Browsing by study line "Farmakologi"

Alpha-beta-hydrolase domain containing proteins (ABHD) are involved in lipid metabolism and its regulation in human and animal cells. Approximately 50 of these proteins have been identified and their physiological and pathophysiological functions are still further investigated. ABHD2, ABHD3, ABHD4, ABHD5, ABHD6, ABHD12 and ABHD16 are involved in the metabolism of glycerin esters and phospholipids, in particular lipid mediator 2-arachidonoylglycerol (2-AG) and its metabolites have a significant effect on neuroinflammation via the endocannabinoid system. ABHD12 and ABHD16A were at the center of focus in this thesis as enzymes regulating phosphatidylserine and pro-inflammatory lysophosphatidylserine. In this study, toxicity of five (5) abietane terpene derivatives was tested using mouse E15/16 prenatal cortical neurons, cultured in 96-well plates. There were totally 8 plates cultured in three different batches, 60 cell containing wells per each plate. Wells from each plate were divided into treatment groups of 17, three concentrations of every five compound, control and VEH groups. Those concentrations were 0,1 µM, 1 µM and 10 µM, and for the last two plates 1 µM, 10 µM and 100 µM. Treatment was also separated into three batches like the cell culturing. After treatment, number of living cortical neurons in each treatment groups were counted. For that, cells were treated with immunofluorescent NeuN and DAPI antigens and the fluorescence was imagined with automated microscope. CellProfiler was used to recognize and count the number of living cortical neurons. Immunofluorescent MAP2 antigens were also used but because the shape of MAP2-fluorescing cells, CellProfiler could not recognize them. Intensity of MAP2-fluorescence were measured from those imaged, so the work would not be wasted. One-way ordinary variance analyze ANOVA was carried out for the data to figure out if there were statistically significant results. For compound TAC174, there were several significant results with different concentrations but unfortunately, some results showed signs of toxicity and others improved cell-growth. Some significant results were also found with compounds TAC121, TAC147 and TAD40 showing sings of toxicity, but reliability of those results was questioned. Only one compound, TAC150 was not showing clear toxicity towards mouse cortical neurons, at least with lower concentrations. For conclusion, there were no clear or significant results if these compounds are toxic for cortical neurons. TAC150 showed the least sings of toxicity, therefore it could possibly be considered for further studies in medical field.

Introduction: When people age, the composition of sleep changes and sleep becomes more sensitive to external disturbances, making insomnia also more common. Medication is not the first-line treatment option for insomnia. Benzodiazepines or benzodiazepine receptor agonists for the treatment of insomnia have been in the focus of past studies. The content of the dosing instructions for the supplied medicines has not been studied. The dosing instructions should provide clear instructions on how to dose the medicine prescribed to the patient. The aim of this study was to investigate the content and quality of dosing instructions prescribed for the treatment of insomnia for Finns aged ≥75 years in 2020 based on the prospective reimbursement register data by the Social Insurance Institution of Finland (Kela). Materials and methods: The reimbursed purchases of all medicines by persons aged ≥75 years from 1.1.2020 to 31.12.2020 were selected by ATC code from the medicines data according to the Insomnia: Current Care guidelines. The data was gathered from Kela’s dispensations reimbursed under the National Health Insurance scheme. The dataset consisted of 1,080,843 delivery lines, which were screened, and 328,285 lines were included in the analyses. Dosage instructions were reviewed according to the following predetermined five categories: frequency of use, dose, timing, warnings or remarks, and inappropriate instructions. In addition, 1000 dosing instructions were randomly derived to study the phrasing and appropriateness of the dosing instructions in more detail. Results: In 2020, an average of 3.8 reimbursed hypnotic drug deliveries were made per elderly person. Of the deliveries, 68% were for women. 52% of drug deliveries were partially made later and not by original prescription. In the hypnotic dataset, the three most administered drugs were zopiclone (41%), mirtazapine (34%) and zolpidem (12%). The dosage was prescribed in 98–99% of the dosage instructions. Dosing schedule was reported in 83% of dosing instructions and regularity of use was reported in 57% of them. Only 3-6% of the dosing instructions had comments or warnings. 1–2% of the dosing instructions were vague. The duration or regularity of use was clearly indicated in 5% of the dosing instructions. Only 0.1% of dosage instructions contained instructions for discontinuation or reduction. Discussion: The dose and timing of administration were well reported, but the frequency of use was reported in only about half of the dosing instructions. Only few dosing instructions contained remarks or warnings even though hypnotics are at risk for the elderly. Among the three most administered drugs for the treatment of insomnia were two benzodiazepine receptor agonists, zopiclone and zolpidem. However, they may not be suitable for the elderly according to Beers criteria and their use should be avoided. During 2020, an average of four drug deliveries were made per elderly person for the treatment of insomnia, which may indicate prolonged hypnotics use. In addition, more than half of the deliveries were partially made later and not by original prescription. Thus, several drug packages are prescribed for prescriptions, although the drug-based treatment of insomnia should only be short-lived. Conclusions: There are significant deficiencies in the contents and quality in the dosing instructions for drugs delivered to the elderly for insomnia. Minimum information on dose, timing and duration of use was not found in all dosing instructions in this study. Understandable dosing instructions and the reduction in the amount of medication in the prescription could have a further effect on reducing the long-term use of hypnotics, also increasing the safety of medicine use in the elderly.

Binge eating disorder (BED) is the most common eating disorder characterized by compulsive recurrent binge eating episodes with the sense of a lack of control. During a binge eating episode, one eats a larger amount of food, typically high in fat and/or sugar, than would normally be eaten in a discrete period of time. After the episode, negative emotions, such as shame and self-disgust, are present. However, BED does not include compensatory behavior, such as vomiting or excessive exercise. Due to compulsive and uncontrollable eating behavior, it has been suggested that BED represents a food addiction. Eating energy-dense food activates the dopamine, opioid, and endocannabinoid systems in the brain. This elicits the activation of the reward process. Some drugs and medications affect the same neurotransmitter systems, which may produce neuronal alterations in the reward process, leading to an addiction. Several studies have found that cannabidiol (CBD) reduces the self- administration of cocaine, morphine, alcohol, and sucrose in rodents, suggesting an effect on the reward-response. Some of these effects have been shown to be mediated by cannabinoid receptor 2 and TRPV1 receptor. However, the effects of CBD on bingeing behavior have not been studied up to date. The aim of the study was to investigate the effect of CBD on homeostatic feeding and binge eating behavior in C57BL/6 mice. Five separate experiments were conducted. The first experiment investigated the effect of CBD (15, 50, and 150 mg/kg, i.p.) on locomotor activity in a modified open field test over a 2-hour period. In the second test, the effect of CBD (15, 50, and 150 mg/kg, i.p.) on homeostatic feeding was monitored in non-bingeing mice. Next, a limited intermittent access binge eating model without food deprivation or stressors was inducted. Mice had access to laboratory chow ad libitum, but a high energy diet (high in fat, HED) was presented in 24-hour periods every 5-8 days. Then the effect of CBD (15, 50, and 150 mg/kg, i.p.) on HED and chow intake in bingeing mice was investigated. In the fourth experiment, seven days following the administration, the after effect of CBD was studied by monitoring food intake without CBD treatment. Finally, it was investigated whether the effect of CBD can be inhibited by TRPV1 receptor antagonist AMG9810 (1 mg/kg, i.p). In each test, the food intake was monitored at the time point 0,5, 2,5, and 24 h after CBD treatment. Also, water consumption was measured in each experiment. The results revealed that CBD does not affect locomotor activity or homeostatic feeding at a dose of 15, 50, or 150 mg/kg (i.p). However, the results showed that CBD reduces the intake of HED in a dose-dependent manner (15, 50, or 150 mg/kg; i.p.) and, possibly, increases chow intake. No after effect was observed seven days following the administration. Most likely, TRPV1 does not mediate the effect of CBD on HED intake. Furthermore, no significant effects on water intake were observed. In this study, the core aims were to evaluate whether CBD affects homeostatic feeding or binge eating behavior in mice. The results provided a novel insight into the effects of CBD. The findings indicate that the acute systemic administration of CBD reduces HED intake, and possibly, simultaneously increases chow intake, suggesting a balancing effect on feeding in bingeing mice. However, the role of TRPV1 in this effect remains unclear, and further studies are needed.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons in brain and spinal cord. The degeneration of motor neurons leads to muscle atrophy and paralysis. Currently there is no cure for ALS. Available drugs for ALS can lengthen the survival time by a couple of months. Several factors involve the pathophysiology of ALS, such as endoplasmic reticulum stress and neuroinflammation. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a protein which has shown neuroprotective effects on animal models of Parkinson disease and brain ischemia. C-terminal fragment of MANF can cross the blood-brain barrier, allowing it to be administered subcutaneously instead of injected directly into the brain. The experimental part consists of two parts. The aim of the first part was to study the pharmacokinetic properties of next generation MANF (C-MANF). The aim of the second part was to elucidate the effect of twice a week administered subcutaneous injection of C-MANF in genetic SOD1-G93A mouse model and its neuroprotective effects by assessing protection of lumbar motor neurons. Pharmacokinetic properties of C-MANF were determined in wild type mice after a single subcutaneous injection of C-MANF at different time points by using indirect ELISA assay. The effects of C-MANF in SOD1-G93A mouse model were assessed by subcutaneous injection of either C-MANF or PBS twice a week and by monitoring clinical score and motor behavior of mice from 10 weeks of age to clinical endpoint. Hematoxylin eosin staining was used to study neuroprotective effects of C-MANF. C-MANF administered subcutaneously is absorbed into the blood circulation and the highest serum concentration of C-MANF is after 60 minutes of dosing. Subcutaneously injected C-MANF also crosses the blood-brain barrier and reach the brain in 120 minutes. C-MANF did not preserve motor function or ameliorated ALS symptoms in SOD1-G93A mouse model. In this study C-MANF did not increase the survival of SOD1-G93A mice. C-MANF did not significantly protect motor neurons from degeneration even though there was a slight trend between the groups. No beneficial effects were observed with C-MANF in SOD1-G93A mouse model and therefore the dose and frequency of administration of C-MANF were not optimal. Subcutaneously injected C-MANF provides a safer dosing option for neurodegenerative disorders.

Parkinson´s disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer´s disease. There is still no drug that alters the state of the disease. It has been found that Endoplasmic reticulum (ER) stress is one mechanism in PD. ER stress occurs due to accumulation of unfolded proteins. ER stress triggers Unfolded protein response (UPR) that protects against ER stress by decreasing unfolding of proteins. In the beginning, UPR has protective effect, but in prolonged ER stress UPR triggers apoptotic cell death. There are several key mediators in the UPR pathway. Characterisation of ER stress in PD models may be important for the current and future drug development of PD. If ER stress is a significant factor that affects the disease development, it would be important to find a drug that alters these mechanisms and UPR. This may be a way to halt the disease development. Different animal models of PD, like 6-OHDA (6-hydroxydopamine) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model, have similarities in their mechanisms. It has been found that ER stress occurs both in the brain of PD patients and animal models of PD. That is why studying and further characterisation in animal models is relevant. The aim of this study was to characterize ER stress in 6-OHDA rat model. The expression of some key mediators of the UPR were determined in this study. There were male and female Spraque Dawley rats in this experiment. 6-OHDA or saline was injected intrastriatally in 3 spots by stereotaxic surgery. Two weeks after 6-OHDA lesions, amphetamine-induced rotation test was conducted to the rats. The rats were divided into groups based on lesion size according to the results. For this study, the rats were euthanised at week 2 or week 4 post lesion. The rats were euthanised by carbondioxide, and the death was confirmed by decapitation. The brains were collected and stored in -80°C. Striatum and substantia nigra were collected later. Total RNA was isolated from these samples. Part of the RNA sample was used to conduct cDNA synthesis. Finally, the gene expression of Atf4, Ire1α, Xbp1s, Xbp1t, Grp78 and Chop was measured from these cDNA samples by qPCR (quantitative polymerase chain reaction). The qPCR data describes the expression of exact gene. The data was processed prior to statistical analysis. By statistical analysis, it was possible to compare the expression of these genes between 6-OHDA group and vehicle group. In addition, comparison was made between 6-OHDA treated groups at week 2 and 4. According to the results, only Chop expression had increased in 6-OHDA lesioned rats at week 2 compared to the vehicle group. In other genes there were no statistical differences, unlike in several other studies where the expression was found to be increased. Thus, the characterisation of this model requires further studying, possibly by increasing the sample size and studying later time points as well.

Mild traumatic brain injury (TBI) is defined as an injury that disrupts the normal functioning of the brain and is the result of external force to the head. It is the most common type of traumatic head injury, and it is common especially in contact sports and within military personnel. Mild TBI typically causes no clear structural changes to the head, but it can induce persistent clinical symptoms, as well as microscopic pathological changes to the brain that may eventually lead to neurodegeneration and increase the risk for several diseases. Mild TBI is a risk factor for several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy. The primary objective of this study was to develop a repetitive mild TBI mouse model for future research purposes in the field of head trauma and neurodegeneration. The injury was induced as a closed head injury with an electromagnetic impactor. Literature and pilot experiments were used to define the parameters of the impactor required to induce a brain injury of desired severity. The characterization criteria of the mild TBI model considered the criteria used to define human mild TBI, as well as long term effects often reported after repetitive mild TBI: neurodegeneration as tau protein related pathology, neuroinflammation, and memory deficits. The secondary objective of this study was to tentatively test a prolyl oligopeptidase (PREP) inhibitor on the behavioral and histological effects of mild TBI. The functioning of the mild TBI model was studied by histopathological and behavioral assessments. After baseline behavioral assessment and repetitive (1 injury every 24 hours altogether 5 times) mild TBI inductions, the mice were monitored for approximately 3 months, during which several rounds of behavioral tests were performed. Barnes maze and novel object recognition tests were used to assess memory functions, and locomotor activity test was used to assess general locomotor activity. After euthanasia, brain histopathology was performed to study the amount of tau protein and the level of neuroinflammation. Due to the low number of animals in the study, the results are directional and need to be confirmed in subsequent studies. The histopathology showed greater amount of neuroinflammation and tau protein in the brains of injured mice, but statistical evaluations could not be made. Memory functions were slightly worse in the injured mice compared to controls, but significance of the results is unclear. Locomotor activity was not influenced by the mild TBIs. PREP inhibition treatment increased the locomotor activity of the mice, but the significance is unclear. The mild TBI model seems promising and the characterization criteria were partially met. The results of the study need to be verified in subsequent studies with a greater amount of animals. The model developed here can be used to study the involvement of head trauma in neurodegeneration, as well as treatment alternatives to changes caused by mild TBIs. As there currently are no curative treatments to neurodegenerative diseases, research regarding neurodegeneration and its risk factors is highly important.

Neurodegenerative diseases and neuronal injury after trauma are common causes of neuronal loss. Adult brain has only a limited regenerative capability to replace the lost neurons caused by several distinct brain diseases. Direct reprogramming of brain resident cells into neurons could provide a promising strategy for efficiently replacing non-functional neurons. To date, the focus has been put largely on astrocyte-to-neuron reprogramming despite the relatively low yield of newly generated neurons reported in vivo. According to our hypothesis oligodendrocytes possess a more diverge transcriptomic profile when compared to neurons and astrocytes thus allowing better cell-specific targeting of reprogramming. Here, we establish the molecular tools for direct neuronal reprogramming of human oligodendrocytes to neurons. We investigate whether the expression of a known neural fate specification factor under selected oligodendrocyte-specific promoters is sufficient to induce oligodendrocyte-to-neuron transformation. Furthermore, we test the established tools in vitro using an immortalized human oligodendrocyte cell line. Our preliminary data shows that the human ERBB3 promoter and a single transcription factor transfected cells express doublecortin (DCX), an early marker of neuronal identity. Only recently, the direct in vitro reprogramming of human oligodendrocyte precursor cells into functional neurons has been reported. The direct reprogramming of oligodendrocytes into neurons provides an exciting alternative of neuronal replacement for astrocyte-to-neuron reprogramming. Overall, the field of direct reprogramming offers interesting possibilities for regenerative medicine providing a method for the production of newly generated disease and patient-specific cells.

Ischemic heart disease (IHD) and subsequent heart failure are caused by irreversible loss of contractile cardiomyocytes due to low oxygen supply to the heart. As the leading cause of death worldwide, IHD raises an urgent need for regenerative therapies that prevent or reverse loss of cardiomyocytes. The fetal mammalian heart grows by cardiomyocyte proliferation and utilizes glycolysis as main energy metabolism pathway, until it is introduced to increased oxygen and fatty acid supply at birth. Subsequently, cardiac energy metabolism shifts from glycolysis to β-oxidation of fatty acids and cardiomyocytes exit the mitotic cell cycle. Due to cessation of proliferation the heart can no longer regenerate after ischemic injury and responds to it by introduction of maladaptive pathological processes leading to heart failure. To gain deeper insight on the roles of cardiac metabolism pathways and hypoxia in cell cycle activation, we evaluated the effects of pharmacological metabolic modulation and oxygen supply on cardiomyocyte phenotype and hypoxia response. Furthermore, we studied the changes in the metabolic genotype of cardiomyocytes under alterations of oxygen supply. We utilized quantitative reverse transcription PCR (qRT-PCR) to evaluate the effects of hypoxia and metabolic maturation on the expression of genes involved in hypoxia signaling and metabolism of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Additionally, we investigated the effects of five metabolism-modulating compounds on cell cycle and phenotype of both metabolically matured and unmatured hiPSC-CMs, by utilizing high content analysis. We observed presence of hypoxia signaling as an increase in vascular endothelial growth factor A (VEGFA) expression following 3-hour hypoxic exposure. High expression of succinate dehydrogenase complex flavoprotein subunit A (SDHA) in hiPSC-CMs, which was downregulated at hypoxia, confirmed occurrence of oxidative metabolism induced by metabolic maturation. Surprisingly, metabolic maturation tended to increase proliferation and decrease stress response signaling of hiPSC-CMs. Introduction of the TCA cycle intermediate succinate decreased proliferation of metabolically unmatured hypoxic hiPSC-CMs by 8.2 %. Finally, inhibition of the mevalonate pathway and ketogenesis caused no alterations in hiPSC-CM phenotype or cell cycle, but introduction of the ketone body β-hydroxybutyrate tended to increase proliferation, supporting current evidence that ketogenesis plays a role in cardiomyocyte cell cycle regulation. Our observations suggest that hypoxic hiPSC-CMs can be useful in investigating gene expression and phenotype. Even so, additional methodologies are needed for in-depth evaluation of metabolic reprogramming and its effects on cardiomyocyte phenotype.

Farmasian ammattilaiset ovat lääkealan asiantuntijoita, joilta vaaditaan uudenlaista osaamista muun muassa teknologiakehityksen myötä. Nykypäivän asiantuntijuus edellyttää alakohtaisen eli sisällöllisen osaamisen lisäksi geneerisiä eli yleisiä taitoja ja ammatti-identiteetin muodostumista. Geneerisillä taidoilla tarkoitetaan yleishyödyllisiä taitoja, kuten ongelmanratkaisu- ja kommunikointitaitoja. Ammatti-identiteetillä tarkoitetaan käsitystä omasta työminästä, jonka avulla omaa roolia ja työnkuvaa järkeistetään. Näiden elementtien muodostamaa osaamisen kokonaisuutta kutsutaan kompetenssiksi. Asiantuntijoilta vaadittavan osaamisen muutos on ohjannut yliopistoja vastaamaan paremmin työelämän tarpeisiin. Helsingin yliopistossa toteutettiin Iso Pyörä -koulutusuudistus, jossa koulutusohjelmia uudistettiin komeptenssilähtöisesti. Kaikkiin koulutusohjelmiin ja opintojaksoihin lisättiin osaamistavoitteet, jotka opiskelijoiden tulisi saavuttaa valmistumiseensa mennessä. Osaamistavoitteiden täyttymistä edistää esimerkiksi portfoliotyöskentely, minkä avulla opiskelijat pääsevät hyödyntämään ja kehittämään reflektiotaitojaan. Opiskelijat voivat tuoda opetuksen kehittämiseen aivan uudenlaista näkökulmaa avatessaan käsityksiään esimerkiksi hyvistä opetusmenetelmistä, mitkä ovat auttaneet heitä saavuttamaan laaditut osaamistavoitteet. Toisaalta opiskelijoiden näkökulmasta saadaan tietoa, mikä osaaminen voidaan kokea puutteelliseksi, jolloin opetuksen kehittäminen on mahdollista. Tutkimuksen tavoitteena oli selvittää opiskelijoiden käsityksiä omasta osaamisestaan ja ammatti-identiteetistään sekä millä tasoilla opiskelijat reflektoivat osaamistaan. Tutkimuksessa analysoitiin vuoden 2017 kolmannen vuosikurssin kandiportfolion loppureflektioesseet käyttäen aineistolähtöistä sisällönanalyysimenetelmää. Esseissä opiskelijat reflektoivat osaamistaan suhteessa farmaseutin tutkinnolle asetettuihin osaamistavoitteisiin ja pohtivat omaa ammatti-identiteettiään. Tulosten mukaan opiskelijat saavuttivat monipuolista osaamista lääkkeiden ja lääkehoitojen näkökulmasta sekä kehittivät geneerisiä taitojaan. Puutteellisesti hallittiin useimmiten kielitaito sekä yrityksen ja yhteiskunnan taloudelliset periaatteet. Opiskelijoiden mukaan farmaseutin ammatti-identiteettiä määrittelevät erityisesti lääkeosaaminen ja terveydenhuolto sekä ammatin arvostaminen. Opiskelijoiden pohtimat valmiudet mukailivat osaamistavoitteita. Opiskelijat osasivat arvioida omaa osaamistaan ja nostaa esille vahvuuksiaan ja heikkouksiaan. Opetussuunnitelmaan on onnistuttu sisällyttämään geneeristen taitojen opetus, sillä opiskelijat kokivat saavuttaneensa näitä taitoja pääasiassa hyvin. Opetusta tulisi kehittää kielitaidon ja liiketalouden kohdalla, sillä nämä koettiin usein puutteellisesti hallituksi. Ammatti-identiteettikäsitykset mukailivat kirjallisuutta, sillä muissa tutkimuksissa on saatu samankaltaisia tuloksia.

Current treatments for major depressive disorder have notable limitations including the delay achieving the therapeutic effect. Ketamine has been shown to alleviate the symptoms of depression rapidly and promising findings have also been found when using nitrous oxide. However, the mechanisms behind rapid antidepressant effect are not fully discovered. It seems that rapid-acting treatments alter brain energy metabolism, enhance synaptic plasticity, and repair neuronal dysfunction connected to depression. Particularly, the activation of brain derived neurotrophic factor (BDNF) mediated tropomyosin receptor kinase B (TrkB) signaling has been connected to rapid antidepressant effect. Fasting is also known to induce BDNF production and it is thought to activate BDNF-TrkB signaling. In addition, both of these treatments alter the brain energy metabolism. The objective of this study was to find out how fasting and nitrous oxide alone and in combination affect the rapid antidepressant effect and synaptic plasticity related BDNF-TrkB signaling in mice. Another aim of the research was to determine whether the body temperature changes after these treatments as a marker of metabolic rate. The analyzed brain samples of the mice were collected 15 minutes after cessation of nitrous oxide administration. As a result, it was found that the fasting protocol used in this study did not activate the studied BDNF-TrkB signaling. However, after nitrous oxide administration, the studied signaling and markers related to synaptic plasticity were partly activated. The results from the combination of nitrous oxide and fasting were similar compared to nitrous oxide administration only. It is therefore conceivable, that the effects were caused exclusively by nitrous oxide. Furthermore, a fascinating finding related to energy metabolism was that nitrous oxide reduced the body temperature of the mice significantly 15 minutes after cessation of the gas administration. Overall, these results are promising and consistent with previous research indicating that nitrous oxide administration could be related to induced synaptic plasticity and therefore have antidepressant associated effects. Nitrous oxide could be used to understand the mechanisms behind rapid antidepressant effect and it could be a potential option to treat depression in the future. Based on these results, it seems that energy metabolism could be related to rapid antidepressant effect. It also supports the observations that all different rapid-acting treatments alter the brain energy metabolism.