The functional and biological properties of whey proteins : prospects for the development of functional foods

Selostus: Heraproteiinit terveysvaikutteisten elintarvikkeiden kehittämisessä

Biological contamination of barley and carrots by pathogens in soil fertilized with anthropogenic nutrients

Conference publication in Kalmar ECO-TECH’07. International Conference on Technologies for Waste and Wastewater Treatment, Energy from Waste, Remediation of Contaminated Sites and Emissions Related to Climate November 26-28 2007, Kalmar, Sweden.

Food Safety Testing: Rapid Molecular Methods for Chemical and Biological Hazards

The central goal of food safety policy in the European Union (EU) is to protect consumer health by guaranteeing a high level of food safety throughout the food chain. This goal can in part be achieved by testing foodstuffs for the presence of various chemical and biological hazards. The aim of this study was to facilitate food safety testing by providing rapid and user-friendly methods for the detection of particular food-related hazards. Heterogeneous competitive time-resolved fluoroimmunoassays were developed for the detection of selected veterinary residues, that is coccidiostat residues, in eggs and chicken liver. After a simplified sample preparation procedure, the immunoassays were performed either in manual format with dissociation-enhanced measurement or in automated format with pre-dried assay reagents and surface measurement. Although the assays were primarily designed for screening purposes providing only qualitative results, they could also be used in a quantitative mode. All the developed assays had good performance characteristics enabling reliable screening of samples at concentration levels required by the authorities. A novel polymerase chain reaction (PCR)-based assay system was developed for the detection of Salmonella spp. in food. The sample preparation included a short non-selective pre-enrichment step, after which the target cells were collected with immunomagnetic beads and applied to PCR reaction vessels containing all the reagents required for the assay in dry form. The homogeneous PCR assay was performed with a novel instrument platform, GenomEra^™, and the qualitative assay results were automatically interpreted based on end-point time-resolved fluorescence measurements and cut-off values. The assay was validated using various food matrices spiked with sub-lethally injured Salmonella cells at levels of 1-10 colony forming units (CFU)/25 g of food. The main advantage of the system was the exceptionally short time to result; the entire process starting from the pre-enrichment and ending with the PCR result could be completed in eight hours. In conclusion, molecular methods using state-of-the-art assay techniques were developed for food safety testing. The combination of time-resolved fluorescence detection and ready-to-use reagents enabled sensitive assays easily amenable to automation. Consequently, together with the simplified sample preparation, these methods could prove to be applicable in routine testing.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.

The oxygenation in biological wastewater treatment of pulp and paper industry

The UPM-Kymmene Oyj Pietarsaari pulp and paper Mill biological wastewater treatment plant was built in the 1980's and the plant has been in use ever since. During the past years there have been problems with deviations. The wastewater treatment plant needs update, especially the aeration basin, where the old surface aerators cannot produce enough mixing and indroduce oxygen enough to the wastewater. In this thesis how extra aeration with oxygen affects the wastewater treatment plant effluent was studied. In the literature part the main focus is in aeration devices, which can be used in biological wastewater treatment. The target is to compare different kind of aerators, which are suitable for pulp and paper wastewater treatment. Studies show, that EDI-aerators are commonly used and also most suitable. In the experimental part, the focus is on the Pietarsaari Mills wastewater treatment plant and oxygen aeration during autumn 2008. This thesis presents the results of the trial run. Studies show, that extra oxygen devices can produce lot a of mixing and the oxygenation capacity was more than what the micro-organisms needed. The effect on sludge quality could not been seen during the trial runs.

Disturbance and ecosystem functioning : the role of changes in benthic biological traits

Rapid changes in biodiversity are occurring globally, as a consequence of anthropogenic disturbance. This has raised concerns, since biodiversity is known to significantly contribute to ecosystem functions and services. Marine benthic communities participate in numerous functions provided by soft-sedimentary ecosystems. Eutrophication-induced oxygen deficiency is a growing threat against infaunal communities, both in open sea areas and in coastal zones. There is thus a need to understand how such disturbance affects benthic communities, and what is lost in terms of ecosystem functioning if benthic communities are harmed. In this thesis, the status of benthic biodiversity was assessed for the open Baltic Sea, a system severely affected by broad-scale hypoxia. Long-term monitoring data made it possible to establish quantitative biodiversity baselines against which change could be compared. The findings show that benthic biodiversity is currently severely impaired in large areas of the open Baltic Sea, from the Bornholm Basin to the Gulf of Finland. The observed reduction in biodiversity indicates that benthic communities are structurally and functionally impoverished in several of the sub-basins due to the hypoxic stress. A more detailed examination of disturbance impacts (through field studies and -experiments) on benthic communities in coastal areas showed that changes in benthic community structure and function took place well before species were lost from the system. The degradation of benthic community structure and function was directed by the type of disturbance, and its specific temporal and spatial characteristics. The observed shifts in benthic trait composition were primarily the result of reductions in species’ abundances, or of changes in demographic characteristics, such as the loss of large, adult bivalves. Reduction in community functions was expressed as declines in the benthic bioturbation potential and in secondary biomass production. The benthic communities and their degradation accounted for a substantial proportion of the changes observed in ecosystem multifunctionality. Individual ecosystem functions (i.e. measures of sediment ecosystem metabolism, elemental cycling, biomass production, organic matter transformation and physical structuring) were observed to differ in their response to increasing hypoxic disturbance. Interestingly, the results suggested that an impairment of ecosystem functioning could be detected at an earlier stage if multiple functions were considered. Importantly, the findings indicate that even small-scale hypoxic disturbance can reduce the buffering capacity of sedimentary ecosystem, and increase the susceptibility of the system towards further stress. Although the results of the individual papers are context-dependent, their combined outcome implies that healthy benthic communities are important for sustaining overall ecosystem functioning as well as ecosystem resilience in the Baltic Sea.

Interpreting marine benthic ecosystem functioning in coastal waters: validating the biological trait concept

Coastal areas harbour high biodiversity, but are simultaneously affected by rapid degradations of species and habitats due to human interactions. Such alterations also affect the functioning of the ecosystem, which is primarily governed by the characteristics or traits expressed by the organisms present. Marine benthic fauna is nvolved in numerous functions such as organic matter transformation and transport, secondary production, oxygen transport as well as nutrient cycling. Approaches utilising the variety of faunal traits to assess benthic community functioning have rapidly increased and shown the need for further development of the concept. In this thesis, I applied biological trait analysis that allows for assessments of a multitude of categorical traits and thus evaluation of multiple functional aspects simultaneously. I determined the functional trait structure, diversity and variability of coastal zoobenthic communities in the Baltic Sea. The measures were related to recruitment processes, habitat heterogeneity, large-scale environmental and taxonomic gradients as well as anthropogenic impacts. The studies comprised spatial scales from metres to thousands of kilometres, and temporal scales spanning one season as well as a decade. The benthic functional structure was found to vary within and between seagrass landscape microhabitats and four different habitats within a coastal bay, in papers I and II respectively. Expressions of trait categories varied within habitats, while the density of individuals was found to drive the functional differences between habitats. The findings in paper III unveiled high trait richness of Finnish coastal benthos (25 traits and 102 cateogries) although this differed between areas high and low in salinity and human pressure. In paper IV, the natural reduction in taxonomic richness across the Baltic Sea led to an overall reduction in function. However, functional richness in terms of number of trait categories remained comparatively high at low taxon richness. Changes in number of taxa within trait categories were also subtle and some individual categories were maintained or even increased. The temporal analysis in papers I and III highlighted generalities in trait expressions and dominant trait categories in a seagrass landscape as well as a “type organism” for the northern Baltic Sea. Some initial findings were made in all four papers on the role of common and rare species and traits for benthic community functioning. The findings show that common and rare species may not always express the same trait categories in relation to each other. Rare species in general did not express unique functional properties. In order to advance the understanding of the approach, I also assessed some issues concerning the limitations of the concept. This was conducted by evaluating the link between trait category and taxonomic richness using especially univariate measures. My results also show the need to collaborate nationally and internationally on safeguarding the utility of taxonomic and trait data. The findings also highlight the importance of including functional trait information into current efforts in marine spatial planning and biomonitoring.

Physical Attractiveness as a Signal of Biological Quality

It has been commonly thought that standards of beauty are arbitrary cultural conventions that vary between cultures and time. In my thesis I found that it is not so. Instead, I show that attractiveness and preferred traits serve as cues to phenotypic qualities that provide selective benefits for those who choose their mates based on these criteria. In the first study I show that attractive men have a stronger antibody response to the hepatitis b vaccine and higher levels of testosterone than their less attractive peers. Men with low levels of testosterone also tend to have high levels of the stress hormone cortisol, suggesting that their immune responses may have been inhibited by stress hormones. Thus, facial attractiveness may serve as an honest cue of the strength of immune defence in men. In the second study, I show that the attractiveness of the male body is also a cue of better immunity. In addition, I show that adiposity, both in men’s faces and bodies, is a better cue of the strength of immunity and attractiveness than of masculinity. In the third study, I test the preferences of women from 13 countries for facial cues of testosterone and cortisol. I show that there is cross-cultural variation in women’s preference for cues of testosterone and cortisol in male faces. I found a relationship between the health of a nation and women’s preferences for cues of testosterone in the male face and the interaction between preferences for cues of testosterone and cortisol. I show also a relationship between preferences for cues of testosterone and a societal-level measure of parasite stress. Thus, it seems that societal-level ecological factors influence the relative value of traits as revealed by combinations of testosterone and stress hormones. In the fourth study, I show that women’s immune responsiveness (amount of antibodies produced) does not predict facial attractiveness. Instead, plasma cortisol level is negatively associated with attractiveness, indicating that stressed women look less attractive. Fat percentage is curvilinearly associated with facial attractiveness, indicating that being too thin or too fat reduces attractiveness. This study suggests that in contrast to men, facial attractiveness in women does not indicate the strength of immune defence, but is associated with other aspects of long-term health and fertility: circulating levels of the stress hormone cortisol and the percentage of body fat. In the last study I show that the attractiveness of men’s body odor is positively correlated with stress hormone levels, suggesting also that the attractiveness of body odors may signal the phenotypic quality of males to females. However, the attractiveness of men’s body odor was not associated with testosterone levels. My thesis suggests that the standard of beauty is not in the eye of the beholder. Instead, our standard of beauty is hardwired in our brains by genes that are selected by natural selection and also influenced by current environmental conditions.

Influence of surface functionalization on the behavior of silica nanoparticles in biological systems

Personalized nanomedicine has been shown to provide advantages over traditional clinical imaging, diagnosis, and conventional medical treatment. Using nanoparticles can enhance and clarify the clinical targeting and imaging, and lead them exactly to the place in the body that is the goal of treatment. At the same time, one can reduce the side effects that usually occur in the parts of the body that are not targets for treatment. Nanoparticles are of a size that can penetrate into cells. Their surface functionalization offers a way to increase their sensitivity when detecting target molecules. In addition, it increases the potential for flexibility in particle design, their therapeutic function, and variation possibilities in diagnostics. Mesoporous nanoparticles of amorphous silica have attractive physical and chemical characteristics such as particle morphology, controllable pore size, and high surface area and pore volume. Additionally, the surface functionalization of silica nanoparticles is relatively straightforward, which enables optimization of the interaction between the particles and the biological system. The main goal of this study was to prepare traceable and targetable silica nanoparticles for medical applications with a special focus on particle dispersion stability, biocompatibility, and targeting capabilities. Nanoparticle properties are highly particle-size dependent and a good dispersion stability is a prerequisite for active therapeutic and diagnostic agents. In the study it was shown that traceable streptavidin-conjugated silica nanoparticles which exhibit a good dispersibility could be obtained by the suitable choice of a proper surface functionalization route. Theranostic nanoparticles should exhibit sufficient hydrolytic stability to effectively carry the medicine to the target cells after which they should disintegrate and dissolve. Furthermore, the surface groups should stay at the particle surface until the particle has been internalized by the cell in order to optimize cell specificity. Model particles with fluorescently-labeled regions were tested in vitro using light microscopy and image processing technology, which allowed a detailed study of the disintegration and dissolution process. The study showed that nanoparticles degrade more slowly outside, as compared to inside the cell. The main advantage of theranostic agents is their successful targeting in vitro and in vivo. Non-porous nanoparticles using monoclonal antibodies as guiding ligands were tested in vitro in order to follow their targeting ability and internalization. In addition to the targeting that was found successful, a specific internalization route for the particles could be detected. In the last part of the study, the objective was to clarify the feasibility of traceable mesoporous silica nanoparticles, loaded with a hydrophobic cancer drug, being applied for targeted drug delivery in vitro and in vivo. Particles were provided with a small molecular targeting ligand. In the study a significantly higher therapeutic effect could be achieved with nanoparticles compared to free drug. The nanoparticles were biocompatible and stayed in the tumor for a longer time than a free medicine did, before being eliminated by renal excretion. Overall, the results showed that mesoporous silica nanoparticles are biocompatible, biodegradable drug carriers and that cell specificity can be achieved both in vitro and in vivo.

Comparing adult-youth and adult-adult online sexual solicitation: Manipulative behaviors, situational factors, and outcomes

Online sexual solicitation (solicitation) of youth has received widespread media and research attention during the last decade. The prevalence rates of youth who have experienced solicitation or solicitation attempts vary between studies depending on the methodology used (e.g., whether youth or adults are the target study group). In studies focusing on youth victims, the prevalence of solicitation attempts made by adults during the past year is typically reported to be between 5 and 9%. Adults who solicit youth online have been found to use deception and other manipulative behaviors to gain access to sexual activities with youth. However, previous studies have lacked a control group of adults who solicit other adults online. Without this comparison, one could argue that deceiving others online about one’s identity, and engaging in manipulative behaviors, is an inherent part of most online sexual interactions with strangers. Additionally, little is known about the associations between manipulative behaviors and the solicitation outcomes. In research concerning offline sexual behaviors, it has been noted that situational factors, such as sexual arousal, may alter both sexual interest and behavior. The effects of situational factors on online sexual behaviors have been less extensively studied (especially so with a quantitative approach); no studies have to date focused on adults’ solicitation of youth. Investigating the role of a lowered sexual age preference and the role of situational factors in the soliciting adults could be an important step in order to receive deeper knowledge of the role of traits and states in the context of solicitation. Additionally, there is a lack of knowledge of the effect of the age of the youth. Although previous studies on solicitation has found that older youth, compared with younger youth and children, are more often solicited, the possible reasons for this have not been investigated. Are adults who solicit youth affected by legal deterrence (through the legal age of consent), is it because older youth are more available online, or are the adults’ age preferences merely a product of a normally distributed age preference in the population? The purpose of the present thesis was fivefold: 1) to obtain an estimate of the frequency of adults’ solicitation of youth as self-reported and observed in actual behavior; 2) to explore whether the legal age of consent (LAC) affects solicitation frequency, or whether a normally distributed sexual age preference more accurately describe the proportion of solicited youth of different ages; 3) to investigate the associations of both traits (e.g., lower sexual age preference) and states (immediate situational factors, such as alcohol intoxication), and the solicitation target; 4) to explore whether adults who solicit youth and adults who solicit adults are equally deceitful and manipulative online, and whether the different solicitation outcomes are as common in both groups; and 5) to investigate whether the deceitful and manipulative behaviors engaged in had different associations with the solicitation outcomes depending on the age of the solicited. In the survey study, a convenience sample of 1393 adult participants (aged 18 years or older) self-reported any online communication with strangers during the past year. Of these, 56% (776 respondents) reported that they had solicited or attempted to solicit at least one stranger. Of the respondents, 453 (58.4%) were men, and 323 (41.6%) were women. Participants with only adult contacts (18 years or older) constituted the majority (640 respondents). In contrast, 136 individuals reported a youth contact (a 13 year old or younger, or a 14 to 17-year old). Approximately half of the participants were men in the adult contact group, while 75% of the participants were men in the youth contact group. Approximately 60% of the participants with youth contacts were recruited from two websites associated with a pedophilic sexual interest. In an online quasi-experimental study, with researchers impersonating youth of different ages (10–18 year olds) in chat rooms, 251 online conversations with chat room visitors made up the entire sample. All chat room visitors alleged to be men. The self-reported frequency of having solicited youth (0–17-year olds) during the past year was approximately 10% in our sample of adults who reported communicating with any strangers online. When we observed this behavior in chat rooms, we found that approximately 30% of the chat room visitors who believed they interacted with a 10 to 14 year old attempted to solicit the youth. We found that solicitation attempts increased equally much when increasing the age of the impersonated youth from 14 to 16, as from 16 to 18. Thus, we concluded that a normally distributed age preference in the population was a more plausible explanation to the effect of the age of the solicited, rather than the LAC (here; 15 and 16). If the chat room visitors would have been deterred only by the LAC, we would have expected that the change in amount of solicitation attempts from an illegal age group to a legal age group would have been significantly stronger than changes between age groups within illegal-illegal and legal-legal groups. Our subsample of survey participants from the pedophilia-related websites expectedly reported that they had solicited youth more often in comparison to the sample gathered through general (i.e., not associated with any particular sexual preference) websites. We also found that participants with a youth contact reported higher levels of sexual arousal and shame before the sexual interaction with their online contact, compared with participants with an adult contact. Additionally, the participants with youth contacts who reported consumption of child- and adolescent pornography also reported being more sexually aroused before the interaction, compared to the participants with youth contacts who did not report consumption of these kinds of pornography. We also found clear indications that the online sexual interaction had an alleviatory effect on reported levels of sadness, boredom and stress, independent of the age of the contact. Generally, the participants with youth and adult contacts reported deceiving their contacts as often and suggesting keeping the communication a secret from someone as often. Participants with a youth contact, however, reported using more persuasion techniques for online sexual purposes or for the purpose of an offline meeting, compared to those with an adult contact. In the chat rooms, we found that more indirect ways of future sexual communication (e.g., continuing chatting) was suggested by the chat room visitors that were under the assumption of interacting with youth aged 10 to 14, compared with more direct means (e.g., meeting offline). Survey participants with youth contacts who had used deception, suggested keeping the interactions a secret, and/or persuaded their contact by appealing to the contacts feelings of love and attachment for the participant had also more often engaged in cybersex with the contact. No other manipulative behaviors were associated with the other investigated solicitation outcomes (receiving a sexual picture, meeting offline, and engaging in sexual contact offline) within this group of participants. However, using deception, suggesting secrecy and using persuasion was also positively associated with certain solicitation outcomes within participants with an adult contact. In summary, adults’ solicitation of youth is much more frequent when observed in chat rooms than self-reported. Additionally, an underlying lowered sexual age preference seems to be a motivating factor on a group level in adults who solicit youth. We concluded that directed prevention efforts should be made on pedophiliarelated websites. Additionally, the role of situational factors, especially sexual arousal in persons with a pedo- or hebephilic sexual interest should be investigated further in the context of online sexual solicitation.

New [18F]Tracers for Alzheimer's Disease Imaging. Labeling Synthesis And Biological Testing

Alzheimer’s disease (AD) is the most common form of dementia. Characteristic changes in an AD brain are the formation of β-amyloid protein (Aβ) plaques and neurofibrillary tangles, though other alterations in the brain have also been connected to AD. No cure is available for AD and it is one of the leading causes of death among the elderly in developed countries. Liposomes are biocompatible and biodegradable spherical phospholipid bilayer vesicles that can enclose various compounds. Several functional groups can be attached on the surface of liposomes in order to achieve long-circulating target-specific liposomes. Liposomes can be utilized as drug carriers and vehicles for imaging agents. Positron emission tomography (PET) is a non-invasive imaging method to study biological processes in living organisms. In this study using nucleophilic 18F-labeling synthesis, various synthesis approaches and leaving groups for novel PET imaging tracers have been developed to target AD pathology in the brain. The tracers were the thioflavin derivative [18F]flutemetamol, curcumin derivative [18F]treg-curcumin, and functionalized [18F]nanoliposomes, which all target Aβ in the AD brain. These tracers were evaluated using transgenic AD mouse models. In addition, 18F-labeling synthesis was developed for a tracer targeting the S1P3 receptor. The chosen 18F-fluorination strategy had an effect on the radiochemical yield and specific activity of the tracers. [18F]Treg-curcumin and functionalized [18F]nanoliposomes had low uptake in AD mouse brain, whereas [18F]flutemetamol exhibited the appropriate properties for preclinical Aβ-imaging. All of these tracers can be utilized in studies of the pathology and treatment of AD and related diseases.