Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.

Factors affecting partitioning of private forest holdings in Finland.

Questions of the small size of non-industrial private forest (NIPF) holdings in Finland are considered and factors affecting their partitioning are analyzed. This work arises out of Finnish forest policy statements in which the small average size of holdings has been seen to have a negative influence on the economics of forestry. A survey of the literature indicates that the size of holdings is an important factor determining the costs of logging and silvicultural operations, while its influence on the timber supply is slight. The empirical data are based on a sample of 314 holdings collected by interviewing forest owners in the years 1980-86. In 1990-91 the same holdings were resurveyed by means of a postal inquiry and partly by interviewing forest owners. The principal objective in compiling the data is to assist in quantifying ownership factors that influence partitioning among different kinds of NIPF holdings. Thus the mechanism of partitioning were described and a maximum likelihood logistic regression model was constructed using seven independent holding and ownership variables. One out of four holdings had undergone partitioning in conjunction with a change in ownership, one fifth among family owned holdings and nearly a half among jointly owned holdings. The results of the logistic regression model indicate, for instance, that the odds on partitioning is about three times greater for jointly owned holdings than for family owned ones. Also, the probabilities of partitioning were estimated and the impact of independent dichotomous variables on the probability of partitioning ranged between 0.02 and 0.10. The low value of the Hosmer-Lemeshow test statistic indicates a good fit of the model and the rate of correct classification was estimated to be 88 per cent with a cutoff point of 0.5. The average size of holdings undergoing ownership changes decreased from 29.9 ha to 28.7 ha over the approximate interval 1983-90. In addition, the transition probability matrix showed that the trends towards smaller size categories mostly involved in the small size categories, less than 20 ha. The results of the study can be used in considering the effects of the small size of holdings for forestry and if the purpose is to influence partitioning through forest or rural policy.

Onnellisten kylät : Vertaileva tapaustutkimus Kumpulan ja Sundsbergin asukkaiden subjektiivisesta hyvinvoinnista sekä onnellisuuden rakentumisesta

The aim of this masters thesis was to examine subjective wellbeing and personal happiness. Empirical study of happiness is part of broader wellbeing reseach and is based on an idea that the best experts of personal wellbeing are the individuals themselves. In addition to perceptions of personal happiness, the aim was also to acquire knowledge about personal values and components personal happiness is based on. In this study, moving into certain community and the charesteristics of neigbourhood contributing happiness, were defined to represent these values. The object was, through comparative casestudy, to obtain knowledge about subjective wellbeing of the inviduals in two different residental areas inside metropolitan area of Helsinki. In comparative case study the intention usually is that the examined units represent spesific "cases" from something broader and therefore the results can be somehow generalized. Consequently the chosen cases in this study were selected due to their image of "urban village" and thus the juxtapositioning was constructed between secluded post-suburban village and more heterogeneous urban village better attached to excisting urban structure. The researh questins were formed as follows: Are there any differencies between the areas regarding the components personal happines is based on? Are there any differencies between the areas regarding the level of residents subjective wellbeing? Based on the residents assesments, what are the most important charesteristics of neigbourhood contributing personal happiness? The data used in order to gain aswers to these questions was obtained from internet-based survey questionnaire. Based on the data residents of post-suburban village Sundsberg seem to share highly family oriented set of values and actualizing these values is ensured with high income, wealth and secure work situation. Insteadt in Kumpula the components of happiness seem place more towards learning and personal developement, interesting leisure and hobbies and specially having an influence regarding communal decisions. Conserning subjective wellbeing of residents there can be seen some differencies as well. Personal life is experienced a bit more happier in Sundsberg than in Kumpula. People are more satified with their personal health and job satisfaction in Sundsberg and additionally feelings of loneliness, inadequancy and frustration are bit more common in Kumpula. Regarding the charesteristics of neigbourhood contributing happiness data suggests that key charesteristics of area are peacefulness and safely, good location and connections and proximity of parks and recreational areas. These charesteristics were concidered highly significant in both areas but they were experienced to actualize better in Kumpula. In addition to these components the residents in Kumpula were overall more satisfied with various charecteristics contiburing happiness in their residental area. Besides these attributes mentioned above residents in Kumpula emphasize also some "softer" elements connecting into social, functional and communal side of area. From Sundsberg point of view residental area best contributing happiness is child friendly and safe community based on likeminded people who share the same sosioeconomical situation. The results of this study can be linked back into the society and metropolitan area, which they were chosen from as a cases to be studied. The results can thereby be seen as an example of differentation of conditions of personal happiness between certain population segments. It is possible to detect an spatial dimetion to this process as well and thereby the results suggests that regional segmentation affects between high-ranking residental areas as well. Thereby the results of this reseach contributes to the debate on innovative, diverse and dynamic urban area and as well cohesion of metropolitan area and the society in whole.

Ideaalin ja todellisuuden välimaastossa - katoavatko perheateriat?

Aims: The recent conversations about disappearing family meals, mental problems of the youngsters and family meals´ protective effect on youngsters problems have been the base of this study. The main aim of this study is to find out, what kind of position family meals do have in the youngsters´lives. Study questions are: 1. What kind of conceptions youngsters have of family meals? 2. What kind of hands-on experience youngsters have about family meals? 3. How youngsters feel family meals on a emotional level? a. Are meals positive moments with the family or do they just limit timetables? b. Do the youngsters need family meals if they do not have those in their families? Why? Why not? Methods: The data was collected using stimulated recall -interviews and basic themed interviews. The data was collected from thirteen 8th graders from Pirkanmaa and Kanta-Häme. The interviews were analyzed with the qualitative content analysis. Results and conclusion: The youngsters´ consepts of the meals and the family meals were quite traditional, assuming that those are warm, varied and eaten with company. The situation of the family meals was good and those were eaten nearly every day in the most of the families. The youngsters thought that the family meals were important social moments with the family and they were able to talk about important things with the family during the meals. They also felt that the daily eating rythm was good and they did not felt that the meals would have been somehow restrictive. Even so that the pupils felt the family meals were important, the pupil whose family did not eat family meals did not long those meals. He felt that he was able to share his thoughts and experiences also in other ways. It is important that youngsters have this channel of communication.

The role of p73 in cancer – a pathway towards more effective cancer therapy

The p53-family consists of three transcription factors, p53, p73 and p63. The family members have similar but also individual functions connected to cell cycle regulation, development and tumorigenesis. p53 and p73 act mainly as tumor suppressors. During DNA damage caused by anticancer drugs or irradiation, p53 and p73 levels are upregulated in cancer cells leading to apoptosis and cell cycle arrest. p53 is mutated in almost 50 per cent of the cancers, causing the cancer cells unable to undergo cell death. Instead, p73 is rarely mutated in cancer cells and because of that could be more viable target for anticancer therapy. The network surrounding the regulation of p73 is extensive and has several potential targets for cancer therapy. One of the most studied is Itch ligase, the negative regulator of p73 levels. Gene therapy directed towards knockdown of Itch ligase is a potential approach but in need for more in vivo proof. p73 has two isoforms, transactivating TA-forms and dominant-negative ΔN-forms. The specific regulation of these isoforms could also offer a possible way for more effective cancer treatment. The literature work includes information of structures, isoforms, functions and possible therapeutic targets of p73. Also the main therapeutic approaches to date are introduced. The experimental part is based on transfection and cytotoxicity studies done e.g. in pancreatic cancer cells (Mia PaCa-2, PANC1, BxPc-3 and HPAC). The aim of the experimental work was to optimize the conditions for effective transfection with DAB16 dendrimer nanoparticles and to measure the cytotoxicity of plain dendrimers and DAB16-pDNA complexes. Also the protein levels of p73 and Itch ligase were measured by Western blotting. The work was done as a part of a bigger project, which was aiming to down regulate Itch ligase (negative regulator of p73) by siRNA/shRNA. Tranfection results were promising, showing good transfection efficacy with DAB16 N/P30 in pancreatic cancer cells (except in BxPc-3). Pancreatic cancer cells showed recovery in 3 days after they were exposed to plain dendrimer solution or to DAB16-pDNA. Measurement of protein levels by Western blotting was not optimal and the proposals for the improvement regarding e.g. the gels and the extracted protein amounts have been done.

Choosing the Arranged One, Arranging the Chosen One – Imagining an Indian Modernity: Reconciling Love, Marriage and Family On and Off the Screen

This is a study on the changing practices of kinship in Northern India. The change in kinship arrangements, and particularly in intermarriage processes, is traced by analysing the reception of Hindi popular cinema. Films and their role and meaning in people´s lives in India was the object of my research. Films also provided me with a methodology for approaching my other subject-matters: family, marriage and love. Through my discussion of cultural change, the persistence of family as a core value and locus of identity, and the movie discourses depicting this dialogue, I have looked for a possibility of compromise and reconciliation in an Indian context. As the primary form of Indian public culture, cinema has the ability to take part in discourses about Indian identity and cultural change, and alleviate the conflicts that emerge within these discourses. Hindi popular films do this, I argue, by incorporating different familiar cultural narratives in a resourceful way, thus creating something new out of the old elements. The final word, however, is the one of the spectator. The “new” must come from within the culture. The Indian modernity must be imaginable and distinctively Indian. The social imagination is not a “Wild West” where new ideas enter the void and start living a life of their own. The way the young women in Dehra Dun interpreted family dramas and romantic movies highlights the importance of family and continuity in kinship arrangements. The institution of arranged marriage has changed its appearance and gained new alternative modes such as love cum arranged marriage. It nevertheless remains arranged by the parents. In my thesis I have offered a social description of a cultural reality in which movies act as a built-in part. Movies do not work as a distinct realm, but instead intertwine with the social realities of people as a part of a continuum. The social imagination is rooted in the everyday realities of people, as are the movies, in an ontological and categorical sense. According to my research, the links between imagination and social life were not so much what Arjun Appadurai would call global and deterritorialised, but instead local and conventional.

Work/Family Reconciliation: Corporate Management, Family Policies, and Gender Equality in the Finnish Context

Work/family reconciliation is a crucial question for both personal well-being and on societal level for productivity and re-production throughout the Western world. This thesis examines work/family reconciliation on societal and organisational level in the Finnish context. The study is based on an initial framework, developing it further and analysing the results with help of it. The methodology of the study is plural, including varying epistemological emphasis and both quantitative and qualitative methods. Policy analysis from two different sectors is followed by a survey answered by 113 HR-managers, and then, based on quantitative analyses, interviews in four chosen case companies. The central findings of the thesis are that there indeed are written corporate level policies for reconciling work and family in companies operating in Finland, in spite of the strong state level involvement in creating a policy context in work/family reconciliation. Also, the existing policies vary in accessibility and use. The most frequently used work/family policies still are the statutory state level policies for family leave, taking place when a baby is born and during his or her first years. Still, there are new policies arising, such as a nurse for an employee’s child who has fallen ill, that are based on company activity only, which shows in both accessibility and use of the policy. Reasons for developing corporate level work/family policies vary among the so-called pro-active and re-active companies. In general, family law has a substantial effect for developing corporate level policies. Also headquarter gender equality strategies as well as employee demands are important. In regression analyses, it was found that corporate image and importance in recruitment are the foremost reasons for companies to develop policies, not for example the amount of female employees in the company. The reasons for policy development can be summarized into normative pressures, coercive pressures and mimetic pressures, in line with findings from institutional theory. This research, however, includes awareness of different stakeholder interests and recognizes that institutional theory needs to be complemented with notions of gender and family, which seem to play a part in perceived work/family conflict and need for further work/family policies both in managers’ personal lives and on the organisational level. A very central finding, demanding more attention, is the by HR managers perceived change in values towards work and commitment towards organisation at the youngest working generation, Generation Y. This combined with the need for key personnel has brought new challenges to companies especially in knowledge business and will presumably lead to further development of flexible practices in organisations. The accessibility to this flexibility seems to, however, be even more dependent on the specific knowledge and skills of the employee. How this generation will change the organisations remains to be seen in further research.

Koti keskellä kaupunkia : Keskusta lapsiperheen asuinalueena, esimerkkeinä Tukholma ja Helsinki

Families with children have traditionally moved to suburbs. In the last 20 years a modest counter process has however been recognized. Families with an urban lifestyle stay in the city centres. This study looks at the phenomenon through two cases, Stockholm and Helsinki. In the first case it has already been observed that the city centre has grown in popularity among families with children. Therefore it serves as a basis for the study and as well as a point of comparison. Stockholm’s city centre is expanding as new neighbourhoods have been built and are being planned. In the city centre of Helsinki the building of two large neighbourhoods for 30 000 inhabitants will start in a few years. The first aim of the study is to look closer at what has really happened in the city centre of Stockholm, why families choose to live there with their children and how the City of Stockholm has reacted to the change. The main sources of information are secondary sources, statistics and interviews with planners, politicians and experts in the field. The main object is to look at the situation in the city centre of Helsinki. Can a preference for urban residential environments be observed in Helsinki? What are the reasons for a family to choose the city centre as a living place? How does the everyday life of a family in the city centre appear? How are these families taken into account in the planning of the city? The main sources of information here are statistics, interviews with dwellers in the neighbourhood Kruununhaka and interviews with planners. In Stockholm the birth rate has grown constantly during the 2000s and is highest in the city centre. Some of the families still move elsewhere, but many of them do not. One of the most important reasons for living in the city centre is short working distances which give working parents more time with their children. Another reason is a preference of an urban, active lifestyle. Families prefer to live close to everything, childcare, schools, shops and entertainments. The popularity of the city centre among families with children has taken politicians and planners by surprise. Helsinki has not experienced a baby boom like Stockholm. However the negative changes in the birth rate have been more modest in the central areas than in the suburbs. Statistics show, that many families move away from the city centre as the children grow. Families who stay in the city centre especially appreciate closeness to public and private services and good public transportation which means that they are not dependent on using the car. Further they find that the city centre has a tolerant climate and is a safe and beautiful place to live in. The families enjoy the social life of the neighbourhood and feel that it makes a good climate to raise children in. However they are concerned with traffic safety and the lack of stimulus in the playgrounds of the neighbourhood parks. Two large neighbourhoods with homes for about 30 000 inhabitants are now planned in the former Port Districts in the city centre of Helsinki. The other one, Jätkäsaari has been planned to become an attractive alternative for families with children. Traffic safety has been one of the main objects for the planning. The other, Kalasatama, has been planned to attract all groups in society.