Molecular Characteristics of Neuroblastoma with Special Reference to Novel Prognostic Factors and Diagnostic Applications

Molecular Characteristics of Neuroblastoma with Special Reference to Novel Prognostic Factors and Diagnostic Applications Department of Medical Biochemistry and Genetics Annales Universitatis Turkuensis, Medica-Odontologica, 2009, Turku, Finland Painosalama Oy, Turku, Finland 2009 Background: Neuroblastoma, which is the most common and extensively studied childhood solid cancer, shows a great clinical and biological heterogeneity. Most of the neuroblastoma patients older than one year have poor prognosis despite intensive therapies. The hallmark of neuroblastoma, biological heterogeneity, has hindered the discovery of prognostic tumour markers. At present, few molecular markers, such as MYCN oncogene status, have been adopted into clinical practice. Aims: The aim of the study was to improve the current prognostic methodology of neuroblastoma, especially by taking cognizance of the biological heterogeneity of neuroblastoma. Furthermore, unravelling novel molecular characteristics which associate with neuroblastoma tumour progression and cell differentiation was an additional objective. Results: A new strictly defined selection of neuroblastoma tumour spots of highest proliferation activity, hotspots, appeared to be representative and reliable in an analysis of MYCN amplification status using a chromogenic in situ hybridization technique (CISH). Based on the hotspot tumour tissue microarray immunohistochemistry and high-resolution oligo-array-based comparative genomic hybridization, which was integrated with gene expression and in silico analysis of existing transcriptomics, a polysialylated neural cell adhesion molecule (NCAM) and poorly characterized amplicon at 12q24.31 were discovered to associate with outcome. In addition, we found that a previously considered new neuroblastoma treatment target, the mutated c-kit receptor, was not mutated in neuroblastoma samples. Conclusions: Our studies indicate polysialylated NCAM and 12q24.31 amplicon to be new molecular markers with important value in prognostic evaluation of neuroblastoma. Moreover, the presented hotspot tumour tissue microarray method together with the CISH technique of the MYCN oncogene copy number is directly applicable to clinical use. Key words: neuroblastoma, polysialic acid, neural cell adhesion molecule, MYCN, c-kit, chromogenic in situ hybridization, hotspot

Biomarkers, medical treatment and fetal intrapartum surveillance in intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder characterized by maternal pruritus and elevated liver enzymes. It usually begins in the third trimester of pregnancy and resolves spontaneously after delivery. ICP is considered benign for the pregnant woman, but it is associated with an increased risk for unexplained term stillbirth and preterm delivery. There are no specific laboratory markers to diagnose ICP. The diagnosis is currently based on the presence of maternal pruritus and elevated values of alanine aminotransaminases (ALT) and serum bile acids (BA). Recently, ursodeoxycholic acid (UDCA) has been used for treatment. Mechanisms leading to intrauterine fetal death (IUFD) may be multifactorial and are unknown at present. For this thesis, 415 pregnant women with ICP were studied. The aim was to evaluate the value of the liver enzyme glutathione S-transferase alpha (GSTA) as a specific marker of ICP and to assess the effect of maternal UDCA therapy on maternal laboratory values and fetal outcome. The specific markers predisposing the fetus to heart arrhythmia were studied by comparing waveform analysis of fetal electrocardiograms (FECG) during labor in pregnancies complicated by ICP with controls. The levels of maternal GSTA were high and the values correlated with the value of ALT in patients with ICP. UDCA therapy reduced the values of the liver enzymes and alleviated maternal pruritus, but it did not influence maternal hormonal values. Although the newborns experienced an uneventful perinatal outcome, severe ICP was still associated with preterm birth and admission to the neonatal intensive care unit (NICU). There were no significant differences in intrapartum FECG findings between fetuses born to ICP women and controls.

University students’ regulation of learning and text processing – examples from medical and teacher education

The general aim of the thesis was to study university students’ learning from the perspective of regulation of learning and text processing. The data were collected from the two academic disciplines of medical and teacher education, which share the features of highly scheduled study, a multidisciplinary character, a complex relationship between theory and practice and a professional nature. Contemporary information society poses new challenges for learning, as it is not possible to learn all the information needed in a profession during a study programme. Therefore, it is increasingly important to learn how to think and learn independently, how to recognise gaps in and update one’s knowledge and how to deal with the huge amount of constantly changing information. In other words, it is critical to regulate one’s learning and to process text effectively. The thesis comprises five sub-studies that employed cross-sectional, longitudinal and experimental designs and multiple methods, from surveys to eye tracking. Study I examined the connections between students’ study orientations and the ways they regulate their learning. In total, 410 second-, fourth- and sixth-year medical students from two Finnish medical schools participated in the study by completing a questionnaire measuring both general study orientations and regulation strategies. The students were generally deeply oriented towards their studies. However, they regulated their studying externally. Several interesting and theoretically reasonable connections between the variables were found. For instance, self-regulation was positively correlated with deep orientation and achievement orientation and was negatively correlated with non-commitment. However, external regulation was likewise positively correlated with deep orientation and achievement orientation but also with surface orientation and systematic orientation. It is argued that external regulation might function as an effective coping strategy in the cognitively loaded medical curriculum. Study II focused on medical students’ regulation of learning and their conceptions of the learning environment in an innovative medical course where traditional lectures were combined wth problem-based learning (PBL) group work. First-year medical and dental students (N = 153) completed a questionnaire assessing their regulation strategies of learning and views about the PBL group work. The results indicated that external regulation and self-regulation of the learning content were the most typical regulation strategies among the participants. In line with previous studies, self-regulation wasconnected with study success. Strictly organised PBL sessions were not considered as useful as lectures, although the students’ views of the teacher/tutor and the group were mainly positive. Therefore, developers of teaching methods are challenged to think of new solutions that facilitate reflection of one’s learning and that improve the development of self-regulation. In Study III, a person-centred approach to studying regulation strategies was employed, in contrast to the traditional variable-centred approach used in Study I and Study II. The aim of Study III was to identify different regulation strategy profiles among medical students (N = 162) across time and to examine to what extent these profiles predict study success in preclinical studies. Four regulation strategy profiles were identified, and connections with study success were found. Students with the lowest self-regulation and with an increasing lack of regulation performed worse than the other groups. As the person-centred approach enables us to individualise students with diverse regulation patterns, it could be used in supporting student learning and in facilitating the early diagnosis of learning difficulties. In Study IV, 91 student teachers participated in a pre-test/post-test design where they answered open-ended questions about a complex science concept both before and after reading either a traditional, expository science text or a refutational text that prompted the reader to change his/her beliefs according to scientific beliefs about the phenomenon. The student teachers completed a questionnaire concerning their regulation and processing strategies. The results showed that the students’ understanding improved after text reading intervention and that refutational text promoted understanding better than the traditional text. Additionally, regulation and processing strategies were found to be connected with understanding the science phenomenon. A weak trend showed that weaker learners would benefit more from the refutational text. It seems that learners with effective learning strategies are able to pick out the relevant content regardless of the text type, whereas weaker learners might benefit from refutational parts that contrast the most typical misconceptions with scientific views. The purpose of Study V was to use eye tracking to determine how third-year medical studets (n = 39) and internal medicine residents (n = 13) read and solve patient case texts. The results revealed differences between medical students and residents in processing patient case texts; compared to the students, the residents were more accurate in their diagnoses and processed the texts significantly faster and with a lower number of fixations. Different reading patterns were also found. The observed differences between medical students and residents in processing patient case texts could be used in medical education to model expert reasoning and to teach how a good medical text should be constructed. The main findings of the thesis indicate that even among very selected student populations, such as high-achieving medical students or student teachers, there seems to be a lot of variation in regulation strategies of learning and text processing. As these learning strategies are related to successful studying, students enter educational programmes with rather different chances of managing and achieving success. Further, the ways of engaging in learning seldom centre on a single strategy or approach; rather, students seem to combine several strategies to a certain degree. Sometimes, it can be a matter of perspective of which way of learning can be considered best; therefore, the reality of studying in higher education is often more complicated than the simplistic view of self-regulation as a good quality and external regulation as a harmful quality. The beginning of university studies may be stressful for many, as the gap between high school and university studies is huge and those strategies that were adequate during high school might not work as well in higher education. Therefore, it is important to map students’ learning strategies and to encourage them to engage in using high-quality learning strategies from the beginning. Instead of separate courses on learning skills, the integration of these skills into course contents should be considered. Furthermore, learning complex scientific phenomena could be facilitated by paying attention to high-quality learning materials and texts and other support from the learning environment also in the university. Eye tracking seems to have great potential in evaluating performance and growing diagnostic expertise in text processing, although more research using texts as stimulus is needed. Both medical and teacher education programmes and the professions themselves are challenging in terms of their multidisciplinary nature and increasing amounts of information and therefore require good lifelong learning skills during the study period and later in work life.

Micronization of Pharmaceuticals and Food Ingredients using Supercritical Fluid Techniques

Micronization techniques based on supercritical fluids (SCFs) are promising for the production of particles with controlled size and distribution. The interest of the pharmaceutical field in the development of SCF techniques is increasing due to the need for clean processes, reduced consumption of energy, and to their several possible applications. The food field is still far from the application of SCF micronization techniques, but there is increasing interest mainly for the processing of products with high added value. The aim of this study is to use SCF micronization techniques for the production of particles of pharmaceuticals and food ingredients with controlled particle size and morphology, and to look at their production on semi-industrial scale. The results obtained are also used to understand the processes from the perspective of broader application within the pharmaceutical and food industries. Certain pharmaceuticals, a biopolymer and a food ingredient have been tested using supercritical antisolvent micronization (SAS) or supercritical assisted atomization (SAA) techniques. The reproducibility of the SAS technique has been studied using physically different apparatuses and on both laboratory and semi-industrial scale. Moreover, a comparison between semi-continuous and batch mode has been performed. The behaviour of the system during the SAS process has been observed using a windowed precipitation vessel. The micronized powders have been characterized by particle size and distribution, morphology and crystallinity. Several analyses have been performed to verify if the SCF process modified the structure of the compound or caused degradation or contamination of the product. The different powder morphologies obtained have been linked to the position of the process operating point with respect to the vapour-liquid equilibrium (VLE) of the systems studied, that is, mainly to the position of the mixture critical point (MCP) of the mixture. Spherical micro, submicro- and nanoparticles, expanded microparticles (balloons) and crystals were obtained by SAS. The obtained particles were amorphous or with different degrees of crystallinity and, in some cases, had different pseudo-polymorphic or polymorphic forms. A compound that could not be processed using SAS was micronized by SAA, and amorphous particles were obtained, stable in vials at room temperature. The SCF micronization techniques studied proved to be effective and versatile for the production of particles for several uses. Furthermore, the findings of this study and the acquired knowledge of the proposed processes can allow a more conscious application of SCF techniques to obtain products with the desired characteristics and enable the use of their principles for broader applications.

Removal of Cadmium, Lead and Arsenic from Water by Lactic Acid Bacteria

A number of contaminants such as arsenic, cadmium and lead are released into the environment from natural and anthropogenic sources contaminating food and water. Chronic oral ingestion of arsenic, cadmium and lead is associated with adverse effects in the skin, internal organs and nervous system. In addition to conventional methods, biosorption using inactivated biomasses of algae, fungi and bacteria has been introduced as a novel method for decontamination of toxic metals from water. The aim of this work was to evaluate the applicability of lactic acid bacteria as tools for heavy metal removal from water and characterize their properties for further development of a biofilter. The results established that in addition to removal of mycotoxins, cyanotoxins and heterocyclic amines, lactic acid bacteria have a capacity to bind cationic heavy metals, cadmium and lead. The binding was found to be dependent on the bacterial strain and pH, and occurred rapidly on the bacterial surface, but was reduced in the presence of other cationic metals. The data demonstrates that the metals were bound by electrostatic interactions to cell wall components. Transmission electron micrographs showed the presence of lead deposits on the surface of biomass used in the lead binding studies, indicating involvement of another uptake/binding mechanism. The most efficient strains bound up to 55 mg Cd and 176 mg Pb / g dry biomass. A low removal of anionic As(V) was also observed after chemical modification of the cell wall. Full desorption of bound cadmium and lead using either dilute HNO₃ or EDTA established the reversibility of binding. Removal of both metals was significantly reduced when biomass regenerated with EDTA was used. Biomass regenerated with dilute HNO3 retained its cadmium binding capacity well, but lead binding was reduced. The results established that the cadmium and lead binding capacity of lactic acid bacteria, and factors affecting it, are similar to what has been previously observed for other biomasses used for the same purpose. However, lactic acid bacteria have a capacity to remove other aqueous contaminants such as cyanotoxins, which may give them an additional advantage over the other alternatives. Further studies focusing on immobilization of biomass and the removal of several contaminants simultaneously using immobilized bacteria are required.

The Future of Mobile Services and Wireless Applications in the Finnish Healthcare Sector

Työn päätavoitteena oli tutkia mobiilipalveluita ja langattomia sovelluksia Suomen terveydenhuollon sektorilla. Tutkimus havainnollistaa avain-alueita, missä mobiilipalvelut ja langattomat sovellukset voivat antaa lisäarvoa perinteiseen lääketieteen harjoittamiseen, ja selvittää, mitkä ovat tähän kehitykseen liittyvät suurimmat ongelmat ja uhkat sekä tutkimustuloksiin pohjautuvat mahdolliset palvelut ja sovellukset 5-10 vuoden kuluttua. Tutkimus oli luonteeltaan kvalitatiivinen ja tutkimuksen toteuttamiseen valittiin tulevaisuudentutkimus ja erityisesti yksi sen menetelmistä, delfoi-menetelmä. Tutkimuksen aineisto kerättiin kahdelta puolistrukturoidulta haastattelukierrokselta. Työn empiirinen osuus keskittyi kuvailemaan Suomen terveydenhuollon sektoria, siinä meneillään olevia projekteja sekä teknisiä esteitä. Lisäksi pyrittiin vastaamaan tutkimuksen pääkysymykseen. Tutkimustulokset osoittivat, että tärkeät alueet, joihin langaton kommunikaatio tulisi vaikuttamaan merkittävästi, ovat ensiaputoiminta, kroonisten potilaiden etämonitorointi, välineiden kehittäminen langattomaan kommunikaatioon kotihoidon parantamiseksi ja uusien toimintamallien luomiseksi sekä lääketieteellinen yhteistyö jakamalla terveydenhuoltoon liittyvät informaation lähteet. Työn tulosten perusteellavoitiin antaa myös muutamia toimenpide-ehdotuksia jatkotutkimuksia varten.

Characterizing Propionibacterium freudenreichii ssp. shermanii JS and Lactobacillus rhamnosus LC705 as a new probiotic combination: basic properties of JS and pilot in vivo assessment of the combination

Characterizing Propionibacterium freudenreichii ssp. shermanii JS and Lactobacillus rhamnosus LC705 as a new probiotic combination: basic properties of JS and pilot in vivo assessment of the combination Each candidate probiotic strain has to have the documentation for the proper identification with current molecular tools, for the biological properties, for the safety aspects and for the health benefits in human trials if the intention is to apply the strain as health promoting culture in the commercial applications. No generalization based on species properties of an existing probiotic are valid for any novel strain, as strain specific differences appear e.g. in the resistance to GI tract conditions and in health promoting benefits (Madsen, 2006). The strain evaluation based on individual strain specific probiotic characteristics is therefore the first key action for the selection of the new probiotic candidate. The ultimate goal in the selection of the probiotic strain is to provide adequate amounts of active, living cells for the application and to guarantee that the cells are physiologically strong enough to survive and be biologically active in the adverse environmental conditions in the product and in GI tract of the host. The in vivo intervention studies are expensive and time consuming; therefore it is not rational to test all the possible candidates in vivo. Thus, the proper in vitro studies are helping to eliminate strains which are unlikely to perform well in vivo. The aims of this study were to characterize the strains of Propionibacterium freudenreichii ssp. shermanii JS and Lactobacillus rhamnosus LC705, both used for decades as cheese starter cultures, for their technological and possible probiotic functionality applied in a combined culture. The in vitro studies of Propionibacterium freudenreichii ssp. shermanii JS focused on the monitoring of the viability rates during the acid and bile treatments and on the safety aspects such as antibiotic susceptibility and adhesion. The studies with the combination of the strains JS and LC705 administered in fruit juices monitored the survival of the strains JS and LC705 during the GI transit and their effect on gut wellbeing properties measured as relief of constipation. In addition, safety parameters such as side effects and some peripheral immune parameters were assessed. Separately, the combination of P. freudenreichii ssp. shermanii JS and Lactobacillus rhamnosus LC705 was evaluated from the technological point of view as a bioprotective culture in fermented foods and wheat bread applications. In this study, the role ofP. freudenreichii ssp. shermanii JS as a candidate probiotic culture alone and in a combination with L. rhamnosus LC705 was demonstrated. Both strains were transiently recovered in high numbers in fecal samples of healthy adults during the consumption period. The good survival through the GI transit was proven for both strains with a recovery rate from 70 to 80% for the JS strain and from 40 to 60% for the LC705 strain from the daily dose of 10 log10 CFU. The good survival was shown from the consumption of fruit juices which do not provide similar matrix protection for the cells as milk based products. The strain JS did not pose

Sensory and Non-sensory Factors Behind the Liking and Choice of Healthy Food Products

A healthy and balanced diet can reduce health problems, such as overweight and metabolic syndrome. In general, people have a considerably good knowledge of what constitutes a healthy diet and how they could achieve it with their food choices. Besides, people argue that health is among their top five food choice motives. Nevertheless, the prevalence of overweight is increasing and other food choice motives, such as taste, seem to conflict with the health. Liking for food does not necessarily determine acceptance alone, thus several non-sensory factors, such as brand, country of origin and nutrition claim, can also influence. Moreover, consumers are individuals in how they prioritize sensory and nonsensory factors of foods, but e.g. increasing age, female gender and health concern have been connected to a more health-oriented dietary behaviour. To sum up, identifying different factors that can increase the liking and consumption of healthy food is essential in order to develop more attractive healthful food products. Adding vitamins, minerals, fibre or other ingredients to a food product can be used to enrich the nutritional quality of the products. However, this may be difficult in practice as regards the sensory quality and pleasantness of the foods. Generally, consumers are not willing to compromise on taste in food. On the other hand, consumers are very heterogeneous in their likings, and their personal values and attitudes may interact with preferences for specific sensory characteristics. The aims of this study were to investigate the effects of intrinsic product characteristics on sensory properties and hedonic responses; to determine the impact of few non-sensory factors; and to examine the interaction between sensory and non-sensory factors with consumers’ demographics, values and attitudes in liking of healthy model foods. The results showed that product composition influenced sensory quality and had an effect on hedonic responses. Adding flaxseed to bakery products showed a significant improvement in the nutritional quality without negative effects on sensory properties. On the other hand, the fortification of wellness beverages with vitamins and minerals may impart off-flavours. In general, sweetness of yoghurts, freshness of wellness beverages and low intensity of rye bread flavour appealed to consumers. Information about the domestic origin of yoghurts and claiming a specific function for wellness beverages enhanced liking. However, consumers who were more concerned about their health and considered natural content as an important food choice motive, rated sourer and less sweet yoghurts and wellness beverages as more pleasant. In addition, interest in health increased the consumption of rye breads and other whole grain breads among adolescents. The results showed that the optimal product quality in terms of intrinsic and extrinsic factors differs between individual consumers, and personal values and food choice motives can be connected to preferences for specific sensory characteristics of foods. This indicates that each food product needs to be considered in relation to its specific market niche, and to which segment of consumer will respond most positively to its characteristics.

Foodstuffs and medicines as legal categories in the EU and China. Functional foods as a borderline case

The thesis discusses the regulation of foodstuffs and medicines, and particularly the regulation of functional foods. Legal systems investigated are the EU and China. Both are members of the WTO and Codex Alimentarius, which binds European and Chinese rules together. The study uses three Chinese berries as case examples of how product development faces regulation in practice. The berries have traditional uses as herbal medicines. Europe and China have similar nutrition problems to be resolved, such as obesity, cardiovascular disease, and diabetes. The three berries might be suitable raw materials for functional foods. Consumer products with health-enhancing functions, such as lowering blood pressure, might legally be classifi ed either as foodstuffs or medicines. The classifi cation will depend on functions and presentation of the product. In our opinion, food and medicine regulation should come closer together so the classifi cation issue would no longer be an issue. Safety of both foodstuffs and medicines is strictly regulated. With medicines, safety is a more relative concept, where benefi ts of the product are compared to side-effects in thorough scientifi c tests and trials. Foods, on the other hand, are not allowed to have side-effects. Hygiene rules and rules on the use of chemicals apply. In China, food safety is currently at focus as China has had several severe food scandals. Newly developed foods are called novel foods, and are specifi cally regulated. The current European novel food regulation from 1997 treats traditional third country products as novel. The Chinese regulation of 2007 also defi nes novel foods as something unfamiliar to a Chinese consumer. The concepts of novel food thus serve a protectionist purpose. As regards marketing, foods are allowed to bear health claims, whereas medicines bear medicinal claims. The separation is legally strict: foods are not to be presented as having medicinal functions. European nutrition and health claim regulation exists since 2006. China also has its regulation on health foods, listing the permitted claims and how to substantiate them. Health claims are allowed only on health foods. The European rules on medicines include separate categories for herbal medicines, traditional herbal medicines, and homeopathic medicines, where there are differing requirements for scientifi c substantiation. The scientifi c and political grounds for the separate categories provoke criticism. At surface, the Chinese legal system seems similar to the European one. To facilitate trade, China has enacted modern laws. Laws are needed as the country moves from planned economy to market economy: ‘rule of law’ needs to replace ‘rule of man’. Instead of being citizens, Chinese people long were subordinates to the Emperor. Confucius himself advised to avoid confl ict. Still, Chinese people do not and cannot always trust the legal system, as laws are enforced in an inconsistent manner, and courts are weak. In China, there have been problems with confl icting national and local laws. In Europe, the competence of the EU vs. the competence of the Member States is still not resolved, even though the European Commission often states that free trade requires harmonisation. Food and medicine regulation is created by international organisations, food and medicine control agencies, standards agencies, companies and their organisations. Regulation can be divided in ‘hard law’ and ‘soft law’. One might claim that hard law is in crisis, as soft law is gaining importance. If law is out of fashion, regulation certainly isn’t. In the future, ‘law’ might mean a process where rules and incentives are created by states, NGOs, companies, consumers, and other stakeholders. ‘Law’ might thus refer to a constant negotiation between public and private actors. Legal principles such as transparency, equal treatment, and the right to be heard would still be important.

Interactions between Polyunsaturated Fatty Acids and Probiotics

The prevalence of inflammatory based diseases has increased in industrialized countries over the last decades. For allergic diseases, two primary hypotheses have been proposed to explain this phenomenon, namely the hygiene and dietary evolution based hypothesis. Particularly, the reduced early exposure to microbes and an increase in the amount of polyunsaturated fatty acids (especially n-6 PUFA) in the diet have been discussed. Often, these two factors have been studied independently, even though both factors have been shown to possess potential health benefits and their mode of action to share similar mechanisms. The hypothesis of the present study was that demonstrate that PUFA and probiotics are not separate entities as such but do interact with each other. In the present study, we investigated whether maternal diet and atopic status influence the PUFA composition of breast milk and serum fatty acids of infants, and whether the fatty acid absorption and utilization of infant formula fatty acids is affected by supplementation of infant formula with probiotic bacteria (Lactobacillus GG and Bifidobacterium lactis Bb-12). Moreover, we investigated the mechanisms by which different PUFA influence the physicochemical and functional properties of probiotics as well as functionality of epithelial cells in vitro. We demonstrated a carry-over effect of dietary fatty acids from maternal diet via breast milk into infants’ serum lipid fatty acids. Our data confirmed the previously shown allergy –related PUFA level imbalances, though it did not fully support the impaired desaturation and elongation capacity hypothesis. We also showed that PUFA incorporation into phospholipids of infants was influenced by probiotics in infant formula in a strain dependent manner. Especially,Bifidobacterium lactis Bb-12 in infant formula promoted the utilization of n-3 PUFA. Mechanistically, we demonstrated that probiotics (Lactobacillus GG, Lactobacillus casei Shirota and Lactobacillus bulgaricus) did incorporate and interconvert exogenous free PUFA in the growth medium into bacterial fatty acids strain and PUFA dependently. In general, high concentrations of free PUFA inhibited the growth and mucus adhesion of probiotics, whereas low concentrations of specific long chain PUFA were found to promote the growth and mucus adhesion of Lactobacillus casei Shirota. These effects were paralleled with only minor alterations in hydrophobicity and electron donor – electron acceptor properties of lactobacilli. Furthermore, free PUFA were also demonstrated to alter the adhesion capacity of the intestinal epithelial cells; n-6 PUFA tended to inhibit the Caco-2 adhesion of probiotics, whereas n-3 PUFA had either no or minor effects or even promote the bacterial adhesion (especially Lactobacillus casei Shirota) to PUFA treated Caco-2 cells. The results of this study demonstrate the close and bilateral interactions between dietary PUFA and probiotics. Probiotics were shown to influence the absorption and utilization of dietary PUFA, whereas PUFA were shown to alter the functional properties of both probiotics and mucosal epithelia. These findings suggest that a more thorough understanding of interactions between PUFA and intestinal microbiota is a prerequisite, when the beneficial effects of new functional foods containing probiotics are designed and planned for human intervention studies.

Berry polyphenol absorption and the effect of northern berries on metabolism, ectopic fat accumulation, and associated diseases

The prevalence of obesity and type 2 diabetes has increased at an alarming rate in developed countries. It seems in the light of current knowledge that metabolic syndrome may not develop at all without NAFLD, and NAFLD is estimated to be as common as metabolic syndrome in western population (23 % occurrence). Fat in the liver is called ectopic fat, which is triacylglycerols within the cells of non-adipose tissue. Serum alanine aminotransferase (ALT) values correlate positively with liver fat proportions, and increased activity of ALT predicts type 2 diabetes independently from obesity. Berries, high in natural bioactive compounds, have indicated the potential to reduce the risk of obesity-related diseases. Ectopic fat induces common endocrine excretion of adipose tissue resulting in the overproduction of inflammatory markers, which further induce insulin resistance by multiple mechanisms. Insulin resistance inducing hyperinsulinemia and lipolysis in adipocytes increases the concentration of free fatty acids and consequently causes further fat accumulation in hepatocytes. Polyphenolic fractions of berries have been shown to reverse inflammatory reaction cascades in in vitro and animal studies, and moreover to decrease ectopic fat accumulation. The aim of this thesis was to explore the role of northern berries in obesity-related diseases. The absorption and metabolism of selected berry polyphenols, flavonol glycosides and anthocyanins, was investigated in humans, and metabolites of the studied compounds were identified in plasma and urine samples (I, II). Further, the effects of berries on the risk factors of metabolic syndrome were studied in clinical intervention trials (III, IV), and the different fractions of sea buckthorn berry were tested for their ability to reduce postprandial glycemia and insulinemia after high-glucose meal in a postprandial study with humans (V). The marked impact of mixed berries on plasma ALT values (III), as well as indications of the positive effects of sea buckthorn, its fractions and bilberry on omental adiposity and adhesion molecules (IV) were observed. In study V, sea buckthorn and its polyphenol fractions had a promising effect on potprandial metabolism after high-glucose meal. In the literature review, the possible mechanisms behind the observed effects have been discussed with a special emphasis on ectopic fat accumulation. The literature review indicated that especially tannins and flavonoids have shown potential in suppressing diverse reaction cascades related to systemic inflammation, ectopic fat accumulation and insulin resistance development.

The health effects of sea buckthorn berries and oil

Sea buckthorn (Hippophaë) berries are ingredients of the Chinese traditional medicine. In addition to China, they are nowadays cultivated for food in several European countries, Russia, Canada, the USA, and Japan. Sea buckthorn berries are a rich source of flavonoids, mainly flavonol glycosides and proanthocyanidins. Depending on the genetic background, growth conditions, and ripeness of the berries, vitamin C concentrations up to over 1 g/100 ml juice, have been reported. Sea buckthorn berries contain inositols and methyl inositols, components of messenger molecules in humans. Sea buckthorn seed oil is rich in essential aplha-linolenic and linoleic acids, whereas the most abundant fatty acids in the berry oil are palmitoleic, palmitic and oleic acids. Other potentially beneficial lipophilic compounds of sea buckthorn seeds and berries include carotenoids, phytosterols, tocopherols and tocotrienols. The effects of sea buckthorn fractions on inflammation, platelet aggregation, oxidation injuries, the liver, skin and mucosa, among others, have been reported. The aim of the thesis work was to investigate the health effects of sea buckthorn berries and oil in humans. The physiological effects of sea buckthorn berries, berry components, and oil have mostly been studied in vitro and in animal models, leaving a demand for more clinical trials. In the first randomized, placebo-controlled trial of this thesis healthy adults consumed 28 g/day of sea buckthorn berries for three months. The main objective was to investigate the effects on the common cold. In addition, effects on other infections, inflammation and circulating lipid markers associated with cardiovascular disease risk were studied. In the second randomized, placebocontrolled trial participants reporting dry eye symptoms consumed 2 g/day of sea buckthorn oil from the seeds and berries for three months. The effects on symptoms and clinical signs of dry eye were monitored. In addition, the effects on circulating markers of inflammation and liver functions were analyzed. Sea buckthorn berries did not affect the common cold or other infections in healthy adults. However, a decrease in serum C-reactive protein was detected, indicating effects on inflammation. Fasting concentrations of serum flavonols, typical to sea buckthorn berry, increased without affecting the circulating total, HDL, LDL cholesterol, or triacylglycerol concentrations. Tear film hyperosmolarity and activation of inflammation at the ocular surface are among the core mechanisms of dry eye. Combined sea buckthorn berry and seed oil attenuated the rise in tear film osmolarity taking place during the cold season. It also positively affected some of the dry eye symptoms. Based on the tear film fatty acid analysis, the effects were not mediated through direct incorporation of sea buckthorn oil fatty acids to tear film lipids. It is likely that the fatty acids, carotenoids, tocopherols and tocotrienols of sea buckthorn oil affected the inflammation of the ocular surface, lacrimal and/or meibomian glands. The effects on the differentiation of meibomian gland cells are also possible. Sea buckthorn oil did not affect the serum concentrations of inflammation markers or liver enzymes investigated. In conclusion, this thesis work suggests positive effects of sea buckthorn berries and oil on inflammation and dry eye, respectively, in humans.

Identifying and Characterizing New Ingredients in vitro for Prebiotic and Synbiotic use

The endogenous microbiota, constituting the microbes that live inside and on humans, is estimated to outnumber human cells by a factor of ten. This commensal microbial population has an important role in many physiological functions, with the densest microbiota population found in the colon. The colonic microbiota is a highly complex and diverse bacterial ecosystem, and a delicate balance exists between the gut microbiota and its host. An imbalance in the microbial ecosystem may lead to severe symptoms in and also beyond the gastrointestinal tract. Due to the important role of the gut microbiota in human health, means of its modification have been introduced in the dietary concepts of pro-, pre- and synbiotics. Prebiotics, which are usually carbohydrates, strive to selectively influence beneficial microbes resident in the colon with the aim of modifying the composition and functionality of the commensal microbial population towards a purportedly healthier one. The study of prebiotic effects on colonic micro-organisms is typically done by using human faecal material, though this provides relatively little information on bacterial populations and metabolic events in different parts of the colon. For this reason, several in vitro models have been developed to investigate the gut microbiota. The aim of this doctoral thesis was to screen through some of the promising prebiotic candidates, characterize their effects on the microbiota through the use of two in vitro methods (pure microbial cultures and a colon simulator model) and to evaluate their potential as emerging prebiotics or synbiotics when combined with the probiotic Bifidobacterium lactis . As a result of the screening work and subsequent colon simulation studies, several compounds with promising features were identified. Xylo-oligosaccharides (XOS), which have previously already shown promise as prebiotic compounds, were well fermented by several probiotic Bifidobacterium lactis strains in pure culture studies and in the following simulation studies utilizing the complex microbiota by endogenous B. lactis Another promising compound was panose, a trisaccharide belonging to isomalto-oligosaccharides (IMO) that also was also able to modify the microbiota in vitro by increasing the number of beneficial microbes investigated. Panose has not been widely studied previously and therefore, this thesis work provided the first data on panose fermentation in mixed colonic microbiota. Galacto-oligosaccharide (GOS) is an established prebiotic, and it was studied here in conjunction with another potential polygosaccharide polydextrose (PDX) and probiotic B. lactis Bi-07. In this final study, the synbiotics including GOS were more effective than the constituting pro- or prebiotics alone in modulating the microbiota composition, thus indicating a synergy resulting from the combination. The results obtained in this in vitro work can be, and have already been, utilized in product development aimed at the nutritional modification of the human colonic microbiota. Some of the compounds have entered the human clinical intervention phase to nvestigate in more detail the prebiotic and synbiotic properties seen in these in vitro studies.