A platform for anti-biofilm assays combining viability, biomass and matrix quantifications in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Bacteria can exist as planktonic, the lifestyle in which single cells exist in suspension, and as biofilms, which are surface-attached bacterial communities embedded in a selfproduced matrix. Most of the antibiotics and the methods for antimicrobial work have been developed for planktonic bacteria. However, the majority of the bacteria in natural habitats live as biofilms. Biofilms develop dauntingly fast high resistance towards conventional antibacterial treatments and thus, there is a great need to meet the demands of effective anti-biofilm therapy. In this thesis project it was attempted to fill the void of anti-biofilm screening methods by developing a platform of assays that evaluate the effect that screened compounds have on the total biomass, viability and the extracellular polysaccharide (EPS) layer of the biofilms. Additionally, a new method for studying biofilms and their interactions with compounds in a continuous flow system was developed using capillary electrochromatography (CEC). The screening platform was utilized with a screening campaign using a small library of cinchona alkaloids. The assays were optimized to be statistically robust enough for screening. The first assay, based on crystal violet staining, measures total biofilm biomass, and it was automated using a liquid handling workstation to decrease the manual workload and signal variation. The second assay, based on resazurin staining, measures viability of the biofilm, and it was thoroughly optimized for the strain used, but was then a very simple and fast method to be used for primary screening. The fluorescent resazurin probe is not toxic to the biofilms. In fact, it was also shown in this project that staining the biofilms with resazurin prior to staining with crystal violet had no effect on the latter and they can be used in sequence on the same screening plate. This sequential addition step was indeed a major improvement on the use of reagents and consumables and also shortened the work time. As a third assay in the platform a wheat germ agglutinin based assay was added to evaluate the effect a compound has on the EPS layer. Using this assay it was found that even if compounds might have clear effect on both biomass and viability, the EPS layer can be left untouched or even be increased. This is a clear implication of the importance of using several assays to be able to find “true hits” in a screening setting. In the pilot study of screening for antimicrobial and anti-biofilm effects using a cinchona alkaloid library, one compound was found to have antimicrobial effect against planktonic bacteria and prevent biofilm formation at low micromolar concentration. To eradicate biofilms, a higher concentration was needed. It was also shown that the chemical space occupied by the active compound was slightly different than the rest of the cinchona alkaloids as well as the rest of the compounds used for validatory screening during the optimization processes of the separate assays.

Proactive and Preventive Student Welfare Activities in Finnish Preschool and Elementary School: Handling of Transition to Formal Schooling and a National Anti-Bullying Program as Examples

Prerequisites and effects of proactive and preventive psycho-social student welfare activities in Finnish preschool and elementary school were of interest in the present thesis. So far, Finnish student welfare work has mainly focused on interventions and individuals, and the voluminous possibilities to enhance well-being of all students as a part of everyday school work have not been fully exploited. Consequently, in this thesis three goals were set: (1) To present concrete examples of proactive and preventive psycho-social student welfare activities in Finnish basic education; (2) To investigate measurable positive effects of proactive and preventive activities; and (3) To investigate implementation of proactive and preventive activities in ecological contexts. Two prominent phenomena in preschool and elementary school years—transition to formal schooling and school bullying—were chosen as examples of critical situations that are appropriate targets for proactive and preventive psycho-social student welfare activities. Until lately, the procedures concerning both school transitions and school bullying have been rather problem-focused and reactive in nature. Theoretically, we lean on the bioecological model of development by Bronfenbrenner and Morris with concentric micro-, meso-, exo- and macrosystems. Data were drawn from two large-scale research projects, the longitudinal First Steps Study: Interactive Learning in the Child–Parent– Teacher Triangle, and the Evaluation Study of the National Antibullying Program KiVa. In Study I, we found that the academic skills of children from preschool–elementary school pairs that implemented several supportive activities during the preschool year developed more quickly from preschool to Grade 1 compared with the skills of children from pairs that used fewer practices. In Study II, we focused on possible effects of proactive and preventive actions on teachers and found that participation in the KiVa antibullying program influenced teachers‘ self-evaluated competence to tackle bullying. In Studies III and IV, we investigated factors that affect implementation rate of these proactive and preventive actions. In Study III, we found that principal‘s commitment and support for antibullying work has a clear-cut positive effect on implementation adherence of student lessons of the KiVa antibullying program. The more teachers experience support for and commitment to anti-bullying work from their principal, the more they report having covered KiVa student lessons and topics. In Study IV, we wanted to find out why some schools implement several useful and inexpensive transition practices, whereas other schools use only a few of them. We were interested in broadening the scope and looking at local-level (exosystem) qualities, and, in fact, the local-level activities and guidelines, along with teacherreported importance of the transition practices, were the only factors significantly associated with the implementation rate of transition practices between elementary schools and partner preschools. Teacher- and school-level factors available in this study turned out to be mostly not significant. To summarize, the results confirm that school-based promotion and prevention activities may have beneficial effects not only on students but also on teachers. Second, various top-down processes, such as engagement at the level of elementary school principals or local administration may enhance implementation of these beneficial activities. The main message is that when aiming to support the lives of children the primary focus should be on adults. In future, promotion of psychosocial well-being and the intrinsic value of inter- and intrapersonal skills need to be strengthened in the Finnish educational systems. Future research efforts in student welfare and school psychology, as well as focused training for psychologists in educational contexts, should be encouraged in the departments of psychology and education in Finnish universities. Moreover, a specific research centre for school health and well-being should be established.

Nivalalaisen talonpoikaistalon anti suomalaiselle kulttuurille

Kirjallisuusarvostelu

Anti-predator adaptations in aquatic environments

Predation is an important selective force that has led to the evolution of a variety of fascinating anti-predator adaptations, such as many types of protective coloration and prey behaviours. Because the evolution of life has begun in the aquatic environment and many anti-predator adaptations are found already in relative primitive taxa, it is likely that many of these adaptations evolved initially in the aquatic environment. Yet, there has been surprisingly little research on the mechanisms and function of antipredator adaptations in aquatic systems. To understand the function of anti-predator adaptations and natural selection imposed on prey appearance and behaviour, I have investigated how protective coloration can be used, either as such or together with behavioural adaptations, to manipulate predator behaviour and decrease predation risk. To this end I conducted a series of behaviour ecological laboratory experiments in which I manipulated the visual appearance of artificial backgrounds and prey items. In paper I of this thesis, I investigated background choice as an anti-predator strategy, by observing the habitat choice of the least killifish (Heterandria formosa) between pairs of artificial backgrounds, both in the presence and absence of predation threat. It has been suggested that prey could decrease their risk of being detected by predators either by preferring backgrounds into which they blend or by preferring visually complex backgrounds. The least killifish preferred a background that matched their patterning to a background that mismatched it, showing that they are able to respond to cues of visual similarity between their colour pattern and the surrounding environment. Interestingly however, in female least killifish visual complexity of the background was a more important cue for habitat safety and may override or act together with background matching when searching for a safe habitat. It is possible that in females, preference for visually complex backgrounds is associated with lower opportunity costs than preference for matching backgrounds would be. Generally, the least killifish showed stronger preference while under predation threat, indicating that their background choice behaviour is an antipredator adaptation. Many aquatic prey species have eyespots, which are colour patterns that consist of roughly concentric rings and have received their name because they for humans often resemble the vertebrate eye. I investigated the anti-predator function of eyespots against predation by fish in papers II, III and IV. Some eyespots have been suggested to benefit prey by diverting the strikes of predators away from vital parts of the prey body or towards a direction that facilitates prey escape. Although proposed over a century ago, the divertive effect of eyespots has proven to be difficult to show experimentally. In papers II and III, I tested for divertive effect of eyespots towards attacking fish by presenting artificial prey with eyespots to laboratory reared three-spined sticklebacks (Gasterosteus aculeatus). I found that eyespots strongly influenced the behaviour of attacking sticklebacks and effectively drew their strikes towards the eyespots. To further investigate this divertive effect and whether the specific shape of eyespots is important for it, I tested in paper III the response of fish also to other markings than eyespots. I found that eyespots were generally more effective in diverting the first strikes of attacking fish compared to other prey markings. My findings suggest that the common occurrence of eyespots in aquatic prey species can at least partly be explained by the divertive effect of the eyespot shape, possibly together with the relative simple developmental mechanisms underlying circular colour patterns. An eyebar is a stripe that runs through the eye, and this pattern has been suggested to obscure the real eyes of the prey by visually blending parts of the eyes and head of the prey and by creating false edges. In paper III, I show that an eyebar effectively disrupts an eyelike shape. This suggests that eyebars provide an effective way to conceal the eyes and consequently obstruct detection and recognition of prey. This experiment also demonstrates that through concealment of the eyes, eyebars could be used to enhance the divertive effect of eyespots, which can explain the common occurrence of eyebars in many species of fish that have eyespots. Larger eyespots have been shown to intimidate some terrestrial predators, such as passerine birds, either because they resemble the eyes of the predator’s own enemy or because highly salient features may have an intimidating effect. In papers II and IV, I investigated whether the occurrence of eyespots in some aquatic prey could be explained by their intimidating effect predatory fish. In paper IV, I also investigated the reason for the intimidating effect of eyelike prey marks. In paper II, I found no clear intimidating effect of eyespots, whereas in paper IV, using a different approach, I found that sticklebacks hesitated to attack towards eyelike but not towards non-eyelike marks. Importantly, paper IV therefore presents the first rigorous evidence for the idea that eye mimicry, and not merely conspicuousness, underlies the intimidating effect. It also showed that the hesitation shown by fish towards eyelike marks is partly an innate response that is reinforced by encounters with predators. Collectively, this thesis shows that prey colour pattern and the visual appearance of the habitat influence the behaviour of fish. The results demonstrate that protective coloration provides numerous distinctive ways for aquatic prey to escape predation. Thus, visual perception and behaviour of fish are important factors shaping the appearance and behaviours of aquatic prey.

Viitekehyksen kehitys, kehykseen perustuvat tutkimukset ja niiden anti : case: Levers of Control

Tutkielmassa tarkastellaan yrityksen strategian ja ohjauksen yhdistävän Levers of Control -viitekehyksen syntymistä ja kehittymistä nykyiseen muotoonsa. Lisäksi tutkimus perehtyy viitekehystä hyödyntävien tieteellisten tutkimusten antiin strategian ja ohjausjärjestelmien ymmärtämisessä sekä näiden tutkimusten antia itse viitekehykselle. Tutkielma on luonteeltaan käsiteanalyyttinen, analysoiden aiheesta julkaistua aikaisempaa kirjallisuutta. Kirjallisuus on rajattu viitekehyksen kehittäjän Robert Simonsin aiheesta kirjoittamaan materiaaliin sekä eri journaaleista löytyvään viitekehystä hyödyntävään aineistoon. Vaikka viitekehys on luotu myös käytännön toimijoiden avuksi, on tähän liittyvä aineisto kuitenkin rajattu tässä tutkielmassa pois tarkastelusta. Viitekehyksen kehityksen havaitaan noudattavan hermeneuttisen kehän piirteitä, sillä kehityksessä voidaan nähdä neljä erilaista vaihetta: esiymmärrys, esikehys, idea viitekehyksestä ja varsinaisen viitekehyksen vaihe. Kutakin näistä kehistä analysoidaan erikseen ja kaikkia vielä yhdessä. Havaitaan myös, että viitekehyksestä käyty vähäinen diskurssi on pääosin positiivista tunnustaen viitekehyksen ansiot mutta myös ongelmat myönnetään. Viitekehysanalyysin jälkeen siirrytään tarkastelemaan viitekehystä hyödyntävää materiaalia. Huomataan, että nämä tutkimusartikkelit voidaan luokitella neljään pääkategoriaan: empiriapohjaisiin, viitekehystä teoreettisesti hyödyntäviin, viitekehystä kehittämään pyrkiviin sekä opetustarkoituksessa kirjoitettuihin artikkeleihin. Näitä pääkategorioita analysoidaan usein eri tavoin erillisinä sekä yhtenä kokonaisuutena. Eri tutkimusartikkeleiden tuloksia on hankala kuitenkin yhdistää viitekehyksessä olevan ongelman takia: useimpien käytännön pohjalta luotujen selitysmallien tapaan Levers of Control –viitekehyksen käsitteet eivät ole yksiselitteisiä ja sama ongelma on nähtävissä myös viitekehystä hyödyntäneissä tutkimuksissa. Joitakin suuntaa antavia tuloksia interaktiivisuuden roolista ja dynaamisesta jännitteestä voidaan kuitenkin tehdä. Tiedeyhteisö ei ole hyväksynyt Levers of Control –viitekehystä varsinaiseksi teoriaksi. Tutkielmassa todetaan, että teoria-statuksen saaminen ei ole mahdotonta, mutta teoriaksi nousemiseen viitekehyksellä on kuitenkin vielä reilusti matkaa. Ennen sitä on havaitut ongelmat kyettävä ratkaisemaan ja löydettävä hyödyntämisen rajat. Ensimmäisiä askeleita tähän suuntaan ovat viitekehystä hyödyntämään pyrkivät tutkimusartikkelit pyrkineet jo kuitenkin ottamaan. Tutkielmassa tullaan siihen loppupäätelmään, että viitekehys on tällä hetkellä kehityskaarensa toisessa vaiheessa, sillä viitekehystä koskeva kirjoittelu keskittyy perustutkimukseen, arvolatautuneen kriittisen tutkimuksen puuttuessa vielä kokonaan.