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  • Miettinen, Elmo (2019)
    Suomen sorsakannat ovat taantuneet viimeisen parin vuosikymmenen aikana ja suurin osa sorsalajeistamme on jo luokiteltu uhanalaisiksi. Erityisesti rehevien vesien populaatiot ovat taantuneet, minkä on epäilty johtuvan muutoksista lisääntymiselinympäristöissä. Syiksi on ehdotettu muun muassa ylirehevöitymistä ja vieraspetojen runsastumista. Sorsakantojen suojelun ja hoidon kannalta on tunnettava eri tekijöiden vaikutukset sorsien lisääntymiseen ja elinympäristövalintaan. Yksi merkittävä tekijä on elinympäristön laatu. Tärkeimmiksi laatutekijöiksi sorsille ovat tutkimuksissa osoittautuneet selkärangattoman ravinnon määrä sekä kosteikon rakenteelliset ominaisuudet. Laadukkaassa ympäristössä poikaset kasvavat ja selviytyvät huonolaatuisia ympäristöjä paremmin. Toisena merkittävänä tekijänä sorsille ovat pedot, jotka ryöstävät sorsien pesiä. Pesäpredaation riskiin voivat vaikuttaa pesintäalueen ympäristön rakenne ja petoyhteisö sekä pesän sijoituspaikka. Luontaisten petojen lisäksi sorsien pesiä voivat ryövätä myös vieraslajit, kuten supikoira (Nyctereutes procyonoides) ja minkki (Neovison vison). Jos poikueille laadukkaat ja pesille turvalliset ympäristöt eivät kohtaa, sorsaemot joutuvat trade-off -tilanteeseen, jossa toista lisääntymisen vaihetta suositaan toisen kustannuksella. Tarkoituksenani oli selvittää 1) vaikuttavatko alue ja kosteikon ominaisuudet sorsien esiintymiseen, 2) onko alueella ja pesäpaikalla vaikutusta pesän selviytymiseen ja 3) onko vieraspedoilla merkitystä sorsien pesäpredaation määrään. Näihin kysymyksiin hain vastauksia sorsalaskentojen, vesiselkärangatonpyyntien ja pesäpredaatiokokeen avulla. Pesäpredaatiokokeessa tutkimuskosteikoille tehtiin keinotekoisia koepesiä käyttäen tarhattujen sinisorsien (Anas platyrhynchos) munia. Koepesiä seurattiin riistakameroilla, joiden avulla pystyttiin määrittämään pesällä vierailleet lajit ja pesän ryöstön ajankohta. Tutkimukset toteutettiin kahdella tutkimusalueella: maatalousvaltaisella ja vesistöiltään rehevällä Maaningalla sekä metsäisellä ja vesistöiltään karulla Evolla. Maaningalla sorsatiheys oli suurempi kuin Evolla. Vesiselkärangattomien määrä ja rantaviivan pituus vaikuttivat positiivisesti sorsaparien määrään kosteikoilla. Tulvaisuudella oli negatiivinen vaikutus parimääriin. Poikueiden osalta vastaavia tuloksia ei saatu. Tuloksiin saattoi vaikuttaa huonon pesimävuoden vuoksi liian vähäiseksi jäänyt poikuehavaintojen määrä. Vaikka Maaningan sorsatiheydet olivat suurempia, Evolla suurempi osuus pareista tuotti poikueen. Pesäpaikkakohtaisia eroja pesäpredaatioriskissä ei tässä tutkimuksessa saatu esiin, mutta pesien riski tulla ryöstetyksi pieneni pesän alttiinaoloajan myötä. Pesäpredaatioriski oli suurempi Maaningalla kuin Evolla. Harakka (Pica pica) oli yleisin pesiä ryöstänyt laji ja juuri varislintujen määrä vaikuttaa selittävän Maaningan suurempaa pesäpredaation määrää. Supikoira oli toiseksi yleisin pesärosvo, minkä vuoksi vieraspetojen osuus pesäpredaatiosta oli merkitsevä, vaikka minkki ryösti vain yhden pesän. Tämä tutkimus antaa viitteitä siitä, että sorsat joutuvat trade-off -tilanteeseen aluetasolla. Rehevien kosteikkojen Maaningalla on enemmän vesiselkärangatonravintoa ja tiheämpi sorsakanta, mutta myös suurempi pesäpredaatioriski. Sorsien voi siis päätellä suosivan poikasille suotuisaa aluetta pesien selviytymistodennäköisyyden kustannuksella. Vähäisempi pesäpredaatiopaine voi selittää miksi karujen kosteikkojen Evolla parikohtainen poikuetuotto oli parempi. Tämä taas voi osaltaan selittää sitä, miksi karujen vesien sorsapopulaatiot eivät ole taantuneet rehevien vesien populaatioiden lailla. Menetelmällisesti tämä tutkimus korostaa riistakameran hyödyllisyyttä tutkimuksessa. Riistakameroiden ansiosta tutkimus muun muassa vahvistaa supikoiran kiisteltyä merkitystä pesärosvona. Tulevaisuudessa vieraslajien torjunnan ja uhattujen kosteikkoelinympäristöjen turvaamisen lisäksi tarvitsemme lisää tutkimustietoa muun muassa erilaisten kosteikoiden merkityksestä väheneville sorsapopulaatioille.
  • Rikberg, Jon (2018)
    The Red List Index (RLI) has widely been recognized as a useful tool in keeping track of extinction risk trends of large taxa. The RLI is an index based on IUCN’s threat categories. Functional diversity (FD) is a way of measuring biodiversity that describes species´ traits that are linked to species´ ecological roles. In this work I have mapped the spatial distribution of the RLI and functional diversity for Finnish vascular plants. I first produced species distribution models (SDMs) for all 1194 species of vascular plants in the Finnish Red List 2010 based on records from the Kastikka and Hertta databases and environmental data. A functional tree incorporating 971 of those species was calculated using seven functional traits. The traits that I used were life form, maximum plant height, seed mass, seedbank longevity, life span, specific leaf area (SLA), and leaf dry matter content (LDMC). The trait data was gained from the databases Leda and TRY. Based on the SDMs, the functional tree, and the Finnish Red List, taxonomic and functional diversity and RLI were mapped for the whole of Finland using 10 x 10 km cells. This was the first time FD and RLI were mapped for vascular plant species across Finland. Null models were used to compare observed values with the ones expected if species (and consequently traits) distributions were random accross the country. Taxonomic diversity (TD) was higher than expected in southern Finland and lowest in northern Finland, suggesting a strong latitudinal gradient. TD correlated with the same environmental variables as FD. Thus, it is likely that both TD and FD are dirven by the same environmental variables. FD was higher than expected in southern and western Finland and lower in the northern and eastern parts of the country. A strong environmental filtering in the north might cause low FD by limiting species´ distributions within many clades and favouring species with similar traits that allow them to survive in extreme conditions. In southern Finland, competitive exclusion might limit the co-existence of species with similar traits, thus increasing trait divergence. The RLI values were lowest in the Åland islands, along the southern coast, in a few sites in eastern Finland (e.g. Koli and Kuusamo areas), around Kemi and the Gulf of Bothnia and in Kilpisjärvi. Thus, these sites have high concentrations of threatened species. The low RLI sites correspond well with areas with either limestone or dolomite deposits, which explains why many of these areas are floristically unique and present high concentrations of threatened species. In addition, many of the sites with low RLI are geographical extreme areas in Finland, corresponding to the distribution limits of many species. The RLI was high in Ostrobothnia and in large parts of Lapland. In Ostrobothnia, centuries of forest management and a homogenous bedrock and topography have resulted in a vascular plant community based mostly on common species. It is possible, that regional extinctions have happened in Ostrobothnia already before red listing measures began, thus explaining the high RLI values today. On sites with more variety among habitats and bedrock, the RLI values were significantly lower than in the rest of Lapland, suggesting that the high RLI values for parts of Lapland are due to homogeneous plant communities in the northern boreal forests that host only few threatened species. The spatial distribution of the RLI and functional diversity for vascular plants in Finland were mapped for the first time. A strong latitudinal gradient was found for TD and FD. Low RLI values were found on calcareous soils and on geographic extremes in Finland. To track possible changes in the RLI it would be crucial to remap the RLI in 2019 when the next Finnish Red List is published. A comparision between this work and the remapping based on the 2019 assessment would track changes in the extinction risk across Finland. The current limitation with RLI is that it only considers taxonomic diversity. However, in future work it is possible to incorporate the functional tree used in this thesis into RLI to calculate a functionally weighted RLI.
  • Asikainen, Arsi (2018)
    Breast cancer is the most common cancer in women worldwide and the number of new events is on the increase. Like many other serious diseases, breast cancer reduces patient’s health related quality of life (HRQoL) and breast cancer treatment burdens our society. Examination of breast cancer patient’s HRQoL makes it possible to calculate how effective breast cancer treatments are. Nevertheless, only cost-effectiveness analysis would further help us allocate the resources of our society in the best way possible. The aim of this study was to produce research about breast cancer treatment’s effects on patient’s HRQoL and to compare generic 15D- and EQ-5D-5L-instruments. The results can be used in the future research and the study might be useful, when it’s time to develop international protocol for measuring HRQoL. The study population included 152 breast cancer patients who were treated in HUCH and whose HRQoL were measured by 15D-, EQ-5D-5L- and VAS-instruments. All measurements were done twice, first before the treatments and then six months after the beginning of the treatments. 89 (58.6 %) patients answered both 15D-questionnaires and 81 (53.3 %) patients answered to both EQ-5D-questionnaires. 57 (37.5 %) patients didn’t respond to any questionnaire. Only some background information was available of this population. The average HRQoL for breast cancer patients’ was 0.92 before the treatments and 0.88 six months after the beginning of the treatments when measured by 15D. The same average HRQoL was 0.86 before the treatments and 0.80 six months after the beginning of the treatments when measured by EQ-5D-5L. During six months’ period, patients HRQoL reduced (-0.04) when it was measured by 15D and (-0.06) when it was measured by EQ-5D. The changes of HRQoL were clinically important (The minimum important change, MIC > ± 0,015) when measured by 15D. HRQoL reduced more with patients who received a mastectomy than with patients, who received a breast conserving surgery according to both instruments. According to the results, the chosen instrument has an effect of breast cancer patients’ HRQoL. It means that the chosen instrument also has an effect of treatment’s effectiveness. 15D offers higher HRQoL values, but EQ-5D offers a greater change in patient’s HRQoL. HRQoL was measured by two different generic instruments in two different times, which was assumed to be the strength of this study. The new 5L-version of EQ-5D-instrument was also used. This is possibly the first time, when 5L is used in this type of study.
  • Kontola, Sandra (2018)
    Flowability is an important powder character and, despite decades of research, there are still issues in finding a suitable measurement method. Common challenges are sample size and methodology’s suitability for cohesive powders due to their ability to form vault structures. Powder flowability properties depend strongly on particle features such as size and shape. As particles are in contact with other particles and materials, they receive electric charge and form bonds. In addition to these variables, the gravity and shear stress affect the powder. A combination of all these determine the powder properties such as flowability. Besides the particle properties, process and preservation conditions and especially humidity affects the powder properties significantly. Hence, the powder’s flow behavior varies in different conditions. There are several measurement devices available but none of them is able to yield intrinsic values. Reliability of the measurements presents another challenge as the measured values cannot be directly compared with published literature. Moreover, the flow measurement of cohesive powders is either impossible or extremely difficult with the devices currently available and the sample size needs to be sufficient. Hence, there is a need for new devices, which measure powder flow easily in small-scale. Small sample size is important especially when developing new, expensive drugs since their properties need to be explored in order to develop a new formulation. The aim of the empirical study was to develop a device, which measures particularly the flowability of cohesive powders in small-scale. A ground for the study was a device developed at University of Helsinki, which measures powder flowability by utilizing horizontal movement. In addition, the device breaks the problematic vault formation of cohesive powders by jolts. In the study a cuvette, which utilizes the horizontal movement and measures the powder flow, was developed. Flowability tests were run with five powders – Acetaminophen, Pharmatose 80M, Pharmatose 200M, Emcompress®, Avicel PH-101, Avicel PH-102, Avicel PH-200 and Maize Starch. The results were promising and the device was capable of classifying the powders by their flowabilities but more research is still needed.
  • Solansuu, Kati (2018)
    Formulation development for protein drugs should base on the knowledge of the mechanism of protein degradation. Excipients and formulation can be chosen to stabilize the protein and prevent decomposition. Stability testing is important to identify the likely degradation routes and provide information for formulation development and stability-indicating analytical method development. Gonadotropin-releasing hormone (GnRH) is a neuropeptide hormone that regulates the synthesis and release of gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FHS). Analogs of the endogenous GnRH have been developed to achieve more potent and longer-acting agonists or antagonists. GnRH agonists degrade in several pathways. The primary degradation routes are hydrolysis/backbone cleavage, oxidation, isomerization and aggregation. The stability of GnRH agonists in solid dosage forms has not been studied as excessively as in solutions. The objective of this study was to evaluate the stability of a GnRH agonist (API) at different storage conditions in powder form and in tablet formulations with maize starch or hydroxypropyl methylcellulose (HPMC). The samples were stored for three months at 5 °C (common refrigerator conditions) 25 °C/58 %RH (long-term conditions), and 40 °C/75 %RH (accelerated storage conditions). The samples were analyzed using high performance liquid chromatography. Additionally, the mechanical properties of the formulations and tablets were studied. The stability of API was confirmed in tablet dosage form, when maize starch or HPMC were used as excipients. No degradation products of API were found. As a pure powder API did not degrade either, but it did not stay physically stable at 40 °C/75 %RH. Stressed conditions could be used to find out degradation products in solid state that were not found in this study. Further, the formulations were not ideal, because neither of the studied excipient produced tablets with desirable properties.
  • Tamminen, Matti (2016)
    Membrane-bound pyrophosphatases (mPPases) are a potential target for drugs against many neglected protozoan diseases, such as malaria, leishmaniasis, toxoplasmosis and trypanosomiasis. New drugs against these diseases are urgently needed, as the clinically used ones are either not effective, suffer from side effects, or resistance against them is developing. The mPPases of these protozoans are genetically conserved, while mammalian DNA does not encode them. A drug development project to find mPPase inhibitors was started, based on mPPase structures solved through X-Ray crystallography. Four hit compounds were identified. The aim of this study was to investigate the binding of these hit compounds at the mPPase binding site, and based on these results, to develop and synthesize novel compounds with higher affinity. A hit compound with an isoxazole ring was chosen as the model compound to be developed further. These novel compounds were evaluated by docking them into the binding site. Eight compounds were chosen to be synthesized and four to be purchased. The Suzuki-Miyaura cross-coupling reaction was used to couple the isoxazole core to different aromatic substituents, producing 3,5-disubstituted isoxazoles. The reactions mostly succeeded, but the yields were uniformly low. Developing the reaction using different solvents and reaction conditions did not produce clear results. Thirteen compounds were tested for activity, including an intermediate product of the synthesis. Two of the compounds showed increased inhibition activity compared to the hit compound, with approximated IC50 values of 10 and 40 μM, respectively. The knowledge gained from these studies can be used to further develop more efficient inhibitors.
  • Kaipainen, Suvi (2018)
    In this study 39 methicillin-resistant Staphylococcus aureus CC398 isolates from Finnish pigs and pork were sequenced by whole-genome sequencing. Also, phenotypic antimicrobial resistance of the strains was examined. The strains originated from the culture collection of Finnish Food Safety Authority (Evira) and they were isolated between the years of 2008 and 2017. MRSA strains were isolated from both asymptomatic and infected pigs and pork. The strains represented the four most common spa types in Finnish pigs (t034, t2741, t011 and t108). DNA extraction method for S. aureus was optimized by comparing automatic DNA extraction robot and manual DNA extraction method. Extracted DNA samples were sent to Germany to be sequenced with Illumina sequencing. Paired-end raw read sequences were assembled using three different algorithms: Spades, Idba and Velvet. The assembled sequences were analysed by bioinformatics tools. Possible antimicrobial resistance genes, virulence genes, plasmids and SCCmec cassettes were identified. The strains were also typed by spa typing and sequence typing. spa typing was already performed in Evira using PCR and the new spa typing results were compared to the PCR results. Using the assembled sequences, a phylogenetic tree was also constructed. Phenotypic antimicrobial susceptibility testing was performed using broth microdilution panel. The manual extraction method was slightly better since the extracted DNA was purer. The best assembly algorithm was Spades and because of that all sequences were assembled with that algorithm for further analyses. The strains carried only a few virulence genes and many genes that are important for human infection were missing. All the strains carried the same SCCmec cassette type and sequence type ST398 both of which are known to be typical for pig related MRSA. Illumina sequencing and spa typing were not working perfectly since the used program was not able to distinguish spa types t034 and t011 from each other. The problem was probably due to very similar short tandem repeats that these two spa types share. Also, Illumina sequencing is known to produce short reads that could be problematic for sequence assembly. All the strains were resistant to beta-lactams and tetracycline which is typical for MRSA CC398 strains. Many strains were also resistant to clindamycin and trimethoprim. Antimicrobial resistance seemed to be carried by plasmids in some cases because the strains carried several plasmids that are known to contain antimicrobial resistance genes. The antimicrobial resistance profiles were different between different spa types since some spa types were more resistant to certain antimicrobials than others. According to the phylogenetic tree, the strains seemed to be a very heterogenic group. The strains that belonged in the same spa type were most related to each other as expected. There were no differences between strains originating from pigs and strains originating from pork which strongly indicates that bacteria could have spread from pigs to pork. Asymptomatic pigs also carried similar strains as infected pigs. The strains that were causing infections in pigs did not contain any more virulence factors compared to the other strains. The study gave plenty of new information concerning MRSA CC398 in Finnish pigs and pork. The antimicrobial resistance and virulence patterns in addition to other qualities of the strains were very similar that was described in the literature before. The strains seemed not to be very virulent for humans, but the situation must be observed in case of increasing resistance and virulence. The best way to prevent spreading of MRSA is to decrease the amount of MRSA in pig farms since MRSA bacteria are most likely to spread from pigs to the environment and to the people who work in pig farms and from there to the rest of the population.
  • Savolainen, Roosa (2018)
    Liposomes are nano-sized vesicles in which the aqueous phase is surrounded by lipid-derived bilayer. They are excellent drug vehicles for example in ocular drug delivery because they can, among other things, increase the bioavailability and stability of the drug molecules and reduce their toxicity. Liposomes are known to be safe to use, because they degrade within a certain period of time and they are biocompatible with the cells and tissues of the body. Owing to its structure, the surface of liposomes can also be easily modified and functionalized. Light-activated ICG liposomes allow drug release in a controlled manner at a given time and specific site. Their function is based on a small molecule called indocyanine green (ICG) which, after being exposed to laser light, absorbs light energy and thereby locally elevates the temperature of the lipid bilayer. As a result, the drug inside is released into the surroundings. The blood circulation time of liposomes has often been prolonged by coating the liposomes with polyethylene glycol (PEG). Although PEG is generally regarded as a safe and biocompatible polymer, it has been found to increase immunological reactions and PEG-specific antibodies upon repeated dosing. Conversely, hyaluronic acid (HA), is an endogenous polysaccharide, which is present in abundance for instance in vitreous. Thus, it could serve as a stealth coating material which extends the otherwise short half-life of liposomes. One of the main objectives of this thesis was to find out whether HA could be used to coat liposomes instead of PEG. In order to prepare HA-coated liposomes, one of the lipid bilayer phospholipids, DSPE, had to be first conjugated with HA. For the conjugation, potential synthesis protocols were sought from the literature. Ultimately two different reductive amination-based protocols were tested. Consequently, the protocol in which the conjugation was achieved via the aldehyde group of HA, proved to be working. Thereafter, HA-coated liposomes were prepared by thin film hydration from the newly synthesised conjugate as well as DPPC, DSPC and 18:0 Lyso PC. Calcein was encapsulated in the liposomes. HA-covered liposomes were then compared with uncoated and PEGylated liposomes by examining their phase transition temperatures, ICG absorbances, sizes, polydispersities, and both light and heat-induced drug releases. The aforementioned tests were also conducted when the effects of the HA and ICG doubling were examined and the possibility to manufacture HA liposomes with small size was assessed. HA-liposomes showed similar results as PEG-coated liposomes. In addition, successful extrusion of HA-liposomes through a 30 nm membrane was also demonstrated in the results. Doubling of HA did not significantly affect the results. In contrast, increasing the molar amount of ICG by double caused spontaneous calcein leakage even before any heat or light exposure. Based on these findings, HA could work as a coating material instead of PEG, yet further studies are required for ensuring this conclusion. The other key objective was to evaluate the stability of four different formulations, named as AL, AL18, AL16 and AL14, in storage and biological conditions. Based on the differences in the formulation phospholipid composition, the assumption was that AL would be the most stable of the group and that the stability would decrease so that AL18 and AL16 would be the next most stable and eventually AL14 would be the least stable formulation. As in the previous study, the liposomes were prepared by thin film hydration with calcein being encapsulated inside the liposomes. In the storage stability test, liposomes were stored in HEPES buffer at either 4 °C or at room temperature for one month. In the test conducted in physiological conditions, the liposomes were added either to porcine vitreous or fetal bovine serum (FBS) and the samples were incubated at 37 ºC for five days. Regardless of the experiment, phase transition temperatures as well as light and heat-induced drug releases were initially measured. As the test progressed, calcein release, ICG absorbance, size, and polydispersity were measured at each time point. The initial measurements confirmed the hypothesis about the stability differences of tested formulations. In the storage stability test, all formulations, except AL14, appeared to be stable throughout the study and no apparent differences between the formulations or temperatures were observed. On the other hand, the stability of liposomes stored in biological matrices varied so that the liposomes were more stable in vitreous than in FBS and the stability decreased in both media as expected.
  • Kinnunen, Oona (2018)
    The insulin/insulin-like growth factor signaling pathway plays an important role in the regulation of growth and development, metabolism, lifespan, reproduction and stress response of an organism. As the pathway is evolutionarily conserved, Drosophila melanogaster serves as a powerful model for characterizing the underlying molecular mechanisms and genetic components, which may further appear as potential therapeutic targets for metabolic disorders. Drosophila has eight genes encoding insulin-like peptides (DILPs), which display stage- and tissue-specific expression patterns. Furthermore, the expression of DILPs is regulated by nutritional status. To date, only few transcription factors have been shown to regulate the expression of the dilp genes: for instance Dachshund and Eyeless are known transcriptional activators of dilp5 and dFOXO is a known transcriptional activator of dilp6. Since transcription factors directly regulating the expression of dilps are not yet well known, there is a need to study them further. The aim of this master’s thesis was to screen for novel transcriptional regulators of the dilp genes and study their phenotypic effects in vivo. For this purpose Drosophila S2 cells were transfected with vectors containing the promoter regions of all the dilp genes and with a library of 822 transcription factors. Promoter activity of the dilp genes was further measured by using the Dual-Luciferase® Reporter Assay System. Some of the revealed potential regulators were further studied in vivo to confirm their regulatory functions by using RNA interference to silence these factors or overexpressing them and by performing quantitative real-time PCR to measure dilp expression levels. Subsequently, organismal growth and development were assessed and effects on larval metabolic profiles were monitored. The screen revealed a few potential transcriptional regulators, among which Spalt-related (Salr) was selected for further study. It was shown to act as an activator of dilp6 expression. dilp6 is an interesting target among the dilp network due to its central role as a communicator between two of the important organs involved in insulin signaling: the brain and the fat body. Subsequently, both Salr and DILP6 were shown to have a role in controlling growth and development of larvae. Based on the experiments in this thesis Spalt-related seems to have a broader role as a metabolic regulator, which is not limited to its function as a regulator of dilp6. Thus, it might act as an important link between the insulin signaling pathway and other pathways regulating growth and metabolism in Drosophila.
  • Lukkarinen, Iiris (2018)
    The aim of this study was to provide information about using citizen science in biology education. Citizen science is a form of scientific research, where public can participate in data collection or other phases of research. It is important to study educational use of citizen science, because citizen science has a lot of potential to increase students´ interest towards biology and knowledge about scientific research. In addition, citizen science could provide meaningful ways to use information and communication technology (ICT) in education. Using ICT, such as computers and smartphones, has become very common way to collect data in citizen science projects. This is a case study about using citizen science project called Matoseuranta, as part of biology education. Research questions were: (1.) How the teachers that participated in Matoseuranta experience the educational use of citizen science? (1.1) What benefits and challenges there is in the educational use of citizen science, according to the teachers? (2.) How well data collecting with mobile application is suited in citizen science project that is used in education? (2.1) What challenges and benefits there were related to collecting data with Matoseuranta-application according to the teachers? (2.2) How the teachers evaluate their skills and knowledge executing fieldwork that includes ICT? Survey was used to collect data from teachers that participated in Matoseuranta. 49 teachers answered to the survey (n=49). Open-end questions were used to collect qualitative data about teachers´ experiences about educational use of citizen science (1.1) and mobile app (2.1). This qualitative data was then categorised using qualitative content analysis. Likert-scaled questionnaire based on TPACK (=technological pedagogical content knowledge), was used to collect quantitative data about teachers´ self-efficacy related to executing fieldwork that includes using ICT (2.2). According to teachers, the most important benefit of educational use of citizen science was the autenthic research context, which increased student motivation. In addition, teachers mentioned that Matoseuranta increased students´ knowledge about scientific research. The unreliability of collected data was the most often mentioned challenge. Teachers felt that collected data was not reliable because students had no prior experience in executing scientific research. The mobile app was suited well in data collecting, because it provided an easy way to send data to researchers. In addition, few teachers mentioned that the app motivated students. Unfortunately, the app had many technical issues, which was the most often mentioned challenge about using the app. Most of the teachers felt that they master the use of technology and the skills needed in executing fieldwork. However, only few teachers felt that they have sufficient skills to execute fieldwork that icludes using ICT in a meaningful way. Based on the results of this study, it would be important to offer more citizen science projects that are suited in educational use to teachers. It would be beneficial to study what types of projects (theme, execution, ICT) teachers would want to use in their teaching. Results confirm prior assumption, that the task value is a very important factor influencing student motivation. It is important to make sure that students feel their work in school is meaningful. This study also suggest, that there is a need to provide training and materials about how ICT can be used in biology fieldwork.
  • Savolainen, Heikki (2018)
    Tablet manufacturing requires both high-quality equipment and powder blend with high flowability and compactability and low segregation tendency. The process is complex and tablet formation process still remains not fully understood. Adequate powder flow is a necessity for the pharmaceutical manufacturing process, i.e., powder flowability and flow properties play a great role when designing manufacturing processes for solid dosage forms. As such, the powder characteristics need to be investigated. However, one property is seldom enough to predict the flowability of a powder in specific processes and different test methods need to be used to fully understand the tableting performance of a particular powder. It is crucial to know how the assessed properties reflect the manufacturing conditions. The need for test batches and the use of empirical testing still exists despite the numerous powder characterization tests available. The main aim of the study was to understand the influence of material properties, flow properties and segregation tendencies on both the processability of a formulation during tablet compression and the critical quality attributes, such as mass, tensile strength and dose uniformity of the final drug product. Additionally, testing of an in-line NIR method to observe the homogeneity of the powder inside the force feeder right before the compression step and transmission Raman as an at-line method for tablet content were also evaluated. A number of powder characterization tests were employed in order to fully understand the impact of the formulation on the process performance. Three formulations with different particle size of the active substance and mannitol were used throughout the study. Both the sifting segregation and fluidization segregation tests’ results predicted the formulations’ tabletability particularly well. Fluidization segregation test predicted the changing composition of the formulation throughout tableting whereas sifting segregation results showed the constantly fluctuating API concentration in the manufactured tablets. Moreover, the Raman results confirmed the tablets of variable content despite the offset caused by the different particle size of the raw materials used. The functionality of the NIR in the force feeder was tested successfully. The residence time distribution could be determined at a sufficient level to point out tablets of a bad quality from the batch on grounds of the NIR data. Results from the powder flow property tests were rather conflicting. Angle of repose, Carr’s index and volume flow rate gave the best characterizing results, whereas the mass flow rate, shear test with higher normal stress in pre-shear gave the worst results, considering the experienced flow character of the formulations. As stated above, different flow property tests may give conflicting result, and hence, it is crucial to know which results are the most relevant ones. Furthermore, the right settings for the test should be known to gain applicable results, best exemplified by the shear cell test.
  • Tarkiainen, Susanna (2018)
    The motivation of this study was to find new treatment options for the rare cancer pseudomyxoma peritonei (PMP). PMP is a slowly progressing mucinous adenocarcinoma that originates from the appendix and disseminates into the peritoneum where the cancer cells secrete large amounts of MUC2, the main component of intestinal mucus, into the peritoneum. The disulfide isomerase AGR2 helps MUC2 with forming the correct intramolecular disulfide bonds prior secretion, and is essential to MUC2 protein production. The mucus build-up into the peritoneum causes stress on vital organs, and eventually death. Therefore, inhibiting MUC2 production in PMP cancer cells might slow down the disease progression significantly. MAPK/ERK and cAMP/PKA signaling pathways stimulate MUC2 production, and activating mutations in KRAS and GNAS of these pathways are common in PMP. The aim of this study was to elucidate how MUC2 and AGR2 affect each other’s expression levels, and how the MAPK/ERK pathway and the cAMP/PKA pathway targeting substances caffeine, theophylline, cromolyn, fudosteine, octreotide, and lanreotide, affect MUC2 expression in vitro. This study was conducted on human colorectal adenocarcinoma cell lines LS174T, LoVo, and HT29 that all produce large amounts of MUC2. In addition, LS174T and LoVo cell lines carry activating heterozygous mutations in their KRAS genes. MUC2 and AGR2 expression levels were measured on mRNA level with real-time quantitative reverse transcriptase polymerase chain reaction. The effects of MUC2 on AGR2 expression, and vice versa, were tested by silencing each at a time with the appropriate siRNA. MUC2 siRNA suppressed both MUC2 and AGR2 mRNA levels down to 50 % in LS174T cells and down to 40 % in LoVo cells in respect to their control groups. AGR2 siRNA suppressed AGR2 mRNA levels down to 50 % in LS174T cells and even down to 30 % in LoVo cells in respect to their control groups, while there were no statistically significant changes in MUC2 mRNA levels. Caffeine and theophylline inhibit phosphodiesterase of the cAMP/PKA signaling pathway, and in consequence, the hydrolysis of the secondary messenger cAMP, prolonging the activation of the pathway. Caffeine stimulated MUC2 production in LS174T and LoVo cells. In addition, AGR2 mRNA levels increased in LoVo cells, in which the fold change in MUC2 mRNA levels was much greater. Theophylline, a compound found in tea, and used for the treatment of asthma, did not affect MUC2 production. PMP cancer cells express protein S100P. Cromolyn is a pharmaceutical substance used for the treatment of asthma, and it inhibits S100P, and thereby S100P/RAGE signaling -dependent activation of MAPK/ERK pathway. Fudosteine, on the other hand, is a mucoactive pharmaceutical substance that lowers the production of MUC5AC, the main component of lung mucus. Since activating GNAS mutation stimulates the production of both MUC2 and MUC5AC in HT29 cells, the expression of both must be at least partially controlled by same mechanisms. In addition, Somatostatin analogues octreotide and lanreotide inhibit ERK1/2 of the MAPK/ERK pathway, as well as protein kinase A of the cAMP/PKA pathway, and hence cAMP production. However, none of these four tested pharmaceutical substances were able to inhibit MUC2 production in the cell lines used this study in vitro.
  • Rahaman, Atikur (2018)
    Some flavonoids are thought to have antioxidant functions in plants, but this is still controversial because of a lack of in planta evidence. Methyl viologen (MV) sensitivity was assayed in sterile culture by growing seven day old Arabidopsis seedlings on 0.5x MS media supplemented with and without MV. Seedlings grew for 9 days after that, and then were photographed and root growth measured. We used several flavonoid deficient Arabidopsis transparent testa (tt) mutants and found they were hypersensitive to MV that produces reactive oxygen species (ROS) in chloroplasts and mitochondria. Flavonoids are transported into different compartments of the cell via a specific flavonoid transport system but no chloroplastic or mitochondrial transporters are currently known. To identify potential chloroplastic and mitochondrial transporters, knockout mutants of candidate MATE and ABC transporters have been screened for MV sensitivity. Flavonoids modulate auxin transport, which raises the possibility of passive effects through auxin during ROS sensitivity. To test whether auxin or the inhibitions of auxin transport have a role in protecting against ROS stress, MV sensitivity was assayed under exogenous treatment of artificial auxin (NAA) and an auxin transport inhibitor (NPA) during MV stress. From this study, we obtained data about the role of flavonoids and auxin in ROS signalling. The inhibition of auxin transport modulated MV sensitivity. We found that NAA had no effect or very modest effect. However, a protective effect of NPA was observed that helps against ROS formation, mimicking the effect of flavonoids. NPA induced more protection. One MATE transporter also gave the mimic of (tt) phenotype, however their role in the movement of flavonoids into or out of the chloroplast or mitochondria remains unclear, as they may be transporting some other protective agent that helps against oxidative stress. However, At1g54350 is a candidate for further testing.
  • Karhunen, Emilia (2018)
    Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.
  • Merenheimo, Salla (2018)
    Nuorten kiinnostus luonnontieteiden opiskelua kohtaan on heikentynyt sekä Suomessa että kansainvälisesti ja nuoria hakeutuu luonnontieteellisille aloille yhä vähemmän. Samaan aikaan Eurooppaan tarvitaan yli 700 000 uutta tutkijaa. Eräänä syynä nuorten kiinnostuksen puutteeseen luonnontieteiden opiskelua kohtaan pidetään nuorten stereotyyppistä tutkijakuvaa. Tutkijakuvalla tarkoitetaan henkilön mielikuvia ja käsityksiä tutkijoista. Stereotyyppiseen tutkijakuvaan kuuluvat käsitykset laboratoriotakkiin ja silmälaseihin pukeutuneista miehistä, jotka työskentelevät laboratoriossa tehden kokeita ympärillään monenlaisia tutkimusvälineitä. Stereotyyppisen tutkijakuvan väitetään vaikuttavan negatiivisesti nuorten asenteisiin ja kiinnostukseen luonnontieteiden opiskelua kohtaan ja jopa heidän halukkuuteensa hakeutua opiskelemaan luonnontieteellisille aloille. Suomalaisten lasten ja nuorten tutkijakuvaa ei ole aiemmin kartoitettu. Tämän tutkimuksen tavoitteena oli selvittää suomalaisten lasten ja nuorten käsityksiä tutkijoista. Tutkimuskohteena olivat Helsingin yliopiston LumA-tiedekasvatuskeskuksen kesän 2017 tiedeleireille osallistuneet lapset ja nuoret (N=455). Tutkimuksessa selvitettiin sukupuolen, iän sekä tiedeleirin teeman yhteyttä leiriläisten tutkijakuvaan. Tutkimusmenetelmänä käytettiin kyselylomaketutkimusta, jossa vastaajat piirsivät omiin mielikuviinsa pohjautuvan kuvan tutkijasta. Piirroksia käsiteltiin määrällisenä aineistona. Jokainen piirustus analysoitiin tarkistuslistan avulla, johon listattiin kirjallisuudesta ja aineistosta nostettuja stereotyyppiseen tutkijakuvaan liittyviä piirteitä. Tulokset analysoitiin deskriptiivisellä analyysillä. Sukupuolten välisiä eroja tarkasteltiin khiin neliö -testien ja Mann-Whitneyn U-testin avulla. Iän yhteyttä tutkijakuvaan selvitettiin Kruskal-Wallis -testillä. Tutkimuksen luotettavuutta tarkasteltiin validiteetin ja reliabiliteetin avulla. Tutkimuksessa selvisi, että suomalaisilla lapsilla ja nuorilla on stereotyyppisinä pidettyjä käsityksiä tutkijoista, vaikkakin stereotyyppisiä piirteitä esiintyi piirroksissa vähemmän aiempiin tutkimuksiin verrattuna. Tyypillisimmät piirroksissa esiintyneet stereotyyppiset piirteet olivat työskentely sisällä, miestutkija sekä erilaiset tutkimuksen symbolit ja teknologiavälineet. Sukupuolella oli yhteys tutkijakuvaan tyttöjen piirtäessä merkitsevästi enemmän naistutkijoita ja hymyileviä tutkijoita kuin pojat. Vastaajan ikä ei ollut yhteydessä stereotyyppisten piirteiden määrään, toisin kuin aiemmissa tutkimuksissa. Leirin teema oli vahvasti yhteydessä niihin tarvikkeisiin, joita tutkijan ympärille piirrettiin. Tutkimuksessa havaittiin piirrosten tulkinnan olevan subjektiivista ja vaikuttavan tutkimuksen tuloksiin. Siten nuorten tutkijakuvaa tulisi jatkossa kartoittaa useiden metodien avulla. Tämä tutkimus antaa viitteitä tiedeleirien potentiaalista lasten ja nuorten tutkijakuvan muovaamisessa. Tutkimuksen tuloksia voidaan hyödyntää luonnontieteiden opetuksen kehittämisessä sekä formaalissa että nonformaalissa tiedekasvatuksessa: tiedostamalla nuoren oman tutkijakuvan muodostuminen voidaan opetuksessa tarjota oppijalle mahdollisuuksia muodostaa realistisempaa ja monipuolisempaa tutkijakuvaa esimerkiksi tutkijavierailujen ja toiminnallisten aktiviteettien kautta. Tämä voi lisätä nuorten kiinnostusta ja minäpystyvyyttä tutkijan uraa kohtaan.
  • Sainio, Aki (2018)
    In this master’s thesis I examine transdisciplinarity in theory and practice. Transdisciplinarity is seen as an inevitable approach when we strive to understand and solve many of the complex problems of the modern world e.g. the environmental crisis. For this reason it has also quickly increased in popularity in the research funding. With this premise and despite the diverse theory on transdisciplinarity the implementation in practice is often relatively flimsy. This gap between theory and practice has acted as a motivation to the thesis, in which I am interested in how the transdisciplinary expectations based on the theoretical ideals and set on the cross-disciplinary environmental research intersect with the realistic transdisciplinary advancements in the research programs. In addition, I am interested to know why the practice doesn’t meet the expectations. The thesis is solidly theory-oriented. First I shed light on the background, characteristics and motivations of transdisciplinarity as well as disciplines, after which I present an array of the complex challenges of transdisciplinarity. In the results section I examine the manifestations of the challenges on a practical level that is based on three environmental research programs organized over the course of 2011–2014 by HENVI, the Helsinki University Centre for Environment (now HELSUS). In addition to the qualitative analysis based on the interview data, the results section also examines theory on a more practical level of implementing transdisciplinary research programs in general. This presentation thus enables a closer dialogue between the analysis data and practical theory. The primary results of this thesis are five problematics that are the manifestations of theory not meeting the practice properly. They are organized into the themes of funding, knowledge production, administration, socio-culture and education and help to organize and ultimately face the challenges of transdisciplinary research. From a comprehensive set of perspectives the problematics display strongly the highly disciplinary nature of the modern academia. This is the most significant single reason for the difficulties to accomplish transdisciplinarity. In addition, all the five problematics reflect the temporal restrictions that also undermine the potential of an effective transdisciplinary interaction. The essential measures to create solutions and hence also the ideal implementation of transdisciplinarity are mainly beyond the focus of the thesis. However, the problematics provide a problem-based framework that strives to contribute to the further studies focused more profoundly on the actual solutions
  • Lehtola, Minna (2018)
    Tramadol products for cats are not commercially available. Problems may occur when dividing a tablet registered for humans due to uneven distribution of active ingredient within a tablet and bitter taste of tramadol. Minitablets have multiple benefits, including small size, better uniformity of content, coatability and fast administration, in comparison to a divided conventional tablet. The purpose of this study was to develop minitablets which are possible to coat with a taste masking coating. Physical and chemical properties of tramadol hydrochloride, such as water solubility, temperature behavior and hygroscopicity were studied. Additionally, compatibility of tramadol hydrochloride with excipients was studied by a 3-month stability exam. The pre-tests of granulation were carried out by using lactose or ascorbic acid as an active ingredient to model tramadol hydrochloride. The granulation was performed with high shear granulator and tableting with a rotary tablet press. The only variable factor between the granulation batches was the amount of granulation fluid. The impact of the amount of granulation fluid to the tableting properties was examined by determining particle size distribution, Carr index and Hausner ratio. Uniformity of mass, uniformity of content, hardness, disintegration time and dissolution were examined. The study revealed that tramadol hydrochloride did not have incompatibilities with the examined excipients. Tramadol hydrochloride was not hygroscopic even though it was found out to be freely soluble in water. Tablets with adequate hardness were successfully compressed of both granulated masses and the direct compression mass. However, the direct compression mass had more undesirable properties regarding the processes. Most batches fulfilled the requirements set for uniformity of mass and uniformity of content. Although the purpose of this study was to develop a tablet for veterinary medicine, the results in this study may be utilized in developing a formulation for pediatric medicine.
  • Johansson, Anna (2018)
    Artificial light that produces some adverse effects is called light pollution. Light pollution has only recently been recognized as an environmental problem – it has been noticed to have adverse effects to organisms, ecosystems, human health and their well-being. The purpose of this study is to combine two aspects of light pollution that have so far gained little attention: the public’s point of view and the lighting solutions of municipalities and forest industry enterprises. These two aspects can be connected to each other through a concept called condition of lighting. Here the conditions of lighting consist of three areas with notable differences in lighting: city centers, countryside and forest industry production plants along with their surroundings. The study was conducted in South Karelia, Finland, and it was divided into two sections. The first section dealt with the public’s point of view on light pollution. The objective was to find out how South Karelians perceive the effects and obtrusiveness of artificial light both generally and also within areas of differing conditions of lighting. In addition, the intention was to uncover other factors that might influence the opinions of people. The second section of the study concerned the lighting solutions of municipalities and forest industry enterprises. The objective was to examine their lighting planning and implementation as well as the role of light pollution in the process. Interview was selected as the method of research in both sections of the study. The public considered electronic billboards and car headlights as the most obtrusive sources of light pollution. Bright lights and glare, on the other hand, were considered the most obtrusive types of light pollution. Most valued benefits of artificial light were its influence on safety, crime prevention and its positive effect on mood. Not being able to see the stars or experience natural darkness were considered as the biggest disadvantages of artificial light. The conditions of lighting in different areas also influenced the respondents’ experiences: people living in the countryside did not feel insecure in dark places outside the cities and did not consider abundant lighting pleasant. They also felt that being able to experience natural darkness was important. Among other factors, nature orientedness, light sensitivity, environmental attitudes and gender strongly affected the views of the respondents. In the municipalities and forest industry enterprises, the lighting planning concentrated on territorial plans. All but one of the municipalities and enterprises had taken light pollution into consideration at some level at least, but the forms and means varied. In the future both municipalities and forest industry enterprises will invest more in LED technology. The results show that artificial light can cause inconvenience to people who live outside the brightly lighted areas and who were not particularly interested in light pollution. Behind the inconvenience and disturbance were mostly the experiences, habits, values and attitudes of the respondents. The municipalities and enterprises have a lot to improve when it comes to light pollution. They should, for example, invest in comprehensive lighting planning, avoid over-illumination and pay attention to suitable direction and positioning of the light fixtures. In the future, it is important to examine the public’s opinion and to map the landscape of lights at a local scale. This would help in finding the most suitable lighting solutions for different areas. In the opinion polls, the subjective characteristics affecting the views of the respondents should be taken into consideration. Research regarding the municipalities and enterprises should be directed to the areas where light pollution is not regulated, in order to gather information on motivation and implementation of the voluntary activities reducing light pollution.
  • Hietala, Ville (2018)
    Antibioottiresistenssi yleistyy infektioita aiheuttavien bakteereiden keskuudessa. Infektioita hoitaessa joudutaan siten miettimään uusia tapoja infektioiden parantamiseksi. Yhden vaihtoehdon tarjoaa bakteriofagiterapia. Siinä infektiota sairastavalle annetaan bakteriofageja, jotka infektoivat bakteereita, lisääntyvät niissä ja tuhoavat ne. Kirjallisuudessa on runsaasti lupaavia esimerkkejä tästä terapiamuodosta. Kuitenkin bakteriofagituotteiden on oltava tarpeeksi puhtaita, erityisesti siinä tilanteessa, jos niitä annetaan suonensisäisesti. Gramnegatiivisten bakteereiden tärkeä rakenneosa on lipopolysakkaridi eli LPS, joka ihmisen verenkiertoon päästessään voi olla vaarallinen. Siksi, mikäli bakteriofagit on tuotettu gramnegatiivisissa bakteereissa, LPS:t on kyettävä poistamaan näytteistä. Tässä Pro Gradu-työssä tutkittiin viidessä eri koejärjestelyssä, kuinka erilaiset tekniikat ja niiden yhdistelmät vaikuttivat bakteriofagien ja LPS:n määrään näytteissä. Lisäksi arvioitiin SDS-PAGE-geelissä näytteiden proteiinikoostumusta. Näytteistä mitattiin myös dsDNA-pitoisuus. Tutkitut tekniikat olivat ultrafiltraatio, anioninvaihtokromatografia, oktanoliuutto ja kaupalliset endotoksiininpoistotuotteet EndoTrap- ja Pierce high endotoxin removal-pylväät. Koejärjestelyssä 1 suodatettua bakteriofagilysaattia puhdistettiin ensiksi oktanoliuutolla, sitten ultrafiltraatiolla, anioninvaihtokromatografialla ja lopuksi uudella ultrafiltraatiolla. Endotoksiinipitoisuus laski eniten kromatografiassa mutta toisen ultrafiltraation jälkeen se pysyi varsin samana. Tiitteri pieneni vain oktanoliuutossa ja kromatografiassa (jossa osittain laimenemisen takia), kun ultrafiltraatioissa muutos edeltävään tiitteriin oli varsin pieni. Lopuksi näytteissä oli keskimäärin n. 15 EU (endotoksiiniyksikköä)/10^9 pfu (plaque forming unit). Toisessa koejärjestelyssä aloitettiin ultrafiltraatiolla ja sen jälkeen näytteet puhdistettiin joko EndoTrap- tai Pierce-pylväällä. Kaikki vaiheet poistivat endotoksiineita. Ultrafiltraatio pienensi hieman tiittereitä mutta EndoTrap-pylväs ei. Pierce-pylväässä tiitterin aleneminen oli huomattavaa, jolloin EndoTrap-pylvään jälkeen näytteissä oli keskimäärin 0,0574 EU/10^9 pfu ja Pierce-pylvään jälkeen 33700 EU/10^9 pfu eli suhde oli jopa enemmän kuin lysaatissa. Kolmannessa koejärjestelyssä aloitettiin ultrafiltraatiolla, jonka jälkeen oli kromatografia ja uusi ultrafiltraatio. Ensimmäisessä ultrafiltraatiossa tiitterit pienenivät jonkin verran mutta kromatografian ja toisen ultrafiltraation jälkeen se oli varsin sama, kuin ensimmäisen ultrafiltraation jälkeen. Endotoksiinipitoisuus pieneni taas eniten kromatografiassa ja se oli varsin sama toisen ultrafiltraation jälkeen, jolloin puhdistetuissa näytteissä oli keskimäärin 57,1 EU/10^9 pfu. Neljännessä koejärjestelyssä lysaattia ultrafiltroitiin ensiksi, sen jälkeen ruiskutettiin kromatografialaitteeseen, ultrafiltroitiin uudestaan ja sen jälkeen puhdistettiin joko EndoTrap- tai Pierce-pylväällä. 1. ultrafiltraatio pienensi taas hieman tiitteriä mutta se oli varsin sama EndoTrap-pylväällä puhdistetussa näytteessä. Pierce-pylvään kanssa tiitteri pieneni taas huomattavasti. Endotoksiinipitoisuudet pienenivät taas kaikissa vaiheissa, jolloin EndoTrap-pylväällä puhdistetussa näytteissä oli keskimäärin 0,0839 EU/10^9 pfu ja Pierce-pylväällä 5480 EU/10^9 pfu. Viides koejärjestely oli muuten sama, kuin koejärjestelyn 2 Pierce-pylväs järjestely mutta korkeamman NaCl-pitoisuuden eluutiopuskurilla ja suuremmalla näytetilavuudella. Tällöin tiitteri ei pienentynyt yhtä paljon, endotoksiinipitoisuus oli lopussa vain hieman korkeampi kuin koejärjestelyssä 2 ja loppuun asti puhdistetussa näytteessä oli keskimäärin 71,21 EU/10^9 pfu. SDS-PAGE-tutkimuksessa ja dsDNA-mittauksissa näkyi, että ultrafiltraatio yksin riittää poistamaan muut bakteeriperäiset proteiinit ja dsDNA:n. Tosin koejärjestelyssä 1 käytetty oktanoli saattaa vaikuttaa dsDNA:n puhdistumiseen ultrafiltraatiossa. Töissä onnistuttiin tuottamaan näytteitä, joissa sekä riitti bakteriofageja, että endotoksiinipitoisuus oli pudonnut huomattavasti. Kun huomioidaan käytännöllisyys ja nopeus, parhaaksi tavaksi puhdistaa LPS:t valikoitui ultrafiltraatio yhdistettynä EndoTrap-pylvääseen. Johtuen bakteriofagien ja LPS:ien monimuotoisuudesta, ei toisella bakteriofagilla välttämättä päästä samoihin
  • Valle, Jenni (2018)
    Gut inflammation and permeability is speculated to play a major role in the pathophysiology of several human diseases. Signs of a low-grade gut inflammation in patients with type 1 diabetes (T1D) have been found. Focus of this study was to understand the role of gut inflammation and increased gut permeability in the development of diabetic complications, especially nephropathy. Approximately, one-third of Finnish patients with T1D develop kidney disease during their lifetime. Inflammatory mechanisms may have an essential role in the pathophysiology of the disease. Lipopolysaccharide, LPS, is found in the outer membrane of gram-negative bacteria. LPS activates innate immune system and triggers the activation of inflammatory cytokines, neutrophils and macrophages as well as many pathophysiological processes in vivo, for instance fever and endotoxic shock. Aim of this study was to establish a zebrafish gut inflammation model using fluorophore conjugated endotoxin, LPS. We hypothesized that delivery of LPS in addition to EDTA in the gut of zebrafish triggers inflammation and increased gut permeability which may lead to leakage of LPS to blood stream and potentially kidney injury. This novel zebrafish inflammation model could possibly be used for studying the pathophysiological mechanisms of gut inflammation and possible kidney injury as well as for screening new anti-inflammatory drugs. In addition, this animal model can be used for studying intestinal alkaline phosphatase (IAP) in reducing gut permeability and LPS-mediated kidney damage. IAP is an enzyme produced in small-intestinal epithelium. IAP can detoxify several bacterial endotoxins including LPS and thus protect against the induction of intestinal inflammation. LPS and EDTA were delivered in the gut of 6 days old zebrafish larvae using microgavage injection. Fluorescence microscopy imaging of live zebrafish enabled following the same individual at different timepoints after injections. Paraffin sectioning of the small larvae was promising for investigating the morphology and permeability of the gut as well as possible immunostaining for detection of IAP. L-phenylalanine was used for inhibition of IAP enzyme. Using the novel method of microinjection to gut on zebrafish larvae the timing and amount of delivered materials to gut can be controlled well. The anatomy and function of the gut in zebrafish is very similar to small intestine of mammals and the highly developed vertebrate immune system makes zebrafish an interesting model organism for studying gut inflammation and permeability. In addition, inflammatory processes can be visualized in live, intact transparent zebrafish larvae. However, the technique has a lot of challenges including small size of the fish and possible tissue damage of the fish while performing injection. More experiments need to be carried out to establish the model for drug screening. Also, along with microscopy images, a more precise way for quantification the gut permeability is needed. Based on the images it’s not yet possible to conclude whether LPS increased gut permeability or if IAP inhibition with L-phenylalanine worked in zebrafish larvae. Using adult zebrafish in the future will give more information about the chronic gut inflammation and development of possible kidney injury.