Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Reconstructions of past climates from documentary and natural sources in Finland since the 18th century

In this paper both documentary and natural proxy data have been used to improve the accuracy of palaeoclimatic knowledge in Finland since the 18th century. Early meteorological observations from Turku (1748-1800) were analyzed first as a potential source of climate variability. The reliability of the calculated mean temperatures was evaluated by comparing them with those of contemporary temperature records from Stockholm, St. Petersburg and Uppsala. The resulting monthly, seasonal and yearly mean temperatures from 1748 to 1800 were compared with the present day mean values (1961-1990): the comparison suggests that the winters of the period 1749-1800 were 0.8 ºC colder than today, while the summers were 0.4 ºC warmer. Over the same period, springs were 0.9 ºC and autumns 0.1 ºC colder than today. Despite their uncertainties when compared with modern meteorological data, early temperature measurements offer direct and daily information about the weather for all months of the year, in contrast with other proxies. Secondly, early meteorological observations from Tornio (1737-1749) and Ylitornio (1792-1838) were used to study the temporal behaviour of the climate-tree growth relationship during the past three centuries in northern Finland. Analyses showed that the correlations between ring widths and mid-summer (July) temperatures did not vary significantly as a function of time. Early (June) and late summer (August) mean temperatures were secondary to mid-summer temperatures in controlling the radial growth. According the dataset used, there was no clear signature of temporally reduced sensitivity of Scots pine ring widths to mid-summer temperatures over the periods of early and modern meteorological observations. Thirdly, plant phenological data with tree-rings from south-west Finland since 1750 were examined as a palaeoclimate indicator. The information from the fragmentary, partly overlapping, partly nonsystematically biased plant phenological records of 14 different phenomena were combined into one continuous time series of phenological indices. The indices were found to be reliable indicators of the February to June temperature variations. In contrast, there was no correlation between the phenological indices and the precipitation data. Moreover, the correlations between the studied tree-rings and spring temperatures varied as a function of time and hence, their use in palaeoclimate reconstruction is questionable. The use of present tree-ring datasets for palaeoclimate purposes may become possible after the application of more sophisticated calibration methods. Climate variability since the 18th century is perhaps best seen in the fourth paper study of the multiproxy spring temperature reconstruction of south-west Finland. With the help of transfer functions, an attempt has been made to utilize both documentary and natural proxies. The reconstruction was verified with statistics showing a high degree of validity between the reconstructed and observed temperatures. According to the proxies and modern meteorological observations from Turku, springs have become warmer and have featured a warming trend since around the 1850s. Over the period of 1750 to around 1850, springs featured larger multidecadal low-frequency variability, as well as a smaller range of annual temperature variations. The coldest springtimes occurred around the 1840s and 1850s and the first decade of the 19th century. Particularly warm periods occurred in the 1760s, 1790s, 1820s, 1930s, 1970s and from 1987 onwards, although in this period cold springs occurred, such as the springs of 1994 and 1996. On the basis of the available material, long-term temperature changes have been related to changes in the atmospheric circulation, such as the North Atlantic Oscillation (February-June).

Food intake, growth and social interactions of signal crayfish, Pacifastacus leniusculus (Dana)

The knowledge about the optimal rearing conditions, such as water temperature and quality, photoperiod and density, with the understanding of animal nutritional requirements forms the basis of economically stable aquaculture for freshwater crayfish. However, the shift from a natural environment to effective culture conditions induces several changes, not only at the population level, but also at the individual level. The social contacts between conspecifics increase with increasing animal density. The competition for limited resources (e.g. food, shelter, mates) is more severe with the presence of agonistic behaviour and may lead to unequal distribution of these. The objectives of this study were to: 1) study the distribution of a common food resource between communally reared signal crayfish (Pacifastacus leniusculus) and to assign potential feeding hierarchy on the basis of individual food intake measurements, 2) explore the possibilities of size distribution manipulations to affect population dynamics and food intake to improve growth and survival in culture and 3) study the effect of food ration and spatial distribution on food intake and to explore the effect of temperature and food ration on growth and body composition of freshwater crayfish. The feeding ranks between animals were assigned with a new method for individual food intake measurement of communally reared crayfish. This technique has a high feasibility and a great potential to be applied in crayfish aquaculture studies. In this study, signal crayfish showed high size-related variability in food consumption both among individuals within a group (inter-individual) and within individual day-to-day variation (intra-individual). Increased competition for food led to an unequal distribution of this resource and this may be a reason for large growth differences between animals. The consumption was significantly higher when reared individually in comparison with communal housing. These results suggest that communally housed crayfish form a feeding hierarchy and that the animal size is the major factor controlling the position in this hierarchy. The optimisation of the social environment ( social conditions ) was evaluated in this study as a new approach to crayfish aquaculture. The results showed that the absence of conspecifics (individual rearing vs. communal housing) affects growth rate, food intake and the proportion of injured animals, whereas size variation between animals influences the number and duration of agonistic encounters. In addition, animal size had a strong influence on the fighting success of signal crayfish reared in a social milieu with a wide size variation of conspecifics. Larger individuals initiated and won most of the competitions, which suggests size-based social hierarchy of P. leniusculus. This is further supported by the fact that the length and weight gain of smaller animals increased after size grading, maybe because of a better access to the food resource due to diminished social pressure. However, the high dominance index was not based on size under conditions of limited size variation, e.g. those characteristic of restocked natural populations and aquaculture, indicating the important role of behaviour on social hierarchy.

Psychosocial processes of health behaviour change in a lifestyle intervention : Influences of gender, socioeconomic status and personality

Background: The onset of many chronic diseases such as type 2 diabetes can be delayed or prevented by changes in diet, physical activity and obesity. Known predictors of successful behaviour change include psychosocial factors such as selfefficacy, action and coping planning, and social support. However, gender and socioeconomic differences in these psychosocial mechanisms underlying health behaviour change have not been examined, despite well-documented sociodemographic differences in lifestyle-related mortality and morbidity. Additionally, although stable personality traits (such as dispositional optimism or pessimism and gender-role orientation: agency and communion) are related to health and health behaviour, to date they have rarely been studied in the context of health behaviour interventions. These personality traits might contribute to health behaviour change independently of the more modifiable domain-specific psychosocial factors, or indirectly through them, or moderated by them. The aims were to examine in an intervention setting: (1) whether changes (during the three-month intervention) in psychological determinants (self-efficacy beliefs, action planning and coping planning) predict changes in exercise and diet behaviours over three months and 12 months, (2) the universality assumption of behaviour change theories, i.e. whether preintervention levels and changes in psychosocial determinants are similar among genders and socioeconomic groups, and whether they predict changes in behaviour in a similar way in these groups, (3) whether the personality traits optimism, pessimism, agency and communion predict changes in abdominal obesity, and the nature of their interplay with modifiable and domain-specific psychosocial factors (self-efficacy and social support). Methods: Finnish men and women (N = 385) aged 50 65 years who were at an increased risk for type 2 diabetes were recruited from health care centres to participate in the GOod Ageing in Lahti Region (GOAL) Lifestyle Implementation Trial. The programme aimed to improve participants lifestyle (physical activity, eating) and decrease their overweight. The measurements of self-efficacy, planning, social support and dispositional optimism/pessimism were conducted pre-intervention at baseline (T1) and after the intensive phase of the intervention at three months (T2), and the measurements of exercise at T1, T2 and 12 months (T3) and healthy eating at T1 and T3. Waist circumference, an indicator of abdominal obesity, was measured at T1 and at oneyear (T3) and three-year (T4) follow-ups. Agency and communion were measured at T4 with the Personal Attributes Questionnaire (PAQ). Results: (1) Increases in self-efficacy and planning were associated with three-month increases in exercise (Study I). Moreover, both the post-intervention level and three-month increases (during the intervention) in self-efficacy in dealing with barriers predicted the 12-month increase in exercise, and a high postintervention level of coping plans predicted the 12-month decrease in dietary fat (Study II). One- and three-year waist circumference reductions were predicted by the initial three-month increase in self-efficacy (Studies III, IV). (2) Post-intervention at three months, women had formed more action plans for changing their exercise routines and received less social support for behaviour change than men had. The effects of adoption self-efficacy were similar but change in planning played a less significant role among men (Study I). Examining the effects of socioeconomic status (SES), psychosocial determinants at baseline and their changes during the intervention yielded largely similar results. Exercise barriers self-efficacy was enhanced slightly less among those with low SES. Psychosocial determinants predicted behaviour similarly across all SES groups (Study II). (3) Dispositional optimism and pessimism were unrelated to waist circumference change, directly or indirectly, and they did not influence changes in self-efficacy (Study III). Agency predicted 12-month waist circumference reduction among women. High communion coupled with high social support was associated with waist circumference reduction. However, the only significant predictor of three-year waist circumference reduction was an increase in health-related self-efficacy during the intervention (Study IV). Conclusions: Interventions should focus on improving participants self-efficacy early on in the intervention as well as prompting action and coping planning for health behaviour change. Such changes are likely to be similarly effective among intervention participants regardless of gender and educational level. Agentic orientation may operate via helping women to be less affected by the demands of the self-sacrificing female role and enabling them to assertively focus on their own goals. The earlier mixed results regarding the role of social support in behaviour change may be in part explained by personality traits such as communion.

Linguistic and perceptual processing of communicative cues in Asperger Syndrome

Asperger Syndrome (AS) belongs to autism spectrum disorders where both verbal and non-verbal communication difficulties are at the core of the impairment. Social communication requires a complex use of affective, linguistic-cognitive and perceptual processes. In the four studies included in the current thesis, some of the linguistic and perceptual factors that are important for face-to-face communication were studied using behavioural methods. In all four studies the results obtained from individuals with AS were compared with typically developed age, gender and IQ matched controls. First, the language skills of school-aged children were characterized in detail with standardized tests that measured different aspects of receptive and expressive language (Study I). The children with AS were found to be worse than the controls in following complex verbal instructions. Next, the visual perception of facial expressions of emotion with varying degrees of visual detail was examined (Study II). Adults with AS were found to have impaired recognition of facial expressions on the basis of very low spatial frequencies which are important for processing global information. Following that, multisensory perception was investigated by looking at audiovisual speech perception (Studies III and IV). Adults with AS were found to perceive audiovisual speech qualitatively differently from typically developed adults, although both groups were equally accurate in recognizing auditory and visual speech presented alone. Finally, the effect of attention on audiovisual speech perception was studied by registering eye gaze behaviour (Study III) and by studying the voluntary control of visual attention (Study IV). The groups did not differ in eye gaze behaviour or in the voluntary control of visual attention. The results of the study series demonstrate that many factors underpinning face-to-face social communication are atypical in AS. In contrast with previous assumptions about intact language abilities, the current results show that children with AS have difficulties in understanding complex verbal instructions. Furthermore, the study makes clear that deviations in the perception of global features in faces expressing emotions as well as in the multisensory perception of speech are likely to harm face-to-face social communication.