength of the association between the Harm Avoidance HA4 subscale and pain-related anxiety. Higher pain intensity was associated with stronger association between the variables. Likewise, pain intensity influenced the association between anger management and depression. A tendency to inhibit angry feelings was associated the somatic and physical symptoms of depression, measured by a two-factor model of the Beck Depression Inventory (BDI). The association was stronger in patients with higher pain intensity. Chronic pain patients with current MDD scored higher in both the somatic and cognitive-emotional subscales of the BDI compared with those without MDD. However, the somatic-physical-related items were more strongly associated with the diagnosis of MDD. Psychiatric disorders are common in chronic pain patients. Because of their influence on the chronic pain treatment and outcome, their thorough assessment is important. The linking mechanisms between chronic pain and the psychiatric disorders and symptoms are complicated and there is an overlap between the constructs. Clear boundaries between pain, anxiety and depression can be difficult to draw, which can be a reflection of the common background mechanisms such as common neural circuits and neurotransmitters.
  • Louhimo, Riku (Helsingin yliopisto, 2015)
    Cancer is one of the leading causes of death in industrialized nations and its incidence is steadily increasing due to population aging. Cancer constitutes a group of diseases characterized by unwanted cellular growth which results from random genomic alterations and environmental exposure. Diverse genomic and epigenomic alterations separately and jointly regulate gene expression and stimulate and support neoplastic growth. More effective treatment, earlier and more accurate diagnosis, and improved management of cancer are important for public health and well-being. Technological improvements in data measurement, storing and transport capability are transforming cancer research to a data-intensive field. The large increases in the quality and quantity of data for the analysis and interpretation of experiments has made employing computational and statistical tools necessary. Data integration - the combination of different types of measurement data - is a valuable computational tool for cancer research because data integration improves the interpretability of data-driven analytics and can thereby provide novel prognostic markers and drug targets. I have developed two computational data integration tools for large-scale genomic data and a simulator framework for testing a specific type of data integration algorithm. The first computational method, CNAmet, enhances the interpretation of genomic analysis results by integrating three data levels: gene expression, copy-number alteration, and DNA methylation. The second computational method, GOPredict, uses a knowledge discovery approach to prioritize drugs for patient cohorts thereby stratifying patients into potentitally drug-sensitive subgroups. Using the simulator framework, we are able to compare the performance of integration algorithms which integrate gene copy-number data with gene expression data to find putative cancer genes. Our experimental results indicate in simulated, cell line, and primary tumor data that well-performing integration algorithms for gene copy-number and expression data use and process genomic data appropriately. Applying these methods to diffuse large B-cell lymphoma, integrative analysis of copy-number and expression data helps to uncover a gene with putative prognostic utility. Furthermore, analysis of glioblastoma brain cancer data with CNAmet suggests that a number of known cancer genes, including the epidermal growth factor receptor, are highly expressed due to co-occuring alterations in their promoter DNA methylation and copy-number. Finally, integration of publicly available molecular and literature data with GOPredict suggests that treating patients with FGFR inhibitors in breast cancer and CDK inhibitors in ovarian cancer could support standard drug therapies. Collectively, the methods developed here and their application to varied molecular cancer data sets illustrates the benefits of data integration in cancer genomics.
  • Peuralinna, Terhi (Helsingin yliopisto, 2015)
    This thesis project aimed to study the genetic background of common neurodegenerative diseases such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and amyotrophic lateral sclerosis (ALS) in the Finnish population. In the first study, we analysed the role of the amyloid precursor protein (APP) and apolipoprotein E (APOE) genes in AD neuropathology. Mutations in the APP gene cause early-onset familial AD and there is some evidence that APP gene variants would play a role in late-onset AD, too. We genotyped more than 50 common variations in the APP gene and sequenced the APP promoter area to detect rare variations in the area, both known and new. In the Finnish elderly population we did not find any of the APP variations to clearly associate with neuropathologically diagnosed AD or with any of the neuropathological features of AD such as cortical β-amyloid, cerebrovascular β-amyloid or neurofibrillary tangles. In addition, the APOEε4 results were updated, using the whole Vantaa85+ cohort. APOE ε4 is currently the strongest known risk factor for late-onset AD. We found a very strong association of APOE ε4 to all neuropathological features of AD. In the second study, we investigated how the common variants in the α-synuclein (SNCA) gene affect different brain pathologies. -synuclein is the main component of the Lewy bodies, which are the pathological hallmarks of PD and DLB, and which are sometimes found also in AD. We genotyped 11 SNPs from the SNCA gene region. We found no association with the cortical β-amyloid and only a faint one with Lewy related pathology. However, we found an association with neurofibrillary tangles. The SNP (rs2572324) associated with a p=0.004 after the Bonferroni correction. The two-locus analysis suggest an independent effect of APOE ε4 (OR=8.67) and rs2572324 (OR=3.36). In the subjects with both risk factors the effect was almost multiplicatively increased (OR=23.3). 35 subjects out of 38 with both risk factors had severe tau-pathology. These results demonstrated the first evidence for a role of genetic variation in SNCA in tau pathology. Moreover, β-amyloid pathology was not associated with the SNCA variants, demonstrating a dissection of genetic effect on the two principal pathological features of AD. In the third study, we performed a whole-genome genotyping using Finnish ALS samples. The Vantaa85+ study subjects were used as controls. This was the first ALS genome-wide association study to find a genome-wide significant association. The location associated with familial ALS was on the chromosome 9p21, which had been noticed before, but the area had been much larger. The area has also been associated with frontotemporal dementia (FTD). Now we managed to define the area with a 42 SNP haplotype. This considerably reduced the area of interest (down to 232 kb) and increased the possibility to find the mutation behind the disease. This haplotype was found in around 40% of the Finnish familial ALS cases and likely explains the high rate of ALS as well as FTD in Finnish population. In the fourth study, we performed a genome-wide association study of DLB in the Vantaa85+ cohort and found two novel areas to be associated with DLB: The c2p21 location with 9 SNP haplotype (p=5.2x10-7) and the c6p21 location with 6 SNP haplotype (p=1.3x10-7). The c6p21 was significant at the genome-wide level. The c2orf21 haplotype has two genes on its area, c2orf72 and SPTBN1. SPTBN1 is the candidate gene since it encodes beta-spectrin, a component of Lewy bodies, while c2orf72 is barely expressed in the brain. The c6p21 associated haplotype block is located in the HLA region and includes HLA-DPB1 and -DPA1 genes. These studies show that the Finnish population is well-suited to study the genetics of neurodegeneration. Our results showed that neuropathologically defined parameters and diagnoses are strongly associated with genetic risk factors, even with relatively low numbers of samples. Hence, phenotypic precision (pathology) is an important element of the statistical power of a study.
  • Sivula, Mirka (Helsingin yliopisto, 2015)
    Disseminated intravascular coagulation (DIC) is common in critically ill patients. Patients with disturbed coagulation develop more organ dysfunction and have higher mortality. The pathophysiology behind the increased morbidity and mortality remains unclear. This study assessed the applicability of diagnostic tools for DIC: a modified score for overt DIC and thromboelastometry (TEM), a viscoelastic coagulation monitor. A further aim was to evaluate matrix metalloproteinase-8 (MMP-8), tissue inhibitor of metalloproteinase-1 (TIMP-1), nucleosomal histone-complexed DNA (hcDNA) and high-mobility group box 1 (HMGB1) protein levels in severe sepsis with disturbed coagulation and to assess their association with organ dysfunctions and outcome. Studies I-IV comprised 769 patients. Study I included 494 unselected critically ill patients in whom the incidence of DIC was studied by a modified score for overt DIC. Study II was a prospective pilot study with 28 patients with severe sepsis and ten healthy controls. TEM analyses were performed on admission to intensive care unit (ICU). Study III contained 22 patients with severe sepsis, in whom serum MMP-8 and TIMP-1 concentrations were measured repeatedly. Study IV was a sub-study of a large, prospective, observational, multicentre study conducted in 17 Finnish ICUs (FINNAKI Study). Admission levels of hcDNA and HMGB1 were analysed from 225 consecutive patients. In the university hospital ICU, incidence of DIC ranged from 31% (I) to over 40% (II, III). A multicentre study revealed a lower incidence (33%) of thrombocytopenia in severe sepsis patients. Components of the score discriminated patients with DIC well; only fibrinogen proved useless. Discriminative power of antithrombin was comparable to D-dimer and prothrombin time ratio. TEM trace was hypocoagulable in DIC patients as compared with other sepsis patients and healthy controls. Clot strength and clot formation parameters discriminated DIC patients well from those without DIC. In all patients fibrinolysis was inhibited. The markers speculated to contribute to coagulation disturbance and organ dysfunction, MMP-8, TIMP-1, hcDNA and HMGB1, were higher in patients with disturbed coagulation. HcDNA and HMGB1 were also elevated in patients with acute kidney injury and adverse outcome. HcDNA was an independent predictor of thrombocytopenia.
  • Kulovesi, Pipsa (Helsingin yliopisto, 2015)
    The tear film covers the cornea and conjunctiva providing nutrients to the corneal cells and protecting it from the external environment. Tear film is composed of three intermixed layers. Mucous matrix covers the epithelial cells and is gradually mixed with the aqueous layer which is covered by a thin lipid layer. The lipid layer consists of both polar and nonpolar lipids. Lipids are thought to prevent the collapse of the tear film onto the ocular surface and to retard evaporation from the tears. In this study several in vitro methods were used to study the surface properties and organization of different lipid mixtures resembling those in the tear film lipid layer. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and freefatty acids (FFA) were used as the polar lipids and cholesterol oleate (CO), triglycerides (TG), and wax esters(WE) were used as the nonpolar lipids. Langmuir film technique was used to examine the behavior of the lipid films during compressions and de-compression. Brewster angle microscopy (BAM) and atomic force microscopy (AFM) were utilized for visualizing the films. Grazing incidence X-ray difraction was used for surface structure studies. The results of experimental studies were compared with coarse-grained molecular dynamics simulations. Custom built system was used to evaluate the evaporation retarding effect of several lipid mixtures containing wax esters. Compression isotherms showed one to two kinks in the compression curve that account for the rearrangement of the lipids in the film for all mixtures studied. Hysteresis was small meaning that these films are very stable and little, if any, solubilization of lipids into the aqueous phase takes place. All lipid films studied were more or less inhomogeneous when viewed with BAM, especially in higher surface pressures. This is most likely caused by the nonpolar lipids aggregating on the lipid film surface. This was also seen in the simulation studies where CO and TG formed circular aggregates on top of the polar lipids. Nonpolar lipids stabilized the films under high compression by arranging so that the lipid film could have a lower surface pressure than would be expected for small surface areas. However, an excess of nonpolar lipids caused the films to be more inhomogenous and to have less stable structure. Lipid mixtures that contained wax esters did not retard evaporation, which suggests that lipids' function in the tear film may have more to do with maintaining a thin tear film and preventing its collapse rather than preventing evaporation. The task of TFLL is still under debate, although it has been regarded to be the evaporation barrier, hindering the movement of water molecules out from the tear fluid. This, however has been questioned in this thesis project. Although some WEs have been shown to retard the evaporation of water, this effect is lost when different lipid species are mixed, as was done in this study where several WE species were mixed with PC, CO, and TG. No evaporation retarding effect was detected for any mixtures studied. Evaporation retarding lipid films must be very tightly packed but this is not possible for TFLL-like films because the tear film lipids must also be suitable for the environment they are in where they must adapt to fast changes in surface pressure and must also be fluid. This cannot be achieved by the films that have been shown to retard evaporation as these films are rather stiff and cannot be compressed.
  • Laine, Merja (Helsingin yliopisto, 2015)
    Cardiometabolic health among male former elite athletes Regular physical activity is one of the cornerstones in the prevention of chronic diseases. The aim of this study was to assess whether a former career as a male elite athlete associates with various cardiometabolic disorders and whether it has any effect on leukocyte telomere length in later life. The original study population (N=4136) consists of 2424 male former elite athletes and 1712 matched controls. Of those, 599 (392 former athletes, 207 controls) participated in a clinical study in 2008. The athletes were divided into three groups based upon their previous career: endurance, mixed and power sports. The clinical study in 2008 included a physical examination, laboratory tests, and several questionnaires. Data on use of medication was obtained from the Finnish Social Insurance Institution. Among the participants, the former elite athletes tend to have lower age-adjusted prevalence of type 2 diabetes compared with the controls (odds ratio [OR] 0.68, 95% confidence interval [CI] 0.45-1.01). The male former athletes also had lower age-adjusted risk for hypertension (OR 0.69, 95% CI 0.49-0.98), metabolic syndrome (OR 0.57, 95% CI 0.40-0.81), and non-alcoholic fatty liver disease (OR 0.61, 95% CI 0.42-0.88) compared to the controls. The former athletes had significantly lower age-adjusted body fat percentage compared to the controls (p=0.021) whereas no significant differences in mean age-adjusted leukocyte telomere lenght between the athlete and control groups (p = 0.845) were observed. Moreover, with aging the former athletes maintained their physically active lifestyle better than their controls. A male former elite athlete career seems to protect from type 2 diabetes, hypertension, metabolic syndrome, and non-alcoholic fatty liver disease at older age. It was also associated with a more favorable body composition. The volume of current leisure time physical activity was inversely associated with these cardiometabolic outcomes.
  • Kupari, Jussi (Helsingin yliopisto, 2015)
    The Glial cell line-derived neurotrophic factor (GDNF) family ligands, which include GDNF, neurturin (NRTN), persephin (PSPN) and artemin (ARTN), signal through a glycosyl phosphatidyl inositol (GPI)-linked cognate-receptor (GFRα1-4) and the transmembrane receptor tyrosine kinase receptor RET. The members of the GDNF family play a particularly important role in the development of the peripheral nervous system (PNS). In the autonomic nervous system, GDNF and NRTN regulate important steps in the development of the enteric and parasympathetic nervous systems from migration and proliferation to soma size and target innervation, whereas ARTN takes part in the early phases of sympathetic nervous system development. In the sensory system, GFRα2 ¬ the co-receptor of NRTN has been shown to mediate trophic signaling for nonpeptidergic nociceptive neurons, and is also required for their innervation of the glabrous epidermis. However, several aspects of the role of GFRα2-signaling in normal PNS development and function remain poorly understood. Therefore, the aims of this study were to elucidate (1) the role of GFRα2-signaling in the development of parasympathetic neurons; (2) the role of GFRα2-signaling in two classes of somatosensory mechanoreceptor neurons and their target innervation; and (3) the role of GFRα2-signaling in the cholinergic innervation of the gastric mucosa and the role of this innervation in gastric secretion. We discovered that programmed cell death (PCD) is a normal part of parasympathetic neuron development in mice. GFRα2-signaling was found to regulate parasympathetic neuron survival in pancreatic and submandibular ganglia during late embryonic development; lack of GFRα2-mediated signaling resulted in the loss of intrapancreatic neurons through PCD. In argreement with previous studies, apoptosis in the ENS was found to be rare, and was not increased in the absence of GFRα2, implying that the normal number of enteric neurons is not determined by PCD. In the dorsal root ganglia (DRGs), we found that GFRα2 regulates the cell size, but not the peripheral innervation of hair follicles in both the large early-RET RA Aβ-class low threshold mechanoreceptors (LTMRs) and in the small C-LTMRs. In contrast, GFRα2 was found to regulate both the cell size and the epidermal innervation in the small Mrgprd+ C-nociceptors. We also found evidence that the RA Aβ-LTMRs downregulate GFRα2-expression at some point after birth, suggesting a possible switch in neurotrophic signaling pathways. In the enteric nervous system, we demonstrated that GFRα2-signaling via NRTN is required for cholinergic innervation of the gastric mucosa. Interestingly, this innervation was found to be unnecessary for maintaining the gastric mucosa and for gastrin secretion and basal acid secretion. Even though vagally-stimulated secretion is lost in the GFRα2-KO mice, their ability to secrete acid in response to direct parietal cell stimulation remains in the absence of gastric mucosal innervation.
  • Marinkovic, Ivan (Helsingin yliopisto, 2015)
    Intracerebral hemorrhage (ICH) represents the subtype of stroke carrying the most detrimental outcome and highest mortality. It is clearly underinvestigated and lacks an effective treatment except supportive management measures. The dismal outcomes of ICH are clearly associated with the mass effect of hematoma accompanied with dynamic perihematomal edema (PHE) and potential intraventricular bleeding (IVH), which is often complicated by development of hydrocephalus. In this thesis, we aimed to develop novel therapy modalities, which apart from experimental conditions, might be found effective in clinical scenario, and successfully implemented in ICH treatment clinical protocols in the future. In study (I), we utilized autologous blood injection model and magnetic resonance imaging (MRI) follow-up. Following the induction of deep right hemispheric hematoma of 75uL of volume, we performed decompressive craniectomy (DC) at 1 h, 6 h, and 24 h time points, aiming at diminishing high intracranial pressure (ICP) after the bleeding event. Control group did not undergo DC procedure. Decompressive craniectomy was found effective in reducing mortality and improving overall neurobehavioral outcome and mortality in all the 3 treatment groups. Effect of DC was most pronounced in the early craniectomy group (1 h). In study (II), we aimed to examine how deleterious is the influence of tissue plasminogen activator (tPA) in experimental ICH scenario, and whether the adverse tPA effects could be antagonized by the clinically safe mast-cell stabilizer chromoglycate. Animals were divided in four groups. We utilized intrahemispheric collagenase injection rat ICH model and MRI follow-up imaging up to 72h. Group 1 received tPA only, whereas Group 2 and Group 3 besides tPA received chromoglycate in single or double dose. Group 4 was used as a control having ICH, but receiving saline only. TPA-treated animals did not have significantly larger hematomas or hemispheric expansion even though been characterized by worse overall outcome. Application of chromoglycate in high dose mitigated detrimental outcomes present in the group being treated with tPA only, and diminished the extent of hemispheric expansion. This encourages further research to potentially establish "blind" thrombolysis concept coupled with concomitant application of MC inhibitors, as we could show that injurious tPA effects were successfully abolished by concomitant MC stabilization in experimental ICH scenario. In the third study (III), we decided to develop a novel combined ICH + intraventricular hemorrhage (IVH) model in the rat as such a model does not exist besides the fact that approximately 40% of all ICHs in human patients are complicated by presence of intraventricular hemorrhage which is known to worsen prognosis. We utilized different blood volumes of 100-250uL in modified autologous blood injection model to induce deep intrahemispheric ICH to establish a fully novel experimental approach of ICH followed by IVH. We performed 1-week MRI follow-up and consistent neurobehavioral evaluation at 24 h, 48 h, 72 h, and 1 week after hemorrhage. Using MRI, we identified the presence of hydrocephalus in all experimental subjects, and its progressive tendency during entire follow-up. The most prominent hemispheric expansion and the most pronounced hydrocephalus were present in experimental groups with >200uL blood injection. This model was reliable and highly reproducible, demonstrating promising initial point for ICH+IVH experimental research. These two novel treatment strategies established in our studies give promise for further refinements and potential applications in human patients. The novel ICH+IVH model we established in rat models may serve as an improved model for future experimental studies as it mimics human disease scenarios better than the existing models.
  • Sandboge, Samuel (Helsingin yliopisto, 2015)
    Background. A small birth size, an indicator of a suboptimal intrauterine environment, is a risk factor for several non-communicable diseases (NCDs), a risk that in many cases is modified by childhood growth patterns. Regional variation in NCD prevalence could partly have its origin in early development. Lifestyle factors further influence NCD prevalence. Aims. We aimed to explore the associations between early growth and adult resting metabolic rate (RMR), body composition, non-alcoholic fatty liver disease (NAFLD), and hypertension. We also studied the associations between fructose intake and NAFLD, and differences in birth size between Helsinki and the Åland Islands. Subjects and methods. The Helsinki Birth Cohort Study consists of 13345 individuals born in Helsinki in 1934‒44. Detailed records are available for all participants including information on maternal and birth characteristics and measurements of childhood body size. 2003 individuals participated in a clinical study in 2001‒04 and 1083 of these additionally participated in a follow-up study in 2006‒08. The Åland records include 1697 births for the years 1937‒44. Results. The association between birth weight and RMR was inverse among women and quadratic among men. A higher attained adult weight than expected, based on weight and height measurements before age 11 years and adult height, was associated with higher adult body fat content. The odds ratio (OR) for NAFLD was 18.5 (95% CI 10.1; 33.6) among those who belonged to the lowest BMI tertile at age 2 years and subsequently were obese as adults, compared to those who were still lean or normal weight as adults. NAFLD was most common among individuals with the lowest dietary fructose intake. Systolic blood pressure (SBP) and the presence of hypertension were inversely associated with linear (height) growth between ages 2 and 11 years. Relative weight gain after age 11 years was positively associated with SBP. Ålandic babies born 1937‒44 were 87 grams (95% CI 61; 111) heavier and 0.4 cm (95% CI 0.3; 0.5) longer than their Helsinki peers. Conclusions. A more pronounced increase in relative weight after age 11 years than would be expected from previous body size, was positively associated with body fat content, NAFLD, and hypertension. Conversely, several growth measurements before age 11 years were negatively associated with the outcomes studied. None of the studied individuals were obese in childhood. Instead, a larger relative childhood body size in this group most likely represents a more beneficial childhood environment. Contrary to previous findings, we found that individuals with the highest fructose intake were least likely to suffer from NAFLD. We found a small but significant difference in birth size between the Åland Islands and Helsinki for the years 1937‒44.
  • Lindahl, Jan (Helsingin yliopisto, 2015)
    Jan Lindahl: MANAGEMENT OF PELVIC RING INJURIES Unstable pelvic ring injuries are relatively rare injuries, but they constitute a major cause of death and disability in high-energy polytrauma patients Massive hemorrhage is the leading cause of potentially preventable death following a blunt pelvic trauma. The overall aim of surgical treatment for unstable pelvic ring injuries is to restore the pelvic anatomy and perform neural decompression, thus allowing normal function with a low rate of complications. This doctoral thesis was initiated to investigate the outcomes of acute and definitive management strategies for unstable pelvic ring injuries. The first study investigated the radiological and functional results of treating type B and C pelvic injuries with an anterior external fixation frame. The second study focused on identifying factors for early predictions of mortality-related outcome and prognosis in patients with pelvic fracture-related arterial bleeding that were treated with transcatheter angiographic embolization (TAE). The third study investigated the outcomes of type C pelvic fractures treated with standardized reduction and internal fixation methods. The fourth study evaluated outcomes and identified prognostic factors for operatively-treated, H-shaped sacral fractures with spinopelvic dissociation. Study I showed that an anterior external fixator failed to achieve and properly maintain reduction in 75% of type B open book injuries and in nearly all (95%) type C pelvic ring injuries. Therefore, an external frame is not a suitable method of treatment for the most unstable pelvic ring injuries as a definitive treatment. The current clinical applications of anterior pelvic external fixators comprise the resuscitation phase, initial fracture stabilization phase, and sometimes, in complex injuries (type C), the definitive phase for fixation of the anterior part of the pelvic ring, in conjunction with posterior internal fixation. Study II of pelvic fracture related arterial bleedings showed that the worst prognosis was related to exsanguinating bleeding from the main trunk of the internal or external iliac artery (large pelvic arteries) or from multiple branches of the internal or external iliac vasculature (high vessel size score). Definitive control of arterial bleeding was achieved with TAE in all patients. In massive hemorrhage with several bleeding arteries uni- or bilaterally, it is reasonable to use non-selective embolization by promptly occluding the main trunk of the internal iliac artery, either uni- or bilaterally. Study III of operatively treated type C pelvic fractures revealed that, internal fixation of injuries in the posterior and anterior pelvic ring provided excellent or good radiological results in 90% of cases. Additionally, because a reduction with displacement less than or equal to 5 mm was more often associated with a good functional outcome, that should be the goal of operative management. However, the prognosis is also often dependent on associated injuries, particularly a permanent lumbosacral plexus injury. The results favoured internal fixation of all the injured elements of the pelvis for improved stability and a more accurate anatomical result in the entire pelvic ring. The H-shaped sacral fracture with spinopelvic dissociation is a rare injury pattern. Study IV revealed that lumbopelvic fixation was a reliable treatment method. The study also showed that neurological recovery and clinical outcome were associated with the degree of initial translational displacement of the transverse sacral fracture component. Permanent neurological deficits were more frequent and the clinical outcome was worst in completely displaced transverse sacral fractures. An accurate operative reduction of all sacral fracture components was associated with better neurological recovery and clinical outcome. We conclude, that with appropriate treatment of unstable pelvic ring injuries, and associated injuries in other organs, it is possible to achieve better survival rates and functional results, and to reduce long-term disability.
  • Pekkonen, Pirita (Helsingin yliopisto, 2015)
    Human tumorigenesis is a process in which a normal cell needs to acquire multiple characteristics to become malignant and metastatic. In short, these so called cancer hallmarks include increased proliferation and cell survival, as well as the ability to invade into the surroundings, induce angiogenesis, and finally metastasize to distant sites. These traits are regulated in a variety of different ways. However, some embryonic signaling pathways, including the Notch pathway, are able to regulate many of these processes. Furthermore, it has been shown that these signaling pathways can be deregulated in cancer, and that their untimely activation can lead to malignancies. In this study, Kaposi's sarcoma herpesvirus (KSHV) associated malignancies, namely Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), as well as melanoma have been used as model cancers. In all these malignancies, the tumor cells show alterations in cell identity and lineage marker expression, i.e. signs of cellular de- or transdifferentiation. In addition, the Notch pathway has been shown to be overly active in all of them. Thus, this thesis has focused on how the pro-tumorigenic traits are affected by cell plasticity and reprogramming in these cancers, and how the signaling pathways leading to these phenotypes, most notably Notch, are in turn regulated. Firstly, the results show that in vivo expression of a KSHV oncogene, viral (v-)cyclin, leads to activation of Notch signaling through Notch3 upregulation as well as fine-tuning of the NF-κB pathway through Cdk6 mediated phosphorylation. These changes in turn lead to defects in T-lymphocyte differentiation and immune functions, as well as to the development of T-cell lymphomas. Secondly, this work demonstrates that KSHV infection in primary lymphatic endothelial cells (LECs) in three dimensional (3D) cell culture model leads to activation of a morphogenic process, endothelial to mesenchymal transition (EndMT), and increased invasiveness through activation of the Notch pathway and matrix metalloproteinase MT1-MMP. Lastly, the data show that the changes in cell plasticity contributing to tumorigenic traits are not confined to virally induced cancers. Melanoma cell interaction with LECs leads to activation of the Notch pathway and increased adhesive, invasive, and metastatic properties of the tumor cells. In conclusion, the results show that regulation of cell plasticity through the Notch pathway takes place in different types of cancers, and it can affect several steps of tumorigenesis. A thorough and comprehensive understanding of the processes discovered herein may help develop better and more efficient treatments for these largely fatal malignancies.
  • Bourbia, Nora (Helsingin yliopisto, 2015)
    The central nucleus of amygdala (CeA) is known to be involved in pain and nociception, but the mechanisms or its role in descending control of pain-related behavior is poorly understood. The aim of this study was to investigate the involvement of the neuropeptide corticotropin-releasing factor (CRF) and the glutamatergic system of the CeA in pain and nociception in healthy control animals and in an animal model of chronic neuropathic pain induced by spared-nerve injury (SNI). Two aspects of pain were studied: emotional-like pain behavior was assessed by using the aversive place-avoidance paradigm and sensory-discriminative was assessed by determining the mechanical limb-withdrawal threshold and the thermal (heat) limb-withdrawal latency. Moreover, the aims were to determine whether medullospinal serotoninergic pathways and the midbrain periaqueductal grey (PAG), respectively, were involved in relaying pain-modulation induced by the CeA in SNI and healthy control animals. Additionally, hemisphere of the CeA and submodality of pain stimulus were among studied parameters. Surgical procedures and electrophysiological recordings were performed under general anesthesia. The studies on the role of the CeA in the emotional-like aspect of pain in SNI rats revealed that activation and blocking of the group I metabotropic glutamate receptors (mGluRs) facilitates and inhibits, respectively, the aversive aspect of pain. Furthermore, increase of endogenous CRF as well as blocking glutamatergic N-methyl-D-aspartate (NMDA) receptors in the CeA reduced the aversive aspect of neuropathic pain. The studies on the sensory-discriminative aspect of pain revealed that an increase of endogenous CRF in the CeA is pronociceptive in both control and SNI rats. CeA injection of a high dose of glutamate had a mechanical antinociceptive effect that was mediated by NMDA receptors in healthy but not SNI rats. A low dose of glutamate had a pronociceptive effect mediated by NMDA receptors in SNI rats. Furthermore, tonic descending pronociception induced by NMDA receptors and the mGluR1 in the CeA contributes to the maintenance of neuropathic hypersensitivity. The investigation on the role of serotonergic neurons of the rostroventromedial medulla (RVM) in modulation of spinal nociception by amygdaloid glutamate in SNI rats indicated that the RVM is a relay for both descending pro- and antinociceptive effects from the CeA. The investigation on the role of the PAG in the descending control of nociception induced by glutamate in the CeA of healthy rats indicated that the PAG is a relay in the descending control of nociception induced by amygdaloid glutamate. Furthermore, the right-hemispheric lateralization of the pronociceptive effect by amygdaloid CRF in controls was lost in SNI rats. However, descending antinociception induced by the glutamatergic system of the CeA showed no hemispheric lateralization in healthy controls; a high dose of glutamate in both the left and right CeA induced equal attenuations of mechanical and thermal nociception, which effects were, respectively, NMDA-dependent and NDMA-independent.
  • Parnov Reichhardt, Martin (Helsingin yliopisto, 2015)
    To live a healthy life, humans need to co-exist with foreign organisms. These consist of the thousands of different types of microbes that colonize the human body. But also, in the case of a pregnant woman, the fetus can be viewed as a foreign organism. To avoid disease, the barriers of the human body, e.g. the mucosal surfaces, must be maintained. Here the innate immune defense system plays an important role. The salivary scavenger and agglutinin (SALSA), also known as gp340, DMBT1 and SAG, is a molecule found at most mucosal surfaces. SALSA is associated with the epithelium or secreted into the lining fluids, such as tears, saliva and mucus in the respiratory tract. SALSA is known to bind and agglutinate a broad spectrum of bacteria, as well as viruses, and thus play a role in the innate immune defense against invading microbes. The effect of SALSA is mediated in concert with several other defense molecules such as IgA, surfactant proteins A and D, and the complement component C1q. These have all been shown to be ligands of SALSA. Alongside the role of SALSA in innate immunity, evidence for a function in epithelial and stem cell differentiation has emerged. This thesis work has addressed the function of SALSA in innate immunity, especially in early life. SALSA was found in the amniotic fluid and in meconium and feces of newborns. In fact, SALSA was among the most abundant proteins in the intestines of newborn children. By comparing the SALSA protein in the different samples we found size polymorphisms, varying from one individual to another, but also from compartment to compartment within the same individual. These differences were found to alter the ability of SALSA to bind known endogenous and bacterial ligands. SALSA was also found to be expressed in the human placental and decidual tissues. In the 1st trimester of pregnancy, SALSA was detected sporadically in maternal decidual capillaries. Closer to term SALSA was found to be expressed by the syncytiotrophoblast layer of the placental villous trees. In certain sites, e.g. at disrupted and damaged areas of the syncytium, SALSA was found deposited into fibrinoid formations. It partially co-localized with the fibrinoid component fibronectin. Complement activation has been observed at the feto-maternal interface of both healthy and complicated pregnancies. SALSA had previously been found to bind C1q. Thus, it was of interest to investigate the ability of SALSA to interact directly with the complement system. We found that SALSA bound to both mannan-binding lectin and to some extent to all three ficolins (H, L and M). SALSA activated complement, when it was bound to a surface. In contrast, fluid-phase SALSA was able to inhibit the deposition of complement on SALSA non-binding microbial surfaces. It thus acted in dual fashion to target complement attack. In the human placenta we observed C1q-targeting of the SALSA-positive fibrinoid formations. C1q and complement are known to function in the clearance of apoptotic cells and debris. Thereby SALSA and complement probably have a cooperative function in the containment and clearance of the injured structures, thus linking its innate immune activity with the maintenance of tissue homeostasis.
  • Kanduri, Chakravarthi (Helsingin yliopisto, 2015)
    Music perception and performance form a useful tool for studying the normal functioning of the human brain. The abundance of neuroscientific literature has demonstrated that music perception and performance alter the human brain structure and function and induce physiological changes through neurochemical modulation. Emerging evidence from molecular genetic studies have suggested a substantial genetic component in musical aptitude and related traits like creativity in music. This thesis puts a step forward in understanding the molecular genetic background of music perception and performance, using a combination of genomics and bioinformatics approaches. Specifically, the role of copy number variations (CNVs; a form of genetic variation) in musical aptitude and creativity in music was investigated both in the largest families of the MUSGEN-project and also in sporadic cases. The effects of listening to music and performing music by playing an instrument on human transcriptional responses were also investigated. The genome-wide CNV analysis principally identified genes like GALM, PCDHA1-9 as the possible candidate genes that could affect musical aptitude and creativity in music. PCDHA1-9 and GALM genes are known to regulate the serotonergic system, which is responsible for neurocognitive and motor functions, the essential biological processes of music related traits. Overall, the detected genes affect neurodevelopment, learning, memory and serotonergic functions. The findings also demonstrated that large and rare CNV burden does not affect normal traits like musical aptitude. Both listening to music and performing music enhanced the activity of genes that are known to be involved in dopaminergic neurotransmission, neuroplasticity, learning, and memory. Most importantly, one of the most up-regulated genes in both studies - synuclein alpha (SNCA) and its upstream transcription regulator GATA2, are located on chromosomal regions 4q22.1 and 3q21 respectively linking the strongest linkage and associated regions of musical aptitude together. In addition, several of the up-regulated genes in both the studies (like SNCA, FOS, and DUSP1) have been known to be regulated during song learning and singing in songbirds, suggesting a possible evolutionary conservation of genes related to sound perception and production. These novel findings give preliminary information about the genes associated with musical aptitude and the effect of music on the human body. It is obvious that replication studies are required to confirm the results. These pioneering findings could guide further research on the molecular genetics of music perception and performance in humans. These findings will also enhance our understanding of the genetic bases of cognitive traits, the evolution of music and music therapy.
  • Haapaniemi, Emma (Helsingin yliopisto, 2015)
    Primary immunodeficiency diseases (PIDD) compromise a heterogeneous group of clinical entities, ranging from isolated susceptibility for certain pathogen to widespread, early-onset infections or overwhelming autoimmunity. Although rare, PIDDs cause significant morbidity and mortality. Over 300 genes are associated with PIDD development, and multiple gene defects can cause a similar phenotype. On the other hand, phenotypes caused by the same mutation can vary considerably even between family members. Therefore, genetic diagnostics in PIDD is challenging with standard candidate gene approach. In this study, three novel primary immunodeficiency conditions are characterized: early-onset multiorgan autoimmune disease caused by STAT3 hyperactivity, combined immunodeficiency as due to DOCK2 deficiency, and generalized infection susceptibility caused by recessive HYOU1 mutations. The mutations were discovered using exome sequencing. In the first and second part of this study, the authors studied a cohort of six patients with missense STAT3 mutations that were shown to be activating by luciferase reporter assay. The patients presented with multi-organ autoimmunity that commonly started in infancy. Additionally, some had lymphoproliferation and developed hypogammaglobulinemia in their teens, and one patient had delayed-onset mycobacterial disease. Three patients were immunologically characterized. They showed peripheral eosinopenia and deficiency of regulatory T cells, T helper 17 cells, NK cells, and dendritic cells. Notably, one patient developed large granular lymphocyte (LGL) leukemia at age 14 and another had LGL cells in her bone marrow without clinical disease. In third part of the study, compound heterozygous DOCK2 mutations were identified in a patient that showed mild hematological autoimmunity and susceptibility to bacterial and viral infections. Four additional patients with a similar phenotype and homozygous DOCK2 mutations were subsequently independently identified in four additional research groups in US, Austria, and France. All patients showed profound lymphopenia with defective B, T and NK cell responses, and invasive viral and bacterial infections that were fatal without stem cell transplantation. Finally, a patient with anemia and combined deficiency of granulocytes, B cells and dendritic cells was studied. She presented with severe early-onset bacterial and herpetic infections and stressinduced hypoglycemic episodes. She was found to harbor compound heterozygous mutations in HYOU1, an endoplasmic reticulum chaperone, and the mutations altered HYOU1 substrate binding specificity. The defective HYOU1 function compromised the redox balance in patient neutrophils and skin fibroblasts, and led to altered endoplasmic reticulum stress response. This study shows that next generation sequencing tools such as exome sequencing are powerful in PIDD diagnostics. With these techniques it is possible to identify causal variants even in situations where only a single individual is affected. The results show that monogenic immunological conditions have considerable phenotypic variation even between patients with similar gene defects. The study also identifies novel genes with previously undescribed roles in human immunity, and broadens the general understanding of human immunobiology.
  • Kauppi, Juha (Helsingin yliopisto, 2015)
    Adenocarcinoma (AC) of the esophagus and esophagogastric junction (EGJ) is a disease with poor prognosis and increasing incidence in western countries. Its pathogenesis is associated with oxidative stress (OS) in the esophageal epithelium. Long-term survival is associated with successful radical surgery, early stage of the disease, and successful downstaging with neoadjuvant therapy. As surgery is accompanied with a large number of possible complications, it is essential to identify patients who might not benefit from surgery and on the other hand, could be treated with less invasive options. The aims of this study were 1) to assess the role of OS below EGJ in the pathogenesis of Barrett s esophagus (BE) and AC, 2) to assess the prognoses and causes of death in early esophageal AC, 3) to determine the value of 18F-fluorodeoxy-D-glucose positron emission tomography with computed tomography (FDG-PET-CT) in quantifying the response to neoadjuvant therapy and 4) to compare the novel mini-invasive technique (MIE) to traditional open esophagectomy (OE) in radical surgery. To quantify OS, we measured 8-isoprostane (8-IP), glutathione content (GSH), and 8-OH- deoxyglucose (8-OHdG) values from mucosa below EGJ, BE-mucosa, and AC-tumors from 43 patients with BE and/or AC and compared them to samples from corresponding sites of 15 healthy control patients. To determine the long-term prognosis of patients with early esophageal AC, we studied patient records and causes of deaths for 85 patients, treated with radical esophagectomy over a 27-year time span. To evaluate pre-treatment response to neoadjuvant therapy in locally advanced AC, we recorded FDG-PET-CT results before and after induction therapy in sixty-six consecutive patients who were to be operated on for locally advanced AC. Decrement in radioactive glucose uptake values was associated with survival and histological treatment response. We compared MIE to OE, to see, if minimal invasiveness reduces the rate of complications and if they are comparable in terms of oncologic radicality and survival. Proximal gastric GSH content was lower and 8-IP and 8-OHdG levels higher with statistical significance in the study patients (BE and AC) as compared to healthy controls. In patients with early esophageal AC, overall and long-term (>5 year) survival rates were mostly affected by diseases related to aging. During the first five years after the operations, disease recurrence was the most common cause of death. However, recurrence-free survival was 80% at five years and no new recurrences were detected after that. Microscopic eradication of locally advanced AC was optimally predicted by a 67% decrease in uptake values before and after induction chemotherapy, with a sensitivity of 79% and specificity of 75%. However, this association was not linear and complete eradication of radioactive glucose uptake, was not always associated with a complete histologic response. However, a decrease in glucose uptake was associated with improved overall and recurrence free survival. MIE and OE were equivalent in terms of 90-day mortality, pneumonia-, leak-, and overall complication rates. Also a minimally invasive technique was associated with significantly shorter overall hospital stay and significantly less blood loss during the operations. OS levels are also elevated below the EGJ, as lipid peroxidation can be detected (8-IP) and antioxidant defense (GSH) is reduced. Also the levels of 8-OHdG adducts were higher showing that DNA is being damaged by free radicals. This suggests that inflammation of the proximal gastric mucosa induced by gastroduodenal content, has a role in the pathogenesis of BE and esophageal AC. As the prognoses for patients with early esophageal AC were good, recurrence was still the most important cause of death. The risk was highest for patients with lymph node metastases and deep submucosal infiltration. Therefore radical surgery should be preferred with patients with a low risk of surgical complications and submucosal infiltration. In patients with intramucosal AC, endoscopic ablation should be considered. Evaluation of the patients responses to induction chemotherapy with FDG-PET-CT was not accurate enough to give indications better than the exclusion of metastatic disease. However, a significant decrease in radioactive glucose uptake was associated with improved survival, independently of histopathologic response. This information can be useful when balancing the risks of surgery against expected benefits. The perioperative and oncological results for MIE were comparable to those of the open approach and MIE seems to shorten hospital stay. However, the MIE technique is demanding and its mastery requires a sufficient number of cases and skilled practitioners.
  • Al-Samadi, Ahmed (Helsingin yliopisto, 2015)
    Recurrent aphthous ulcer (RAU) is an ulcerative disease of the oral mucosa characterised by the appearance of ulcerations in the oral mucosa accompanied by an erythematous halo area surrounding the ulcer and showing signs of acute inflammation. While RAU affects approximately 20% of the population globally, its pathogenesis remains poorly understood. Furthermore, most studies concentrate on treatment while few address the pathogenesis of the disease. This project aimed to determine the mechanisms of oral epithelial cell death in RAU, the role of these cells in disease pathogenesis in terms of toll-like receptor (TLR) expression, and the ability of the these cells to produce chemokines, pro-inflammatory cytokines, and antimicrobial peptides. Together these may first aggravate and, then, down-regulate the inflammation and initiate the healing process. For this purpose, we collected 13 aphthae and 11 healthy control biopsies for immunohistochemical staining, immunofluorescence staining, and quantitative PCR. For functional studies, we cultured primary oral keratinocytes and oral squamous cell carcinoma cell-line SCC-25 and tested their responses to different stimuli. Our results highlight the importance of oral epithelial cells in RAU; interestingly, oral epithelial cells in RAU tested positive for apoptosis markers caspase-3, especially at the superficial and spinous layer, and TUNEL, but negative in controls. We also found that TLRs are primarily present in the basal and suprabasal layers of control epithelium, but their expression extends to the superficial layer in RAU epithelium. Additionally, we found significally higher expressions of tumour necrosis factor-α (TNF-α), interleukin-8 (IL-8), IL-17C, and beta defensin 2 (BD-2) in RAU oral epithelium compared with control epithelium. Functional studies on cultured primary oral keratinocytes and SCC-25 supported our results from RAU biopsies since these cells responded to damage-associated molecule patterns (DAMPs), such as self-DNA and pro-inflammatory cytokines including IL-17C, TNF-α, and interferon gamma (IFN-γ), through a significant increase in the expression of selected molecules including TLR2, TNF-α, and BD-2. Based on our findings, RAU may begin with a strong initiating factor activating a self-amplificatory cycle. This cycle is characterised by the induction of epithelial cells apoptosis at the superficial layer down to the basal layer, a change in the pattern of TLR distribution, the up-regulation of several chemokines and pro-inflammatory cytokines, and, finally, the secretion of antimicrobial peptides initating the healing process. As a result of the lack of adaptive immunity in RAU, the cycle recurs when the mucosa is subjected to an initiating factor of the same sequence.
  • Mäkinen, Laura K. (Helsingin yliopisto, 2015)
    Predicting the clinical course of an early-stage oral tongue squamous cell carcinoma (OTSCC) is challenging, as even small tumors can behave aggressively. OTSCC often metastasizes to the cervical lymph nodes, and the presence of lymph node metastasis at the time of diagnosis is considered the most important tumor-related prognostic factor in OTSCC. The mechanisms of this disease progression are poorly understood. Despite slight improvement in the prognosis of OTSCC in recent decades, the outcome of these patients is still modest. Therefore, a deeper understanding of the phenomena behind tumor progression would enable medical professionals to evaluate the aggressiveness of the disease and to adjust its treatment more effectively. The extracellular matrix and basement membrane must be broken down before a tumor can invade surrounding tissues and further spread into blood and lymph vessels. This is a process that involves various proteolytic enzymes, the most important of which are matrix metalloproteinases (MMPs). Over 25 structurally related, but genetically distinct, human MMPs have been identified and characterized: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other MMPs. Toll-like receptors (TLRs) are pattern-recognition molecules involved in innate immunity that are also expressed in many types of cancer. TLRs apparently play a pivotal role in malignant disease: they are related to tumor progression and, conversely, to cancer inhibition. Their expression pattern and role in oral cancer, however, remains unclear. In this thesis we studied the expression of MMPs 2, 7, 8, 9, 13, and 25 and TLRs 2, 4, 5, 7, and 9 in early-stage OTSCC. The study comprised 73 consecutive clinically T1N0M0 and T2N0M0 OTSCC patients treated at the Helsinki University Hospital, Helsinki, Finland in 1992-2002. We prepared tissue array blocks from primary tumors and immunostained them. We also used whole section slides from patients with metastasized or recurrent disease. We compared immunoexpression of MMPs and TLRs to tumor and patient characteristics as well as to patient outcomes. We also used Western immunoblot to examine TLR-2 and -4 expression in the highly invasive and aggressive HSC-3 OTSCC cell line. In addition, we studied the effect of TLR-2 and -4 antagonist GIT27 (4,5-dihydro-5-isoxasoleacetic acid) on the invasion of the HSC-3 cell line in myoma organotypic invasion assay. OTSCC tumors expressed both MMPs and TLRs. Nuclear expression of MMP-13, but not cytoplasmic expression of MMP-2, -8, and -9, associated with deeper invasion and advanced tumor size. Furthermore, high nuclear MMP-13 expression predicted poor disease-specific survival. High MMP-7 protein expression associated with the presence of occult cervical metastases, increased invasion depth, and higher tumor grade, and also predicted poor outcome. Immunostaining of MMP-25 failed to correlate with any clinical parameters. High TLR-2, -4, and -9 expression correlated with deeper tumor invasion. Cytoplasmic expression of TLR-2 and -4 also correlated significantly with higher tumor grade, whereas high TLR-5 expression associated with lower tumor grade. High expression of TLR-9 correlated with advanced tumor size. Negative or mild TLR-5 expression predicted poor disease-specific survival. OTSCC primary tumors, neck metastases and recurrent tumors expressed TLR-2, -4, and -9. TLR-2 and -4 antagonist GIT27 did not affect the invasion of the HSC-3 cell line in myoma organotypic invasion assay. Thus, TLRs may operate under a different mechanism of action depending on whether they are activated by damage-associated molecular patterns in cancer or by pathogen-associated molecular patterns in infection. Our results suggest that MMP-7 and MMP-13 in particular may have prognostic value in OTSCC. Their use as prognostic biomarkers, however, calls for further study. TLR-2, -4, and -9 seemed to predict invasive tumor growth. Primary tumors and neck metastases as well as recurrent tumors of OTSCC express these TLRs, suggesting that TLRs seem to play a role in both the development and progression of tongue carcinoma.
  • Veistinen, Lotta (Helsingin yliopisto, 2015)
    The flat bones of the skull, the calvarial bones, develop by intramembranous ossification during which mesenchymal cells first condense and subsequently differentiate into osteoblasts. Sutures separate the calvarial bones and facilitate the synchronized growth of the underlying brain and the calvaria. Hedgehog (Hh) signalling has an indisputable role in craniofacial development as well as during endochondral ossification. Yet, little is known about its function during intramembranous ossification of the calvarial bones. GLI-Kruppel family member 3 (Gli3) is a zinc-finger transcription factor that mediates Hh signalling. In the absence of Hh ligand Gli3 is proteolytically cleaved into a repressor that inhibits transcription of Hh target genes. Mutations in GLI3 cause Greig cephalopolysyndactyly syndrome in humans, in which an infrequent, but significant feature is premature fusion of the metopic suture (interfrontal suture in mice). We have used Gli3 loss-of- function mouse (Gli3Xt-J/Xt-J) as a model to investigate the effects of aberrant Hh signalling during calvarial development. In my thesis I describe how loss of Gli3 causes craniosynostosis of the lambdoid as well as interfrontal sutures in mice. Elevated proliferation and ectopic differentiation of osteoprogenitors underlies this phenomenon. We were able to rescue craniosynostosis in these mice by two mechanisms. Firstly, by elevating fibroblast growth factor (Fgf) signalling in the suture prior to its fusion by imbedding Fgf2 soaked beads in tissue culture. This induced Twist1 expression, which inhibits function of ectopically expressed Runx2. Secondly, craniosynostosis was prevented by genetically reducing Runx2 activity by generating Gli3Xt-J/Xt-J;Runx2+/- mice, which normalized elevated levels of Bmp signalling in the affected sutures. We also put forward a model of how Hh signalling helps to maintain the integrity of bone margins during calvarial development. The repressor isoform of Gli3 inhibits Runx2 activity in the early osteoprogenitor cells. Runx2, on the other hand, activates Ihh expression in the mature osteoblasts, which then induces osteogenesis by inhibiting the function of Gli3 repressor. Our findings indicate that Gli3 and Hh signalling have an important role in mediating the location of osteoblast differentiation and the speed of bone formation in the developing calvaria. Uncovering the cellular and molecular mechanisms that underlie normal calvarial development, as well as pathological processes, is a vital step in developing treatment strategies for patients with craniosynostosis.