Skip to main content
Login | Suomeksi | På svenska | In English

Browsing by Title

Sort by: Order: Results:

  • Hyttinen, Nea (2023)
    Chronic wounds are a worldwide health problem that produce a lot of costs for society and can have a substantial impact on patients’ quality of life. Human adipose stem cells (hASCs) have been studied as a treatment option for chronic wounds as they can induce wound healing in many ways. Extracellular vesicles (EVs) produced by hASCs are a great solution to acquire the benefits of hASCs while avoiding their problems such as possible mutagenicity. HASC-EVs have been found to induce wound healing by for example enhancing angiogenesis and fibroblast proliferation. HASCs can be grown in 2D where the cells attach to the bottom of the cell culture vessel or in 3D where the cells attach to each other and create a spheroid. 2D cell culturing is easy and inexpensive but 3D cultured cells resemble in vivo –like conditions more. Because of these in vivo -like features, hASCs grown in 3D might produce EVs that resemble the properties of host cells in natural environment more than 2D. The aim of this thesis was to compare 2D culture, matrix-based nanofibrillar cellulose (NFC) hydrogel culture, and matrix-free suspension culture in ultra-low attachment (ULA) wells as growing platforms for hASCs and as continuous EV production methods. During culturing, the conditioned media was collected after which, the EVs were isolated, and the EV concentration and size range was measured with nanoparticle tracking analysis (NTA). After culturing, the metabolic activity of hASCs was measured and the cells were collected for immunocytochemistry (ICC) assay, western blot (WB) assay, and for quantitative PCR (qPCR) to examine the stemness and differentiation of hASCs grown in different cell cultures. The hypothesis of this thesis was that the NFC cell culture would produce the best EV yield and the best EVs for therapeutic use. Based on the acquired results, this hypothesis could not be supported. When visually inspecting the cells, all three cell cultures were viable but the metabolic activity of hASCs in NFC hydrogel was low compared to 2D and suspension cultures. Also, the EV, protein and RNA yield were lower in NFC. ICC, western blotting, and qPCR results were inadequate to make a straightforward implication of what cell culturing condition is the best for EV production and they would need repetition and optimization. Looking at the overall results, 2D cell culturing produced the best EV and RNA yield, had the highest metabolic activity and was least laborious cell culturing method which makes it a good option for continuous EV production. Suspension culture on the other hand resembles in vivo -like environment which could possibly produce better EVs for therapeutic use. The metabolomic assays on the EVs would be interesting to perform in the future to examine if the in vivo –like features affect the quality of EVs.
  • Räisänen, Titvi (2023)
    A clean area is an area isolated from its environment to prevent contamination of final product during aseptic processing. The clean area can be divided into four different grades from A to D, which all have different cleanliness standards. Grade A is the highest grade where preparing products that are not terminally sterilized must be performed. Airlocks are located between different grades to prevent free airflow, and enable necessary precautions, such as putting on protective garments and disinfecting material surface. These procedures reduce risk of contamination of the higher grade. The purpose of this study was to create a protocol to help evaluate material disinfection and transfer processes in the hospital pharmacy of Turku University Central Hospital and to determine surface bioburden of material stored in the grade C area. Surface samples of the examined material were taken in accordance with in-house guidelines by using contact plates and swabs depending on the surface of the material examined. After incubation, colony forming units were counted. Samples were taken from primary packages of ingredients and equipment stored in grade C area, as well as from material transfer boxes and cut flush plastic folders used in the clean area. Samples were taken both before and after routine disinfection of this material. 45 % of the samples taken before disinfection were contaminated. The lowest contamination rates were observed from items made from glass and those that were stored in their secondary package. In five plates grew more than 25 colonies, of which two had biofilm covering the whole surface of the plate. These samples were taken from larger plastic items, such as an infusion bag and a plastic folder. High bioburden is possible on the surface of material stored in grade C clean room, despite precautions. 25 % of the samples taken after routine disinfection were contaminated with a maximum of two colonies per plate. Despite disinfection, viable microbes may remain on the surface of material. Material with risk of high bioburden were selected for the protocol. Items were disinfected and transferred to grade B area as a simulation of normal processes. Different operators performed the protocol a total of eight times. 14 % of samples were contaminated with a maximum of two colonies per plate, except for one plate with 15 colonies. This repetition exceeded the limits set for the protocol. One repetition had zero contaminated samples. The bioburden of material surface after disinfection is affected by operators, cleanliness of the grade C area, and manipulation of the storage. A high bioburden increases risk of unsuccessful disinfection, and recontamination is possible in a non-sterile environment. Bacillus and Staphylococcus -species were identified from the samples taken during the protocol. Bacillus-species are usually isolated from soil, can tolerate harsh and low nutrient environments, and can form spores. Staphylococcus-species are part of the human skin microbiome. Microbes inside clean area are originated from personnel or surface of material transferred there. Material surface bioburden creates a contamination risk of aseptically prepared products, and thus material transfer and disinfection are critical stages during aseptic processing.
  • Pessi, Jenni (2013)
    Polymer microspheres hold great potential as oral drug delivery system for therapeutic proteins. Microspheres prepared with biocompatible and biodegredable polymers have been extensively studied, since the oral delivery of therapeutic proteins is challenging due to the conditions in the GI-tract. The aims of this research were to apply microfluidics on polymeric microsphere preparation process, to determine what kind of formulations are suitable for this technology, to establish a controlled preparation process that produces advanced particles and to create a template for oral protein drug delivery. With microfluidic fabrication it is possible to gain control over the process and content of each droplet. However, finding suitable formulations for microfluidics is demanding. In this study, biphasic flow was employed to successfully produce double (W/O/W) emulsion droplets with ultra thin shells. Once the process and formulation variables were optimized constant droplet production was achieved. Flow rates used were 500 µl/h in the inner and in the middle phase and 2500 µl/h in the outer phase, respectively. Two formulations were selected for further characterization: 5 % poly(vinyl alcohol) in water in the outer phase, 3 % polycaprolactone in ethyl acetate in the middle phase and either 10 % or 20 % poly(vinyl alcohol) and polyethylenglycol (1:4) in water in the inner phase. All the particles were found to be intact and contain the inner phase, as verified by confocal microscopy. Further, the particles were monodisperse and non-porous, as observed by scanning electron microscopy. Particle size was found to be around 20-40 µm, variation in the particle size within one batch was small and the particles were stable up to 4 weeks. The encapsulation efficiency of the particles was remarkable; as high as 85 % loading of the model compound, bovine serum albumin. Particles released 30 % of their content within 48 hours. In conlusion, developing functional formulations for micfoluidic technology was possible, the microparticles encapsulated the model protein extremely well and all in all microfluidic technology had a lot of potential for droplet manufacturing for pharmaceutical applications.
  • Lifländer, Rami (2020)
    Throughout the history, there has been a wide selection of drugs developed for therapy of cardiovascular diseases (CVD). Despite a broad spectrum of different therapeutic strategies to deaccelerate and try to reverse the progression of cardiovascular diseases has been achieved, only a modest amelioration of the health of the CVD patients was achieved, as the mortality remains high by being the cause of nearly one in every three deaths yearly, myocardial infarction being involved in majority of these cases. Novel solutions are being studied to overcome this problem, one of them being nanoparticles, which may provide potential solution by carrying drugs to the desired location. Microfluidics technique may further improve the properties of nanoparticles, being a platform that allows the production of homogenous and repeatable batches that are non-dependent by the operator using it. In this thesis, it is described how microfluidics-based preparation of spermine-functionalised acetalated dextran nanoparticles co-loaded with a trisubstituted isoxazole and curcumin perform in physicochemical and in vitro experiments, in order to evaluate their potential in the application of ischemic myocardial injury therapy.
  • Lehto, Kristiina (2023)
    Migreeni on toistuvia päänsärkykohtauksia aiheuttava neurologinen sairaus, jonka esiintyvyys on hyvin laajaa – Suomessa migreeniä sairastaa lähes joka kymmenes väestöstä. Migreenin puhkeamisella on tutkimusten mukaan vahva yhteys genetiikkaan, ja migreenin hoidossa käytettyjen lääkevalmisteiden metabolia on oleellisesti sidoksissa geeneihin. Farmakogenetiikka tutkii tieteenalana, miten perintötekijät vaikuttavat lääkeaineiden aineenvaihduntaan ja niistä syntyvään lääkevasteeseen. Tässä tutkimuksessa tarkasteltiin migreenin estolääkkeenä käytetyn trisyklisen masennuslääkkeen, amitriptyliinin, metaboliassa esiintyviä mahdollisia geneettisiä eroja itä- ja länsisuomalaisten välillä. Tutkimus toteutettiin tarkastelemalla yli 10 000 Terveystalon Biopankin biopankkinäytettä, joista määritettiin kolmen CYP-entsyymin (CYP2C9, CYP2C19, CYP2D6) fenotyyppien esiintyvyys itä- ja länsisuomalaisissa. Tutkimustulosten mukaan eroavaisuudet fenotyyppien esiintyvyydessä itä- ja länsisuomalaisten välillä olivat maltillisia. Amitriptyliinin metaboliassa erityisen oleellisen CYP2C19 geenin osalta sekä normaalia hitaampi että hidas metabolia olivat yleisempiä idässä kuin lännessä. Hitaan metabolian riskinä on tavallista suuremmat plasmapitoisuudet ja siten lisääntyneet lääkevalmisteen haittavaikutusriskit. Näin ollen amitriptyliiniä tulisi hitailla metaboloijilla käyttää harkiten, aloittaa vaihtoehtoinen lääkitys tai pienentää aloitusannosta puoleen tavanomaisesta. Oikean annostuksen löytämisessä tulisi hyödyntää laboratorion pitoisuusmäärityskokeita. Lisäksi farmakogeneettisillä testeillä voitaisiin havaita mahdollinen geenien tarkempi polymorfia, ja siten varmistaa sekä turvallinen että tehokas yksilöllinen lääkehoito.
  • Peuraniemi, Tuukka (2012)
    The aim of this research was to evaluate the use of microfluidic paper-based devices (µPAD) in drug analysis. Micro total analysis systems (µTAS) channels are in the range of a few micrometers and are capable of performing all steps of a chemical analysis. The advantages of miniaturization are lower sample consumption and faster analysis time. µTASs are usually fabricated of glass, silicon or polymers and their fabrication requires cleanroom facilities and specific equipment. Paper offers an inexpensive and versatile substrate for µTASs. Paper wicks liquids and no external pumps are required. µPADs advantages over µTAS are its ease of use and inexpensive and simple fabrication. µPADs are fabricated by patterning hydrophobic barriers in hydrophilic paper. There are several fabrication methods for µPADs such as photolithography, cutting and methods based on the application of wax (etching, wax printing, wax dipping). In this research wax printing was selected as the fabrication method because it's simple, rapid and inexpensive. Wax was printed using Xerox Phaser 8560DN solid ink printer. After printing the wax was melted through the paper by heating the paper at 150 °C for 120 seconds on a hotplate. Thus the wax creates a hydrophobic barrier on the hydrophilic paper which channels the liquids flow. Owing to papers anisotropic nature the wax also spreads horizontally in the paper when heated, thus reducing the wax patterns resolution and making the pattern coarse. Wax printing is an inexpensive and simple fabrication method suitable for fabricating µPADs. Also liquids flow velocity and methods for controlling the flow rate were studied. By knowing the flow velocity, one can assure that the analytes and reagents reach the reaction site. Controlling the flow velocity enables the use of multiphase reactions or the use of multiple simultaneous reactions on the µPAD. The liquid flow velocity can be controlled by changing the hydrophilic channels width, reducing the average pore size by melting a layer of wax inside the hydrophilic channel or by changing the surface tension or viscosity of the liquid used. Colorimetric assays are the most commonly used detection methods in µPADs, but also electrochemical sensing and detection methods based on fluorescence are used. In this study direct and indirect fluorescence detection methods were studied. In the detection method based on direct fluorescence, fluorescein and coumarine derivates were studied. In indirect fluorescence amino acids fluorescamine conjugates, which were created in the paper, were studied. Level of the analytes detected in direct fluorescence detection was 10-13 mol in the range of visible light and 10-12 mol in the range of UV-light. Level of the amino acids fluorescamine conjugates detected in indirect fluorescence detection was 10-9 mol. According to our results the fluorescence based detection methods used in this study are suitable for drug analysis on µPADs.
  • Pöyhönen, Suvi (2017)
    Cortical stroke induces a chain of events that results in secondary injury in the ipsilateral thalamus. Inflammation is a key player in the delayed injury. Microglia, the resident innate immune cells of the brain, seem to have an important role in the initiation and maintenance of the inflammation. After infarct they are rapidly activated and start to proliferate and release proinflammatory cytokines. They may even phagocytose viable neurons, a phenomenon called "phagoptosis". Many studies, which have aimed at inhibition of the the detrimental function of microglia, suggest that inhibition of microglia might offer promising therapeutical targets. However, microglia are also involved in the resolution and the repair phase after infarct, which makes development of novel therapies challenging. The only approved treatment for ischemic stroke, a fibrinolytic agent, has a very narrow therapeutic time window. Thus, new treatments are urgently needed. Modulation of inflammation may offer a wider therapeutic time window. In this study, we investigated the effects of two potentially neurotrophic factors, CDNF (cerebral dopamine neurotrophic factor) and MANF (mesencephalic astrocyte-derived neurotrophic factor), as well as a specific vitronectin receptor blocker, cRGDfV, on the prevention of neuronal death in thalamus in a transient murine cortical stroke model. MANF and CDNF are proteins released during stress of the endoplasmic reticulum (ER). They have been shown to protect neurons during ER stress and to reduce the production of some proinflammatory mediators. The vitronectin receptor blocker has in vitro inhibited microglial phagoptosis. The treatments were administered as single injections to the thalamus 7 days after the stroke onset. CDNF and MANF alleviated functional deficits, but did not protect thalamic neurons from death or affect the accumulation of phagocytic microglia. cRGDfV neither enhanced functional outcome nor protected neurons from death. The mechanisms of action were not investigated. In addition, we investigated, whether the death of thalamic neurons in the cortical stroke results in sensitization to pain. Central post-stroke pain has been reported on stroke patients and it has been associated with the death or the disturbances in the function of thalamic neurons. However, in spite of significant reduction in the number of neurons in the ipsilateral thalamus and the increase in the accumulation of phagocytic microglia on day 30 after stroke, we did not observe any significant sensitization to pain caused by thermal or mechanical stimuli on days 3, 14 and 28 after stroke. In conclusion, transient ischemic cortical stroke doesn't seem to induce sensitization to pain. MANF and CDNF seem to alleviate functional deficiencies, but they do not protect thalamic neurons from delayed death.
  • Petäjäsuvanto, Piia (2023)
    Microcrystalline cellulose is a compactable, versatile, and popular excipient in tableting. Microcrystalline cellulose is produced using acid hydrolysis where most of the amorphous areas are removed and the crystalline part is left. Particle size affects most on the functionality of microcrystalline cellulose and that can be altered by changing the duration of acid hydrolysis or the drying method. The aim of this Master’s thesis was to compare new microcrystalline materials produced using energy efficient methods, to commercial Avicel-powders. The used formulation consisted of microcrystalline cellulose, hydroxypropyl methylcellulose, magnesium stearate and dried colloidal silicon dioxide. Due to the small particle size of AaltoCell™ samples it was not possible to use it for direct compaction, but with wet granulation this was successful. The tablets were tested by the standards of European pharmacopoeia and the tablets from wet granulated Avicel PH-101, AaltoCell™ sample B and C passed all the tests. Probably the problem with the rejected formulations was poor flowability, which caused poor reproducibility in the experiments with direct compressed tablets. The wet granulated Avicel PH-101 produced the best tablets with the used formulation.
  • Mäkinen, Jarkko (2014)
    Miniaturizing of analytical techniques in mass spectrometry has received a lot of attention amongst scientists. The gains of miniaturization of analytical systems are rapid analyses, lower solvent consumption, the option for automatization and lower costs. A glass-made microchip heated nebulizer and a newer version, steel-made nebulizer, have been recently developed. The aim of this study was to evaluate and compare performances of the nebulizers. Changes in test conditions and effects of different dopants to intensiveness of the analytes' signals were analyzed. Speed of nebulizer gas, speed of analyte flow and magnitude of heating were the parameters of the changes in test conditions. The temperature of the flow from the nebulizers was also measured and analyzed. The intensiveness profiles of the analytes between the nebulizers were unequal, when changes in the speed of nebulizer gas and magnitude of heating were measured. The nebulizers reacted the same way to changes of the speed of analyte flow. The faster the analyte flow was, the more intensive the analytes' signals were. The steel tube nebulizer generated on average more intensive signals of the analytes than glass-made microchip. Temperature of the glass-made nebulizer was considerably higher than that of steel tube nebulizer. The most intensive signals of the analytes were achieved when toluene was used as a dopant. Steel tube nebulizer was more efficient in ionizing analytes than glass-made microchip. However, with steel tube nebulizer it could be difficult to analyze compounds with high boiling point. One goal of this study was to combine the steel tube nebulizer with capLC, but due to technical failures of the capLC equipment this was not possible. In the future, it would be beneficial to improve the steel tube nebulizer's heating mechanism. Also it could be combined with other ionization techniques as has been done with glass-made nebulizer.
  • Kurvonen, Sampo (2019)
    Background: Antibiotics have been an important factor in the dramatic decrease of infectious disease mortality in the 20th century. Bacteria are, however, very quick to respond to the changes in their environment because of their short life cycle. Thus, the development of bacterial antibiotic resistance is a natural consequence of the enormous worldwide antibiotic use. The current situation is that the antibiotic resistance develops faster than novel antibiotics are found and developed. The three main resistance strategies of Gram-negative bacteria are: modification of the antibiotic target, enzymatic inactivation of the antibiotic and reduce of the intracellular antibiotic concentration by changing the function of the outer membrane. To decrease the intracellular antibiotic concentration bacteria use efflux pumps. RND efflux pumps are the most important family of efflux pumps regarding antibiotic resistance. They typically function as a part of a tripartite structure which allows the efflux of antibiotics to the extracellular space. Multiple inhibitors have been developed against RND efflux pumps but none has reached the clinical stage of drug development. Objectives: Development and testing of a 384-well plate method for screening efflux pump inhibitors for E. coli (BAA1161) efflux pumps. Methods: Verifying that the absorbance measurement is a sensitive enough method for measuring the bacterial (BAA1161) growth in 384-well plate format. The antibiotic chosen to be used in the screening method was piperacillin and the positive control efflux pump inhibitor was mefloquine. Determining the minimum growth inhibiting concentrations (MICs) of piperacillin and mefloquine in 96- and 384-well plate formats. Verification of the synergistic growth inhibitory effect of piperacillin and mefloquine with the checkerboard method in 96- and 384-well plate formats. Determining the positional effect in the 384-well plate. Determining the highest DMSO concentration without effect on the growth of BAA1161. Screening of 126 natural compounds in 384-well plates to test the developed method. Screening was done in quadruplicates based on the growth inhibitory effect of the natural compounds when combined with piperacillin. Dose-response assay was conducted in combination with and without piperacillin with the compounds that showed growth inhibiting effect during screening. Results and discussion: Absorbance measurement was sensitive enough method for measuring the BAA1161 growth in the 384-well plate. MIC value of mefloquine was 32 μg/ml in both plate formats. Piperacillin’s MIC was 1024 μg/ml in the 96-well plate, but on the 384-well plate there was variation in the MIC. Piperacillin and mefloquine showed synergistic effect on BAA1161 growth inhibition in the checkerboard assays. Positional effect could not be determined, because of the variation in the BAA1161 growth inhibition effect of piperacillin. This randomly occurring phenomenon were piperacillin inhibited BAA1161 growth completely or almost completely with sub-MIC concentration was encountered in all the subsequent experiments in the 384-well plate format. One possible reason for this phenomenon, occuring in the 384-well plate format, could be piperacillin heteroresistance of BAA1161 strain. In the test screen, four compounds, which all included gallic acid ester, showed promising activity. These compounds were: epigallocatechin gallate, hamamelitannin, isopropyl gallate and octyl gallate. In the dose-response assay, hamamelitannin’s and octyl gallate’s effect was synergistic with piperacillin. Conclusions: The developed method can be used to screen novel efflux pump inhibitors. However, to increase the reliability of the method, further optimization is required to eliminate the variability in the effect of piperacillin. When plate format of a method is changed, factors which could affect the functionality of the method in the new format should be carefully assessed. Based on the test screed, gallic acid esters are interesting compounds which combined effects with antibiotics should be studied in the future experiments.
  • Svanbäck, Sami (2013)
    The dissolution rate is one of the most important physicochemical properties of drug substances. Above all, it demonstrates the energetic interaction between solvent and solute molecules, and is therefore a valuable tool for understanding drug substance properties. Dissolution studies are a widely used method in many areas of the pharmaceutical development process, however, only lately has the value of dissolution testing in drug discovery and early development been assessed. The advantages of dissolution testing over other early screening methods, such as kinetic solubility and in silico screening, lies in the possibility of obtaining solid state dependent quantitative data, from small amounts of drug substances. While the general way of studying drug dissolution has been by the multiparticulate bulk approach, studying the constituent single particles of these systems, could give a deeper understanding of the core factors affecting the dissolution rate of drug substances. The aim of the present study was to develop a static and dynamic method, in which it would be possible to analyze the dissolution process of a single pure drug substance particle, by optical microscopy. Both methods produced practically identical dissolution profiles, for image analysis and UV-spectrophotometric data, from the same systems of a single dissolving particle. The dynamic method developed in the present study is the first flow-through technique, in which it is possible to assess the dissolution of a single freely moving drug particle, by continuous physical analysis. The possibility of using physical analysis instead of chemical analysis poses many advantages. These include reduced materials consumption, reduced experiment times, as well as a reduction in the possible sources of error. Most importantly, the advantage of physical analysis lies in the fact that no prior chemical knowledge about the studied substance is needed. This makes physical analysis an optimal technique for studying new chemical entities. The novel flow-through method succeeded in obtaining the dissolution characteristics and 3D particle morphological data, of a single pure drug substance particle, of sub-milligram initial weight. The theoretical detection limit of 1 pg, poses an intriguing opportunity for further development.
  • Kallio, Sonja (2014)
    Population is aging. Within aging the morbidity and the use of medicines increase. Polypharmacy and the physiologic changes related to the ageing expose to medication-related problems. This has to be taken into consideration when planning the care of the elderly. Multiprofessional cooperation is seen as a solution to optimize the medicines' use among the aged people. Finnish Medicines Agency (Fimea) has started a network with local multiprofessional health care teams. The aim of the network is to make a national guideline for multiprofessional cooperation and optimizing the medicines' use among the aged people. The objective of the study was to clarify multiprofessional working models to optimize the medicines' use that had been carried out or planned by the teams belonging to the network. The models can work as examples when creating standardized practices to multiprofessional cooperation in Finland. Factors that promote or prevent multiprofessional cooperation and the problems of optimizing the medicines' use were clarified as were the possible solutions to solve them. Factors to strengthen cooperation and its effects were clarified on the basis of experience of the multiprofessional teams. As a material of the study were the interviews (n=15) of health care professionals (n=55) invited to Fimea's multiprofessional network. Fimea had collected the material that consists of group discussions (n=10), pair interviews (n=3) and individual interviews (n=2). The interviews that had been recorded were transcribed and analyzed by using a combine of inductive and deductive content analysis. A theoretical framework in the study was multiprofessional teamwork and networking. According to the interviews, multiprofessional cooperation in optimizing the medicines' use among the aged has been carried out in Finland in both public and private health care. The interviewees think that the most important way to optimize the medicines' use is clear division of tasks and responsibilities. Adding more pharmacists to all over the public health services and fostering the role of the community pharmacies as a part of the health care are seen as solutions. Multiprofessional meetings and education can break barriers between different professionals. The most common problems are the challenges related to economic limitations and to the busy work. There are problems in IT systems and information transfer. At the individual level, the most common problems seem to be in communication and the attitudes. The interviewees' experience is that successful multiprofessional cooperation increases medication safety and improves patients' state. The work of all the professions is faciliatated and burden of the public health service decreases. Lighter medication reviews could be used to find the patients who benefit from the comprehensive medication review. Information transfer and the currency of patients' medication should be secured with functioning IT systems. The results of the study can be utilised when developing multiprofessional practices to optimize the medicines' use. More study is needed to show the profitability of medical reviews, dose dispensing and other services.
  • Ojala, Katja (2010)
    Glutamate is the principal excitatory neurotransmitter in the central nervous system. Glutamatergic neurotransmission plays a central role in the development and maintenance of drug addiction. Glutamate interacts with other neurotransmitters such as dopamine in the actions concerning addiction. During the development of drug addiction, plastic changes in the neuronal connections related to memory and learning occur for example in the amount of synapses and in the efficacy of their action. Glutamatergic AMPA receptor and especially its GluA1 subunit are thought to be included in the neurobiological mechanisms related to drug addiction. Compulsive drug craving and relapses to drug use after a period of abstinence are central problems among people suffering drug addiction. Conditioned place preference is a technique that is used to study motivational properties of drugs in experimental animals. The aim of this master's thesis was to examine the importance of glutamatergic AMPA receptor GluA1 subunit in the morphine-induced place preference and in its extinction and reinstatement behaviour. Locomotor activity of mice was studied during all the phases of experiment. Glutamatergic AMPA receptor GluA1 subunit-deficient (GluA1-/-) and their control (wildtype) mice, based on C57BL/6J mouse strain, were used in the experiments. During the conditioning phase, the mice were trained to associate the effects of morphine (20 mg/kg) with a specific environment. After conditioning, the extinction with morphine paired conditioning environment was assessed by giving saline (0,9 % NaCl solution) to mice. The extinction phase was followed by reinstatement test, in which mice were given morphine (20 mg/kg). The seeking of animals with morphine paired conditioning environment described drug-seeking during different phases of experiment. GluA1-/- mice were more hyperactive when placed in the testing environment compared to the wildtype mice. However, the morphine-induced locomotor activity did not differ between genotypes. Locomotor activity of both genotypes was sensitized equally in consequence of repeated morphine exposures. Morphine induced place preference in both genotypes. Furthermore, the extinction of morphine place preference happened in both genotypes. However, the results of reinstatement test differed partly between genotypes. The place preference was reinstated by morphine in wildtype mice, but not in GluA1-/- mice, when using repeated testing extinction method. Instead of place preference, wildtype mice exhibited place aversion, when extinction method was saline conditioning. As a result of these experiments, extinction method can have an impact on the results of reinstatement test and conclusions cannot be done on the importance of GluA1 subunit in morphine reinstatement. In conclusion, the results of place preference experiments support the conception that GluA1 subunit is not significant in morphine conditioning. However, based on these experiments, GluA1 subunit is not important in morphine extinction, as one might assume on the basis of literature. GluA1 subunit may have an importance in morphine reinstatement, although the results of reinstatement test were partly contradictory.
  • Andersson, Charlotta (2023)
    Heart failure is a global health issue that can result from various factors, one of which is myocardial infarction. The adult human heart has limited regenerative capacity to cover the loss of cardiomyocytes after myocardial infarction with new cardiomyocytes. The main responses to the loss of cardiomyocytes are fibrotic scar formation and the hypertrophy of remaining cardiomyocytes. Prolonged hypertrophy eventually leads to heart failure. Current treatments for heart failure only relieve the symptoms. Inducing cardiac regeneration could be one possible way to prevent and treat heart failure. Thus, to develop medical treatments that enhance the regenerative capacity, a comprehensive understanding of precise cellular mechanisms behind heart regeneration is crucial. The objective of this study was to establish a high-content analysis method for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) utilizing the Cell Painting assay to identify and categorize morphological alterations induced by various compounds in hiPSC-CMs. To evaluate the morphological impacts, dozens or even hundreds of cell features were measured at the same time. hiPSC-CMs were exposed to two hypertrophy inducers, endothelin-1 and angiotensin II, and to doxorubicin, which is known to be a cardiotoxic compound. In addition, the effects of a GATA4- targeting compound, C-2021, on hiPSC-CMs were examined. C-2021, was expected to have antihypertrophic effect on the cells. Previously used methods, proBNP staining and qPCR, were used to validate the novel method. According to proBNP staining and qPCR, endothelin-1 induced cardiomyocyte hypertrophy greater than angiotensin II. Compound C-2021 did not show statistically significant antihypertrophic properties after hypertrophic stimuli, but some tendency the alleviate the hypertrophy was noticed. Moreover, by utilizing different data processing programs a novel analysis method was developed. With this method, the different treatment groups were clustered based on the morphological alterations caused by compounds exposures. The hiPSC-CMs exposed to endothelin-1, angiotensin II or doxorubicin showed a different morphological profile compared to the control group hiPSC-CMs. Compound C-2021 was also observed to affect cell morphology. However, the data analysis still needs improvements in order to detect which cell features these compounds affect.
  • Sinisalo, Aino (2015)
    End stage renal disease (ESRD) burdens both society and patient trough lower quality of life and the cost of treatment, as well as through lost productivity. In 2012, the incidence of ESRD was 81 patients per one million inhabitants in Finland. Annual number of kidney transplantations range from 150 to 210. The costs of specialized medical care, adherence to medication and health related quality of life (HRQoL) of kidney transplant patients were analyzed in this study. The aim of the study was to provide research to support the improvement of the kidney transplant patients' health care process and future research on the cost-effectiveness of kidney transplantation. In addition, the aim was to produce information to support health care decision making and resource allocation. The study population included 320 patients who had received a kidney transplant in HYKS. Of the included patients, 198 answered the questionnaire and 122 formed a control population of which only cost data was available. The cost data was collected from the HUS Ecomed-database. Medication adherence was measured with the BAASIS- and VAS-instruments and the HRQoL with the generic 15D-instrument. Forty-three per cent of the patients were non-adherent. There was no statistical difference in the adherence of patients with different dialysis modalities. The correct timing of taking the immunosuppressive medication proved to be its biggest challenge. The average quality of life for kidney transplant patients was measured at 0.87. There were no statistically significant differences in the 15D scores between adherent and non-adherent patients or different dialysis modalities. Instead, there were statistically significant differences between dialysis modalities in some of the 15 dimensions. The 15D score was on average lower among patients with a higher MRCI-score or a longer dialysis period prior to transplantation. The average cost for the specialized medical care of the kidney transplant patients was 34 331 euros on the year prior to the transplant, 52 834 euros one year after the transplant and 8 537 and 7 791 euros on the second and third year after the transplant, respectively. Average costs for all three years after the transplantation combined were 68 932 euros. Based on the results of this study, non-adherence to medication proved to be a considerable issue for kidney transplant patients. The HRQoL after a kidney transplantation was moderately high, although lower than in the age standardized general population. Adherence to medication, HRQoL or the dialysis modality were not associated with cost of the specialized medical care after the kidney transplantation and there was no single factor associated with these post transplant costs. The strength of the study is a comprehensive longitudinal analysis of special care costs and the factors associated with them. On the other hand, health related quality of life is only measured once, which is a limitation. The cost analysis would have been more comprehensive if all the health care cost and other direct costs such as travel and time cost as well as indirect costs such the loss of productivity had been included.
  • Kauppinen, Elisa (2021)
    Polypharmacy in older adults is common and there are many things to be corrected in their medication. Medication reviews can be used to identify and address these problems using interprofessional collaboration. Renal insufficiency is common in older adults and its consideration contributes to medication safety. The aim of this study was to investigate the prevalence of renal insufficiency in Lohja home care clients over the age of 65, for whom medication review or comprehensive medication review had been done. The purpose was to investigate from medication review reports how many observations pharmacists made about the drugs that should be avoided or dose reduced. In addition, it was investigated whether the medications of the subjects could be changed during the intervention and whether the plasma creatinine values correlated with the GFR values. The material consisted of the medication review reports of 60 home care clients in the intervention study launched in Lohja year 2015. Medication reviews were done in 2016–2017. Half (n = 30/60) of the subjects had at least one drug for which pharmacist proposed a medication change due to a reduced GFR. Proposals for changes (n=60) were presented 1–7 per subject. The majority of the proposed changes, (52 %, n= 31/60), concerned dose reduction, and 22 % (n=13/60) discontinuation. Other proposals totaled 26 % (n= 16/60). 42 % (n=13/31) of the dose reduction proposals were implemented. Almost all of the drug discontinuation 92% (n=12/13) proposals were implemented. In total, 47 % (n = 28/60) of the proposals were implemented. Nervous system drugs formed the largest group (30 %, n = 18) for which a change was proposed. The second highest number of proposals was for drugs for cardiovascular system (27 %, n=16) and the alimentary tract and metabolism (27 %, n=16). Based on GFR, 93 % (n = 56) of subjects had declined renal function (GFR <90 ml/min). Mild kidney damage (GFR=89–60 ml/min) was the most common; 73 % of men (n=11) and 47 % of women (n=21). In 65 % (n=39) of subjects, plasma creatinine was within or below reference range. Plasma creatinine was above reference value in 25 % (n=15) of subjects. The study confirms that plasma creatinine is not suitable measure of renal insufficiency in the elderly.
  • Laakso, Riina (2023)
    Diseases caused by foodborne pathogens are a global threat, which is why new bioactive compounds are expected in the food industry. The purpose of this work was to investigate the antimicrobial effects of three different plants, blackcurrant (Ribes nigrum), rhubarb (Rheum spp.), and Scots pine (Pinus sylvestris), against seven pathogenic bacteria. Bioactivity of these plants has been previously shown, but results have varied widely depending for example on the plant part, extraction solvent and pathogen. The plant samples were extracted with 30 % or 80 % ethanol-water solution. There was a total of 12 extracts: rhubarb petiole (dried at 45 °C or lyophilized), rhubarb root (dried at 50 °C), blackcurrant berry (dried at 45 °C or lyophilized) and lyophilized juice of Scots pine needles. Extracts were dissolved in dimethyl sulfoxide and bioactivity screening of the extracts was determined at a concentration of 1,0 mg/ml, after which the active extracts were subjected to minimum inhibitory concentration (MIC) determination (n=2-3) at eight concentrations (0,0625-4,0 mg/ml). Antimicrobial experiments were performed on a 96-well plate following Clinical and Laboratory Standards Institute guidelines. Bioactivity was determined based on absorbance measurements and visual inspection. The extract of rhubarb root showed most potential against tested bacteria. The lowest MIC values (0,25 mg/ml and 0,50 mg/ml) were obtained with rhubarb root extracts (extracted with 80% or 30 % ethanol-water solution) against Staphylococcus aureus and Bacillus cereus and 1,0 mg/ml against Listeria monocytogenes. Based on this study rhubarb root extract could be a potential natural antimicrobial against foodborne pathogens.
  • Kari-Koskinen, Julia (2021)
    Left ventricular noncompaction cardiomyopathy (LVNC) is a unique form of cardiomyopathy, which is believed to arise from arrest in the compaction process during cardiac development. Dysfunctions in cell cycle regulation and increased or decreased proliferation of cardiomyocytes during cardiac development are likely to contribute to the development of LVNC. SCN5A gene encoding the α-subunit of cardiac voltage gated sodium channel Nav1.5 has associated with LVNC- phenotype in a Finnish family. The direct correlation of SCN5A gene mutation and LVNC has not been studied before. There is strong evidence that Nav1.5 channel has an essential role in cardiac development and cardiomyocyte proliferation, therefore perturbed function of the channel might also contribute to the development of LVNC. We used patient-specific human induced pluripotent stem cell -derived cardiomyocytes (hiPSC-CMs), reprogrammed from fibroblasts obtained from LVNC patient carrying SCN5A to study the phenotype of the cells. We utilized immunofluorescent staining in combination with high content analysis (HCA) to investigate the proliferation and Nav1.5 cellular localization. Proliferation potential was assessed at multiple timepoints from three to six weeks. We also investigated the stress response of patient-specific hiPSC-CMs by exposing the cells to mechanical stretch, a hypertrophy inducer, followed by quantitative reverse transcription PCR to study changes in stress biomarker levels. According to our results, the patient-specific hiPSC-CMs have prolonged proliferation compared to control cells as the proliferation peaks towards the last timepoint, whereas in control cells it decreases. Differences were also observed in the hypertrophic gene expression after 24-hour mechanical stretching. An increase in NPPB expression levels caused by stretching was threefold in patient-specific cells to control cells. These results implicate that SCN5A gene has as an important role on cardiomyocyte proliferation. Mutations in SCN5A could correspond to increased proliferation in trabeculations during cardiac development, which might be preventing the compaction process and lead to the development of LVNC. Our results emphasizes that SCN5A has an important role in cardiomyocyte physiology unrelated solely to electrical activity.
  • Kunnari, Mikko (2016)
    Crohn's disease is a type of inflammatory bowel disease. There are no treatment procedures that can cure Crohn's disease, but it is usually controllable with medicinal options. However 70 - 80 % of patients will require surgery and most undergo several during their life, due to weak local potency of drugs and disrupted recovery from surgical treatment. A better method of combined treatment, such as a drug releasing surgical suture, could improve the disease recovery process. One approach would be to coat a surgical suture with nanofibrillar cellulose (NFC) hydrogel containing the active drug ingredient within. NFC is biocompatible, biostable and it can be easily chemically modified. It displays pseudoplastic and thixotropic gel-like behavior in aqueous suspension in addition to high shear thinning properties under low and high shear rates. The shear-thinning behavior is particularly useful in a range of different coating applications. Furthermore, recent studies have shown the potential of NFC in controlled drug release. The aim of this Master's thesis was to investigate the suitability of anionic NFC hydrogel for surgical suture coatings and controlled release applications. The structure of NFC hydrogel was modified with crosslinking cations (Fe3+, Al3+, Ca2+) and alginate. The diffusion studies were performed with two antibiotics, metronidazole and rifaximin together with FITC-dextrans (10 and 250 kDa). The surgical suture was coated with each type of hydrogels (n = 16). Furthermore, the suitability of suture drug formulation for controlled drug release was simulated with STELLA® modeling software. It was shown that the NFC hydrogel structure was stiffened with the use of crosslinking cations; however similar results were not observed with the addition of alginate. Release profiles of model compounds were similar before and after NFC hydrogel crosslinking. At 6 days, 50 - 60 % of 10 kDa dextran (6 µg) was released. For 250 kDa dextran (6 µg) the released amount was 25 - 35 %. During the first 3 days of the diffusion study, all of metronidazole (20 µg) was released. Rifaximin samples were not obtained due to high adsorption to the container surfaces. The release profiles of metronidazole and 10 kDa dextran had linear correlation with square-root diffusion process. 250 kDa dextran followed a near zero-order kinetics after a few hours from the start. The coating was performed successfully with NFC hydrogels except for hydrogels with dextrans or without crosslinking. Metronidazole was predicted to release from the surgical suture almost instantly with STELLA® modeling software. NFC hydrogel shows potential as a matrix for controlled drug release and the coating of surgical sutures. However, the manufacturing method of the NFC hydrogel could be improved with surface modifications of nanofibrils or with the choice of a drug or of its derivatives. With pharmacokinetic simulation models it is possible to predict and estimate different factors which affect drug release from the surgical suture. Furthermore, the simulation models can be used to estimate an effect in the treatment of Crohn's disease.
  • Li, Mingwei (2016)
    Nanofibrillar cellulose (NFC) can form hydrogels with high water content (> 98 %). It has been studied for drug release, and it has been used as a cell culture matrix, due to its similar structure to extracellular matrix (ECM). In addition it has been found that they has no cytotoxicity. Iontophoresis is the application of an electric current over a defined area for the purpose of enhancing permeation across a membrane for ionized drug species. The aim in the experimental work in this Master's thesis is twofold. First, to find out the suitable drug loading concentrations into NFC hydrogels, which can provide a good release profile, a release study with two model drugs, propranolol and ketoprofen, loaded into three types of NFC hydrogels at three different concentrations, was carried out for this purpose. Second, to see if NFC hydrogels are applicable as a drug reservoir in iontophoretic transdermal drug delivery applications, an iontophoresis study was carried out using porcine ear skin model in vitro for human skin with propranolol loaded into NFC hydrogel of type A. In addition, Stella models were used as an aid to find suitable ways to predict the release and permeation behaviour of models drugs in the abovementioned context. The UPLC results from the release study show for both model drugs, the wt. % released had linear correlation with squareroot of time. At 6 hours, more than 70 wt. % propranolol was released from hydrogel reservoir. For ketoprofen, the release varied between 30 - 87 wt. %, where higher initial loading concentrations produced a decrease in the wt. % released from hydrogel. The iontophoresis study did not show a significant difference between the tested current densities (0.50 mA/cm2; 0.25 mA/cm2) produced on the wt. % of drug released. Simulation models could be run with the mathematical equations for diffusion controlled drug release. In conclusion, the NFC hydrogels show potential as drug reservoir for drug release. Additional experimental data using other types of drug reservoirs should be obtained for a better understanding of the suitability of NFC hydrogels as a drug reservoir in iontophoretic transdermal drug delivery.