USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Leader - Follower Relations In An Intercultural Chinese Context - Personal, Interpersonal and Behavioural Influences and Impact on Work Contribution

The successful interaction between leaders and their followers is central to the overall functioning of a company. The increasingly multinational nature of modern business and the resulting multicultural and increasingly heterogeneous workforce imposes specific challenges to the development of high-quality work relationships. The Western multinational companies that have started operations in China are facing these challenges. This study examines the quality of leader-follower relationships between Western expatriate leaders and their Chinese followers as well as between Chinese leaders and their Chinese followers in Western-owned subsidiaries in China. The focus is on the influence of personal, interpersonal and behavioural factors (personality, values, cultural knowledge, perceived and actual similarity, interactional justice, and follower performance) and the work-related implications of these relationships (job attitudes and organisational citizenship behaviour). One interesting finding of this study is that Chinese followers have higher perceptions of their Western than their Chinese leaders. The results also indicate that Chinese and Western leaders’ perceptions of their followers are not influenced favourably by the same follower characteristics. In a similar vein, Chinese followers value different traits in Western versus Chinese leaders. These results, as well as the numerous more specific findings of the study, have practical implications for international human resource management and areas such as selection, placement and training. Due to the different effect of personal and interpersonal factors across groups, it is difficult to achieve the “perfect match” between leader and follower characteristics that simultaneously contribute to high-quality relationships for Chinese and Western leaders as well as for followers. However, the results indicate that the ability of organisations to enhance the quality of leader-follower relations by selecting and matching people with suitable characteristics may provide an effective means for organisations to increase positive job attitudes and hence influence work-related outcomes.

Multidimensionality of Actors in Business Networks: The Influence of Social Action in Pharmacy Networks in Finland

In the markets-as-networks approach business networks are conceived as dynamic actor structures, giving focus to exchange relationships and actors’ capabilities to control and co-ordinate activities and resources. Researchers have shared an understanding that actors’ actions are crucial for the development of business networks and for network dynamics. However, researchers have mainly studied firms as business actors and excluded individuals, although both firms and individuals can be seen as business actors. This focus on firms as business actors has resulted in a paucity of research on human action and the exchange of intangible resources in business networks, e.g. social exchange between individuals in social networks. Consequently, the current conception of business networks fails to appreciate the richness of business actors, the human character of business action and the import of social action in business networks. The central assumption in this study is that business actors are multidimensional and that their specific constitution in any given situation is determined by human interaction in social networks. Multidimensionality is presented as a concept for exploring how business actors act in different situations and how actors simultaneously manage multiple identities: individual, organisational, professional, business and network identities. The study presents a model that describes the multidimensionality of actors in business networks and conceptualises the connection between social exchange and human action in business networks. Empirically the study explores the change that has taken place in pharmaceutical retailing in Finland during recent years. The phenomenon of emerging pharmacy networks is highly contemporary in the Nordic countries, where the traditional license-based pharmacy business is changing. The study analyses the development of two Finnish pharmacy chains, one integrated and one voluntary chain, and the network structures and dynamics in them. Social Network Analysis is applied to explore the social structures within the pharmacy networks. The study shows that emerging pharmacy networks are multifaceted phenomena where political, economic, social, cultural, and historical elements together contribute to the observed changes. Individuals have always been strongly present in the pharmacy business and the development of pharmacy networks provides an interesting example of human actors’ influence in the development of business networks. The dynamics or forces driving the network development can be linked to actors’ own economic and social motives for developing the business. The study highlights the central role of individuals and social networks in the development of the two studied pharmacy networks. The relation between individuals and social networks is reciprocal. The social context of every individual enables multidimensional business actors. The mix of various identities, both individual and collective identities, is an important part of network dynamics. Social networks in pharmacy networks create a platform for exchange and social action, and social networks enable and support business network development.

Exploring Customer Value Formation: A Customer Dominant Logic Perspective

This paper extends current discussions about value creation and proposes a customer dominant value perspective. A customer-dominant marketing logic positions the customer in the center, rather than the service provider/producer or the interaction or the system. The focus is shifted from the company´s service processes involving the customer, to the customer´s multi-contextual value formation, involving the company. It is argued that value is not always an active process of creation; instead value is embedded and formed in the highly dynamic and multi-contextual reality and life of the customer. This leads to a need to look beyond the current line of visibility where visible customer-company interactions are focused to the invisible and mental life of the customer. From this follows a need to extend the temporal scope, from exchange and use even further to accumulated experiences in the customer´s life. The aim of this paper is to explore value formation from a customer dominant logic perspective. This is done in three steps: first, value formation is contrasted to earlier views on the company’s role in value creation by using a broad ontologically driven framework discussing what, how, when, where and who. Next, implications of the proposed characteristics of value formation compared to earlier approaches are put forward. Finally, some tentative suggestions of how this perspective would affect marketing in service companies are presented. As value formation in a CDL perspective has a different focus and scope than earlier views on value it leads to posing questions about the customer that reveals earlier hidden aspects of the role of a service for the customer. This insight might be used in service development and innovation.

Moral Professionalism in Interaction : Educators' Relational Moral Voices in Urban Schools

The aim of this study was to investigate educators relational moral voices in urban schools and to listen to what they told about moral professionalism and moral practices in challenging urban schools. Their relational moral voices were investigated through the following three questions: 1. What are the educators moral voices in relation to themselves and other people? 2. What are the educators moral voices in relation to their work and society? 3. What kind of interaction process lies between the educators moral voices and the urban school context? The research data of this study were gathered in four urban schools in Jyväskylä and Helsinki. The research schools were chosen for this study according to the criteria of the international Socrates Comenius project called Leading Schools Successfully in Challenging Urban Context: Strategies for Improvement. This study formed part of this project, which investigated successful urban schools as challenging learning environments in nine European countries and explored the principals success in leadership in particular. The data, which included 37 narratively constructed interviews with four principals and key informants selected by the principals, were gathered in interviews conducted in 2006. In other words, the data comprised three interviews with each of four principals, and interviews with two teachers, two parents, and two pupils from each school. In addition, the school deacon from one school was also interviewed. Furthermore, part of the data from one of the research schools included a medium report of the school deacon s work. This study combined the case study method, the narrative approach and the critical incident technique as the methodological framework. In addition, all of these methods served as practical tools for both analyzing and reporting the data. The educators' narrations and the results of the study appear in the original articles (Hanhimäki & Tirri 2008; Hanhimäki 2008b; Hanhimäki & Tirri 2009; Hanhimäki 2008a). The educators moral voices in relation to themselves and other people emerged through the main themes of moral leadership, the development and evaluation process, moral sensitivity, gender, values, and student well-being. The educators moral voices in relation to their work and society emerged through the main themes of multiprofessional cooperation, families and parental involvement, and moral school culture. The idea of moral interaction connected moral professionalism and the methodological combination of this study, which together emphasized social interaction and the creation of understanding and meaning in this interaction. The main point of this study was to state that the educators moral voices emerged in the interaction between the educators themselves and the urban school context. In this interaction, the educators moral professionalism was constructed and shaped in relation to themselves, other people, their work and society. The loudest relational moral voices heard through the main themes were those of caring, cooperation, respect, commitment, and professionalism. When the results were compared to the codes of ethics which guided these educators moral professional work, the ethical principles and values of the codes were clearly visible in their moral practices. The loudest message from the educators narration could be summarized in the words caring, respect and cooperation: at its best, there is just a human being and a human being with caring, respect and cooperation between them. The results of this study emphasize the need for practical approaches such as case studies and the narrative approach in teacher education to encourage educators to become moral professionals capable of meeting the needs of people of varied backgrounds. In addition, opportunities for moral, religious and spiritual education should be noticed and utilized in the plural interaction of urban schools when nurturing pupils and creating a moral school culture. Furthermore, multiprofessional cooperation and parents as the school s primary cooperation partner are needed to carry out the shared duty of moral education in urban schools. Keywords: moral professionalism, educator, relational moral voice, interaction, urban school

Carbon dioxide and methane exchange between a boreal pristine lake and the atmosphere

Lakes serve as sites for terrestrially fixed carbon to be remineralized and transferred back to the atmosphere. Their role in regional carbon cycling is especially important in the Boreal Zone, where lakes can cover up to 20% of the land area. Boreal lakes are often characterized by the presence of a brown water colour, which implies high levels of dissolved organic carbon from the surrounding terrestrial ecosystem, but the load of inorganic carbon from the catchment is largely unknown. Organic carbon is transformed to methane (CH4) and carbon dioxide (CO2) in biological processes that result in lake water gas concentrations that increase above atmospheric equilibrium, thus making boreal lakes as sources of these important greenhouse gases. However, flux estimates are often based on sporadic sampling and modelling and actual flux measurements are scarce. Thus, the detailed temporal flux dynamics of greenhouse gases are still largely unknown. ----- One aim here was to reveal the natural dynamics of CH4 and CO2 concentrations and fluxes in a small boreal lake. The other aim was to test the applicability of a measuring technique for CO2 flux, i.e. the eddy covariance (EC) technique, and a computational method for estimation of primary production and community respiration, both commonly used in terrestrial research, in this lake. Continuous surface water CO2 concentration measurements, also needed in free-water applications to estimate primary production and community respiration, were used over two open water periods in a study of CO2 concentration dynamics. Traditional methods were also used to measure gas concentration and fluxes. The study lake, Valkea-Kotinen, is a small, humic, headwater lake within an old-growth forest catchment with no local anthropogenic disturbance and thus possible changes in gas dynamics reflect the natural variability in lake ecosystems. CH4 accumulated under the ice and in the hypolimnion during summer stratification. The surface water CH4 concentration was always above atmospheric equilibrium and thus the lake was a continuous source of CH4 to the atmosphere. However, the annual CH4 fluxes were small, i.e. 0.11 mol m-2 yr-1, and the timing of fluxes differed from that of other published estimates. The highest fluxes are usually measured in spring after ice melt but in Lake Valkea-Kotinen CH4 was effectively oxidised in spring and highest effluxes occurred in autumn after summer stratification period. CO2 also accumulated under the ice and the hypolimnetic CO2 concentration increased steadily during stratification period. The surface water CO2 concentration was highest in spring and in autumn, whereas during the stable stratification it was sometimes under atmospheric equilibrium. It showed diel, daily and seasonal variation; the diel cycle was clearly driven by light and thus reflected the metabolism of the lacustrine ecosystem. However, the diel cycle was sometimes blurred by injection of hypolimnetic water rich in CO2 and the surface water CO2 concentration was thus controlled by stratification dynamics. The highest CO2 fluxes were measured in spring, autumn and during those hypolimnetic injections causing bursts of CO2 comparable with the spring and autumn fluxes. The annual fluxes averaged 77 (±11 SD) g C m-2 yr-1. In estimating the importance of the lake in recycling terrestrial carbon, the flux was normalized to the catchment area and this normalized flux was compared with net ecosystem production estimates of -50 to 200 g C m-2 yr-1 from unmanaged forests in corresponding temperature and precipitation regimes in the literature. Within this range the flux of Lake Valkea-Kotinen yielded from the increase in source of the surrounding forest by 20% to decrease in sink by 5%. The free water approach gave primary production and community respiration estimates of 5- and 16-fold, respectively, compared with traditional bottle incubations during a 5-day testing period in autumn. The results are in parallel with findings in the literature. Both methods adopted from the terrestrial community also proved useful in lake studies. A large percentage of the EC data was rejected, due to the unfulfilled prerequisites of the method. However, the amount of data accepted remained large compared with what would be feasible with traditional methods. Use of the EC method revealed underestimation of the widely used gas exchange model and suggests simultaneous measurements of actual turbulence at the water surface with comparison of the different gas flux methods to revise the parameterization of the gas transfer velocity used in the models.

Therapeutic change in interaction

A process of change within a single case of cognitive-constructivist therapy is analyzed by means of conversation analysis (CA). The focus is on a process of change in the sequences of interaction, which consist of the therapist’s conclusion and the patient’s response to it. In the conclusions, the therapist investigates and challenges the patient’s tendency to transform her feelings of disappointment and anger into self-blame. Over the course of the therapy, the patient’s responses to these conclusions are recast: from the patient first rejecting the conclusion, to then being ambivalent, and finally to agreeing with the therapist. On the basis of this case study, we suggest that an analysis that focuses on sequences of talk that are interactionally similar offers a sensitive method to investigate the manifestation of therapeutic change. It is suggested that this line of research can complement assimilation analysis and other methods of analyzing changes in a client’s talk.