Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Fumarate Hydratase and Succinate Dehydrogenase in Neoplasia

Germline mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell cancer (HLRCC). FH is a nuclear encoded enzyme which functions in the Krebs tricarboxylic acid cycle, and homozygous mutation in FH lead to severe developmental defects. Both uterine and cutaneous leiomyomas are components of the HLRCC phenotype. Most of these tumours show loss of the wild-type allele and, also, the mutations reduce FH enzyme activity, which indicate that FH is a tumour suppressor gene. The renal cell cancers associated with HLRCC are of rare papillary type 2 histology. Other genes involved in the Krebs cycle, which are also implicated in neoplasia are 3 of the 4 subunits encoding succinate dehydrogenase (SDH); mutations in SHDB, SDHC, and SDHD predispose to paraganglioma and phaeochromocytoma. Although uterine leiomyomas (or fibroids) are very common, the estimations of affected women ranging from 25% to 77%, not much is known about their genetic background. Cytogenetic studies have revealed that rearrangements involving chromosomes 6, 7, 12 and 14 are most commonly seen in fibroids. Deletions on the long arm of chromosome 7 have been reported to be involved in about 17 to 34 % of leiomyomas and the small commonly deleted region on 7q22 suggests that there might be an underlying tumour suppressor gene in that region. The purpose of this study was to investigate the genetic mechanisms behind the development of tumours associated with HLRCC, both renal cell cancer and uterine fibroids. Firstly, a database search at the Finnish cancer registry was conducted in order to identify new families with early-onset RCC and to test if the family history was compatible with HLRCC. Secondly, sporadic uterine fibroids were tested for deletions on 7q in order to define the minimal deleted 7q-region, followed by mutation analysis of the candidate genes. Thirdly, oligonucleotide chips were utilised to study the global gene expression profiles of uterine fibroids in order to test whether 7q-deletions and FH mutations significantly affected fibroid biology. In the screen for early-onset RCC, 214 families were identified. Subsequently, the pedigrees were constructed and clinical data obtained. One of the index cases (RCC at the age of 28) had a mother who had been diagnosed with a heart tumour, which in further investigation turned out to be a paraganglioma. This lead to an alternative hypothesis that SDH, instead of FH, could be involved. SDHA, SDHB, SDHC and SDHD were sequenced from these individuals; a germline SDHB R27X mutation was detected with loss of the wild-type allele in both tumours. These results suggest that germline mutations in the SDHB gene predispose to early-onset RCC establishing a novel form of hereditary RCC. This has immediate clinical implications in the surveillance of patients suffering from early-onset RCC and phaeochromocytoma/paraganglioma. For the studies on sporadic uterine fibroids, a set of 166 fibroids from 51 individuals were collected. The 7q LOH mapping defined a commonly deleted region of about 3.2 mega bases in 11 of the 166 tumours. The deletion was consistent with previously reported allelotyping studies of leiomyomas and it therefore suggested the presence of a tumour suppressor gene in the deleted region. Furthermore, the high-resolution aCGH-chip analysis refined the deleted region to only 2.79Mb. When combined with previous data, the commonly deleted region was only 2.3Mb. The mutation screening of the known genes within the commonly deleted region did not reveal pathogenic mutations, however. The expression microarray analysis revealed that FH-deficient fibroids, both sporadic and familial, had their distinct gene expression profile as they formed their own group in the unsupervised clustering. On the other hand, the presence or absence of 7q-deletions did not significantly alter the global gene expression pattern of fibroids, suggesting that these two groups do not have different biological backgrounds. Multiple differentially expressed genes were identified between FH wild-type and FH-mutant fibroids, and the most significant increase was seen in the expression of carbohydrate metabolism-related and hypoxia inducible factor (HIF) target genes.

Diet and the development of atherosclerosis: a whole-diet approach from childhood to adulthood

Cardiovascular diseases (CVDs) are the leading cause of mortality in the world. Studies of the impact of single nutrients on the risk for CVD have often provided inconclusive results, and recent research in nutritional epidemiology with a more holistic whole-diet approach has proven fruitful. Moreover, dietary habits in childhood and adolescence may play a role in later health and disease, either independently or by tracking into adulthood. The main aims of this study were to find childhood and adulthood determinants of adulthood diet, to identify dietary patterns present among the study population and to study the associations between long-term food choices and cardiovascular health in young Finnish adults. The study is a part of the multidisciplinary Cardiovascular Risk in Young Finns study, which is an ongoing, prospective cohort study with a 21-year follow-up. At baseline in 1980, the subjects were children and adolescents aged 3 to 18 years (n included in this study = 1768), and young adults aged 24 to 39 years at the latest follow-up study in 2001 (n = 1037). Food consumption and nutrient intakes were assessed with repeated 48-hour dietary recalls. Other determinations have included comprehensive risk factor assessments using blood tests, physical measurements and questionnaires. In the latest follow-up, ultrasound examinations were performed to study early atherosclerotic vascular changes. The average intakes showed substantial changes since 1980. Intakes of fat and saturated fat had decreased, whereas the consumption of fruits and vegetables had increased. Intake of fat and consumption of vegetables in childhood and physical activity in adulthood were important health behavioural determinants of adult diet. Additionally, a principal component analysis was conducted to identify major dietary patterns at each study point. A similar set of two major patterns was recognised throughout the study. The traditional dietary pattern positively correlated with the consumption of traditional Finnish foods, such as rye, potatoes, milk, butter, sausages and coffee, and negatively correlated with fruit, berries and dairy products other than milk. This type of diet was independently associated with several risk factors of CVD, such as total and low-density lipoprotein cholesterol, apolipoprotein B and C-reactive protein concentrations among both genders, as well as with systolic blood pressure and insulin levels among women. The traditional pattern was also independently associated with intima media thickness (IMT), a subclinical predictor of CVD, in men but not in women. The health-conscious pattern, predominant among female subjects, non-smokers and urbanites, was characterised by more health-conscious food choices such as vegetables, legumes and nuts, tea, rye, fish, cheese and other dairy products, as well as by the consumption of alcoholic beverages. This pattern was inversely, but less strongly, associated with cardiovascular risk factors. Tracking of the dietary pattern scores was observed, particularly among subjects who were adolescents at baseline. Moreover, a long-term high intake of protein concurrent with a low intake of fat was positively associated with IMT. These findings suggest that food behaviour and food choices are to some extent established as early as in childhood or adolescence and may significantly track into adulthood. Long-term adherence to traditional food choices seems to increase the risk for developing CVD, especially among men. Those with intentional or unintentional low fat diets, but with high intake of protein may also be at increased risk for CVD. The findings offer practical, food-based information on the relationship between diet and CVD and encourage further use of the whole-diet approach in epidemiological research. The results support earlier findings that long-term food choices play a role in the development of CVD. The apparent influence of childhood habits is important to bear in mind when planning educational strategies for the primary prevention of CVD. Further studies on food choices over the entire lifespan are needed.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Rakkauden välittäjä vai moraalinvartija : Narratiivinen tutkimus Suomen evankelis-luterilaisen kirkon papeista perhe- ja seksuaalikysymysten äärellä

The research goal was to clarify how ministers in the Evangelical Lutheran Church of Finland construct their identities when they confront family and sexual issues, particularly as seen in their narrations about their work and the factors that influence their ways of working. The approach was a narrative one placing the emphases was on the internal story. The research material consisted of interviews of 19 ministers and the written biographies of 3 ministers who had also been interviewed. The data was analysed narratively (analysis of the narrations and a narrative analysis). The life stories were classified on the basis of the logic in each, and five different internal story types were created: the persons on the road to recovery, those within safe boundaries, those who had learnt to be critical, those with an obligation to help, and those who had grown to be open. In all of the story types was evidented a conflict between the ministry of the Church and the way it was adapted to the lives of individuals experiencing family and sexual issues. On one hand, this was a source of stress at work, but on the other, it offered a chance to create new images of ministers struggling with family and sexual issues. Life experience was found to be important when the study subjects interpreted and dealt with family and sexual issues. Those on the road to recovery felt that becoming a Christian and the personal recovery that faith had made possible had a significant impact on their personal lives. This healing effect of faith was also a strengthening factor for them when they dealt with family and sexual issues. Typically, they approached work situations by taking into account their faith and the type of psychological knowledge that was acceptable within the boundaries set by it. Those within safe boundaries worked within the limits prescribed by the revivalist movement that they had grown up in, from their childhood onwards, and their experiences in adulthood had even strengthened their commitment to the movement. Typically, they were keen to proclaim the views of the movement in public, but they were also prone to stay silent if and when felt those views would cause an uproar. Those who had learnt to be critical had previously been holistically committed to the views expressed by the Church in family and sexual matters. It was their experiences in life that had led them into conflict with the teachings of the Church. Their approach to work was one of ambivalence resulting from a conflict between their current and previous views, which was further exacerbated by their irresolution concerning how a minister should act in these situations. Those with an obligation to help questioned the church teachings marriage as the only family ideal. When they met various kinds of families and sexual identities, and also when they adopted the identity of a helper, the foundation of their ministerial identity was the Two Commandments of Love. Their work was burdened, however, by a fear of how the Church and the parishioners would take their teaching. Those who had grown to be open were more sure of themselves than the other groups. Years in the ministry as well as life-long experience had made them into persons who were following paths of their own. Openly critical of the views on family and sexual issues proclaimed by the Church, they were keen to present their personal convictions and were able to defend these publicly when necessary. Search words: Narrative research, internal story, minister, church, family, sexuality.

Essays on Banking Crises

This thesis aims at finding the role of deposit insurance scheme and central bank (CB) in keeping the banking system safe. The thesis also studies the factors associated with long-lasting banking crises. The first essay analyzes the effect of using explicit deposit insurance scheme (EDIS), instead of using implicit deposit insurance scheme (IDIS), on banking crises. The panel data for the period of 1980-2003 includes all countries for which the data on EDIS or IDIS exist. 70% of the countries in the sample are less developed countries (LDCs). About 55% of the countries adopting EDIS also come from LDCs. The major finding is that the using of EDIS increases the crisis probability at a strong significance level. This probability is greater if the EDIS is inefficiently designed allowing higher scope of moral hazard problem. Specifically, the probability is greater if the EDIS provides higher coverage to deposits and if it is less powerful from the legal point of view. This study also finds that the less developed a country is to handle EDIS, the higher the chance of banking crisis. Once the underdevelopment of an economy handling the EDIS is controlled, the EDIS separately is no longer a significant factor of banking crises. The second essay aims at determining whether a country s powerful CB can lessen the instability of the banking sector by minimizing the likelihood of a banking crisis. The data used include indicators of the CB s autonomy for a set of countries over the period of 1980-89. The study finds that in aggregate a more powerful CB lessens the probability of banking crisis. When the CB s authority is disentangled with respect to its responsibilities, the study finds that the longer tenure of CB s chief executive officer and the greater power of CB in assigning interest rate on government loans are necessary for reducing the probability of banking crisis. The study also finds that the probability of crisis reduces more if an autonomous CB can perform its duties in a country with stronger law and order tradition. The costs of long-lasting banking crises are high because both the depositors and the investors lose confidence in the banking system. For a rapid recovery of a crisis, the government very often undertakes one or more crisis resolution policy (CRP) measures. The third essay examines the CRP and other explanatory variables correlated with the durations of banking crises. The major finding is that the CRP measure allowing the regulation forbearance to keep the insolvent banks operative and the public debt relief program are respectively strongly and weakly significant to increase the durations of crises. Some other explanatory variables, which were found by previous studies to be related with the probability of crises to occur, are also correlated with the durations of crises.