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).

In vitro development of islets from human adult pancreatic tissues

Transplantation of isolated islets from cadaver pancreas is a promising possibility for the optimal treatment of type 1 diabetes. The lack of islets is a major problem. Here we have investigated the possibility of generating islets in tissue culture of human pancreatic cells. We first reproduced a previously reported method of in vitro generation of endocrine cells from human adult pancreatic tissue. By tracing the bromodeoxyuridine-labeled cells in differentiated islet buds, we found that the pancreatic progenitor cells represented a subpopulation of cytokeratin 19 (CK19)-positive ductal cells. Serum-free medium and Matrigel overlay were essential for the endocrine differentiation. We then examined the involvement of preexisting islet cells in islet neogenesis. About 6-10% of endocrine cells dedifferentiated and acquired a transitional phenotype by coexpressing CK19. Significant cell proliferation was only observed in CK19-positive cells, but not in chromogranin A-positive endocrine cells. The in vitro-derived human islets were morphologically and functionally immature when compared with normal islets. Their insulin mRNA levels were only 4-5% of that found in fresh human islets, and glucose-stimulated insulin release was 3 times lower than that of control islets. Moreover, some immature endocrine cells coexpressed insulin and glucagon. After transplantation in nude mice, the in vitro-generated islets became mature with one type of hormone per endocrine cell. In addition, we also found that also in both fresh islet transplants many cells coexpressed endocrine markers and ductal marker CK19 as a sign of ductal to endocrine cell transition. Finally, we studied the effects of clinically used immunosuppressive drugs on precursor cell proliferation and differentiation. Mycophenolate mofetil (MMF) severely hampered duct-cell proliferation, and significantly reduced the total DNA content indicating its antiproliferative effect on the precursors. Tacrolimus mainly affected differentiated beta cells by decreasing the insulin content per DNA as well as the proportion of insulin-positive cells. Sirolimus and daclizumab did not show any individual or synergistic side effects suggesting that these drugs are amenable for use in clinical islet transplantation. In summary, we confirm the capacity of endocrine differentiation from progenitors present in the adult human pancreas. The plasticity of differentiated cell types of human pancreas may be a potential mechanism of human pancreas regeneration. Ductal cell differentiation into endocrine cells in transplanted islets may be an important factor in sustaining the long-term function of islet transplants. The immunosuppressive protocol is likely to be an important determinant of long-term clinical islet graft function. Moreover, these results provide new information on the mechanisms of pancreatic islet regeneration and provide the basis for the development of new strategies for the treatment of insulin deficient diabetes mellitus.

Integrative functional genomics analysis of sustained polyploidy phenotypes in breast cancer cells identifies an oncogenic profile for GINS2

Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and that aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication and possibly through polyploid intermediate states. Here, we used a novel cell spot microarray technique to identify genes with a loss-of-function effect inducing polyploidy and/or allowing maintenance of polyploid cell growth of breast cancer cells. Integrative genomics profiling of candidate genes highlighted GINS2 as a potential oncogene frequently overexpressed in clinical breast cancers as well as in several other cancer types. Multivariate analysis indicated GINS2 to be an independent prognostic factor for breast cancer outcome (p = 0.001). Suppression of GINS2 expression effectively inhibited breast cancer cell growth and induced polyploidy. In addition, protein level detection of nuclear GINS2 accurately distinguished actively proliferating cancer cells suggesting potential use as an operational biomarker.

Activityscape Mapping: Consumer Activity Systems as Service Context

Activity systems are the cognitively linked groups of activities that consumers carry out as a part of their daily life. The aim of this paper is to investigate how consumers experience value through their activities, and how services fit into the context of activity systems. A new technique for illustrating consumers’ activity systems is introduced. The technique consists of identifying a consumer’s activities through an interview, then quantitatively measuring how the consumer evaluates the identified activities on three dimensions: Experienced benefits, sacrifices and frequency. This information is used to create a graphical representation of the consumer’s activity system, an “activityscape map”. Activity systems work as infrastructure for the individual consumer’s value experience. The paper contributes to value and service literature, where there currently are no clearly described standardized techniques for visually mapping out individual consumer activity. Existing approaches are service- or relationship focused, and are mostly used to identify activities, not to understand them. The activityscape representation provides an overview of consumers’ perceptions of their activity patterns and the position of one or several services in this pattern. Comparing different consumers’ activityscapes, it shows the differences between consumers' activity structures, and provides insight into how services are used to create value within them. The paper is conceptual; an empirical illustration is used to indicate the potential in further empirical studies. The technique can be used by businesses to understand contexts for service use, which may uncover potential for business reconfiguration and customer segmentation.

Rethinking Service Companies’ Business Logic: Do We Need a Customer-Dominant Logic as a Guideline?

Purpose –This paper explores and expands the roles of customers and companies in creating value by introducing a new a customer-based approach to service. The customer’s logic is examined as being the foundation of a customer-based marketing and business logic. Design/methodology/approach – The authors argue that both goods-dominant logics and service-dominant logics are provider-dominant. Contrasting the customer-dominant logic with provider-dominant logics, the paper examines the creation of service value from the perspectives of value-in-use, the customer’s own context, and the customer’s experience of service. Findings –Moving from a provider-dominant logic to a customer-dominant logic uncovered five major challenges to service marketers: Company involvement, company control in co-creation, visibility of value creation, locus of customer experience, and character of customer experience. Research limitations/implications – The paper is exploratory. It presents and discusses a conceptual model and suggests implications for research and practice. Practical implications –Awareness of the mechanisms of customer logic will provide businesses with new perspectives on the role of the company in their customer’s lives. We propose that understanding the customer’s logic should represent the starting-point for the marketer’s business logic. Originality/value – The paper increases the understanding of how the customer’s logic underpins the customer-dominant business logic. By exploring consequences of applying a customer-dominant logic, we suggest further directions for theoretical and empirical research.

El Motezuma de Vivaldi

This article explores aesthetic and historical aspects of Antonio Vivaldi's (1678- 1741) Motezuma (1733), in three acts, to celebrate this opera revival after the score's discovery and its first premiere at De Doelen theatre, Rotterdam (2005). Particular attention is paid to cultural aspects, regarding the European interpretation of the Conquest of Mexico.

Activityscape Mapping: Consumer Activity Systems as Service Context

Activity systems are the cognitively linked groups of activities that consumers carry out as a part of their daily life. The aim of this paper is to investigate how consumers experience value through their activities, and how services fit into the context of activity systems. A new technique for illustrating consumers’ activity systems is introduced. The technique consists of identifying a consumer’s activities through an interview, then quantitatively measuring how the consumer evaluates the identified activities on three dimensions: Experienced benefits, sacrifices and frequency. This information is used to create a graphical representation of the consumer’s activity system, an “activityscape map”. Activity systems work as infrastructure for the individual consumer’s value experience. The paper contributes to value and service literature, where there currently are no clearly described standardized techniques for visually mapping out individual consumer activity. Existing approaches are service- or relationship focused, and are mostly used to identify activities, not to understand them. The activityscape representation provides an overview of consumers’ perceptions of their activity patterns and the position of one or several services in this pattern. Comparing different consumers’ activityscapes, it shows the differences between consumers' activity structures, and provides insight into how services are used to create value within them. The paper is conceptual; an empirical illustration is used to indicate the potential in further empirical studies. The technique can be used by businesses to understand contexts for service use, which may uncover potential for business reconfiguration and customer segmentation.

Jac. Ahrenberg ja historian perintö : Restaurointisuunnitelmat Viipurin ja Turun linnoihin 1800-luvun lopussa

Jac. Ahrenberg and Patrimony Restoration Plans for Viipuri and Turku Castles at the End of the 19th Century This dissertation examines the unrealized restoration plans for two castles in the Grand Duchy of Finland one located at Viipuri (Vyborg, nowadays in Russia), the other at Turku (in Swedish, Åbo) during the last decades of the 19th century. Both castles were used as prisons, barracks and warehouses. From the middle of the 19th century on, their restoration and transformation into museums and "national monuments" were demanded in the newspapers. The prison reform in the 1860s stimulated the documentation and debate concerning their future, but it was only at the beginning of the 1880s when their restoration became an official state-run project. The undertaking was carried out by Johan Jacob (Jac.) Ahrenberg (1847 1914), architect of the National Board of Public Buildings. By combining written sources with drawings and photographs, this dissertation examines the restoration projects, the two castles' significance and the ways in which they were investigated by scholars. The plans are analyzed in connection with restoration practices in France and Sweden and in the context of contemporary discussions concerning national art and patrimony. The thesis argues that these former castles of the Swedish crown were used to manifest the western roots of Finnish law and order, the lineage of power and the capacity of the nation to defend itself. However, because of their symbolism, their restoration became a politically delicate question concerning the role of the Swedish heritage in Finland's nation-building process. According to Jac. Ahrenberg's plans, the two castles were to be restored to their assumed appearance at the time of the Vasa dynasty. Consequently, the structures would have resembled castles in Sweden. It is suggested that one aim of the restoration plans was to transform the two buildings into monuments testifying to the common history of Sweden and Finland. They were meant to consolidate the Swedish basis of Finnish culture and autonomy and thus to secure them against the threatening implications of Russian imperialism. It seems that along with the changing ideals of architectural restoration and the need for an original Finnish architectural heritage, the political connotations associated with the castles were one reason why Jac. Ahrenberg's restoration plans were never realized.

How to deal with sparse macroseismic data

This study discusses the scope of historical earthquake analysis in low-seismicity regions. Examples of non-damaging earthquake reports are given from the Eastern Baltic (Fennoscandian) Shield in north-eastern Europe from the 16th to the 19th centuries. The information available for past earthquakes in the region is typically sparse and cannot be increased through a careful search of the archives. This study applies recommended rigorous methodologies of historical seismology developed using ample data to the sparse reports from the Eastern Baltic Shield. Attention is paid to the context of reporting, the identity and role of the authors, the circumstances of the reporting, and the opportunity to verify the available information by collating the sources. We evaluate the reliability of oral earthquake recollections and develop criteria for cases when a historical earthquake is attested to by a single source. We propose parametric earthquake scenarios as a way to deal with sparse macroseismic reports and as an improvement to existing databases.