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Browsing by study line "Farmakologi"

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  • Jäntti, Heli-Noora (2019)
    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.
  • Hedström, Anna (2020)
    The ability to regulate release of noradrenaline, dopamine and GABA is one of the most important roles of the nicotinic receptors. The release of neurotransmitters following stimulation of nicotinic receptors is addressed in the thesis, with focus on dopamine and noradrenaline. Release of neurotransmitters, mediated through nicotinic receptors, has been researched using various methods, including brain slices, microdialysis and synaptosomes. Research using synaptosomes have provided valuable information regarding nicotinic receptors and their ability to regulate neurotransmitter release. Research using receptor specific antagonists have provided information regarding the stoichiometry of nicotinic receptor in different regions of the brain. The primary focus in the thesis, was the characterization of [3H]dopamine release following stimulation of nicotinic receptors with varenicline and acetylcholine, using synaptosomes from mouse striatum. Using a-conotoxin-MII, the [3H]dopamine release was divided into a-conotoxin- MII-resistant and -sensitive release. [3H]Dopamine release was mediated through a6b2*- and a4b2*-receptors from striatal synaptosomes. The involvement of other receptors could not be ruled out, but based on these results and results from previous studies, the involvement of other nicotinic receptors is supposedly low.
  • Järvinen, Janina (2021)
    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.
  • Aromaa, Virve (2022)
    Mild traumatic brain injury (mTBI) is an insult to the brain caused by an external force. Typically contact sport players and military soldiers are prone to mTBI. TBI events trigger pathological processes in the brain and may cause long-term and progressive damages. Increased formation and accumulation of misfolded toxic protein aggregates in the brain leading to neuronal death has been observed after mTBI. In particular, repetitive mTBIs are a risk factor for the development of many neurodegenerative diseases, such as chronic traumatic encephalopathy, Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. As there is no curative treatment to neurodegenerative diseases, research regarding neurodegenerative processes is highly important. Prolyl oligopeptidase (PREP) negatively regulates functions of protein phosphatase 2A (PP2A). It has been shown that PP2A activity is decreased in the brain of those with neurodegenerative diseases and TBI patients, which is thought to be a contributing factor to the development of pathologies of neurodegenerative diseases. The primary objective of this study was to study behavioural changes after repeated mild TBI in PREP knockout mice. The aim was to model mild repeated brain injuries that are common, for instance, in contact sports and that are not accompanied by skull fractures or brain swelling. The intension was to clarify the involvement of the PREP enzyme in behavioural changes induced by repeated mTBI’s and to elucidate long-term pathological changes in the brain. The injury was induced as a closed-head injury with an electromagnetic impactor with one hit every 24 hours and altogether 5 times. A locomotor activity test was performed before the induction of brain injury and was repeated 3 times after mTBI induction. Barnes maze test was used to assess memory and learning functions. In this thesis the brain samples from a previous study were included to also determine the accumulation of total tau protein in wild-type mice. The wild- type mice were administered with either the PREP inhibitor KYP-2047 or HUP-46 10 mg/kg (i.p.) immediately after each hit. After euthanasia, the Western blot assay and immunostaining were performed to study the amount of phosphorylated tau, neuroinflammation, activity of PP2A and autophagy. No differences were found between the sham group and TBI group on the locomotor activity and Barnes maze tests in PREP knockout mice. There was no consistency in total tau protein in wild-type mice treated with PREP inhibitors. In PREP knockout mice there was an upward trend in PP2A levels after mTBI. Repeated mTBI increased markers of phosphorylated tau and neuroinflammation significantly. No significant difference was observed in autophagic function. The results of this thesis are indicative. Due to the low number of animals, the results need to be confirmed in subsequent studies with greater amounts of animals. Based on the results, it seems that absence of the PREP enzyme protects from memory impairments after repeated mTBI. Increased tau protein phosphorylation and neuroinflammation were observed in the TBI group which indicate that PREP alone is not responsible for the development of pathological changes.
  • Artes, Sanna (2020)
    Left ventricular hypertrophy (LVH) takes place when cardiomyocytes respond to excessive stress by growing in size. Cardiomyocytes have a very marginal capability to proliferate, which is why hypertrophic growth is almost their only option to meet the requirements of increased workload. In the long run, however, LVH leads to further problems, such as cardiac failure and an increased risk of myocardial infarction. Hypertension is the most prevalent cause of LVH, and its current treatment relies on antihypertensive drugs. They decrease the workload of the heart and therefore alleviate symptoms but have very little effect on the built damage and remodeling. Understanding the details of cellular level signaling pathways and genetic expression in LVH is crucial for future drug development. Regulation of gene expression is a very complex process, which involves more than just DNA being translated into a protein. In this project, two types of factors participating in this regulation were in focus: long non-coding RNAs (lncRNA) and transcription factors GATA4 and FOG2. LncRNAs are RNA sequences of more than 200 nucleotides that do not code for any protein final products themselves but are involved in chromatin remodeling as well as transcriptional and post-transcriptional gene regulation. They are highly organ-selective, which makes them potential targets for drug development. Our group has previously found a selection of cardiomyocyte-selective lncRNAs, which share a similar expression pattern in neonatal mouse hearts. In this project, three of them were silenced in a primary cardiomyocyte culture while simultaneously hormonally inducing hypertrophy. The goal was to see whether these lncRNAs have an effect on the hypertrophic response and apoptosis in the cardiomyocytes. Transcription factors are proteins with partially similar activities to lncRNAs; they regulate, which genes are expressed under certain circumstances. GATA4 is an important transcription factor in the heart as it targets various developmental and functional genes in cardiomyocytes. FOG2 is a cofactor of GATA4; interaction between them regulates the activity of GATA4. Our group has recently developed a selection of compounds that affect protein-protein interaction between GATA4 and NKX2-5, another important transcription factor. The second part of the project was to set up and optimize a compound screening assay for GATA4-FOG2 interaction. The results showed no change in hypertrophic response when the lncRNAs were silenced. Other experimental designs could still reveal if they have effects that could not be seen with these protocols. The silencing had no effect on apoptosis. As for the GATA4-FOG2 interaction experiments, transfecting COS-1 with GATA4 and FOG2 plasmids in a ratio of 10:1 resulted in a signal suitable for compound screening. Initial compound screening results indicated the compounds may have an effect on GATA4-FOG2 interaction, but further studies are needed before drawing conclusions.
  • Nohynek, Risto (2023)
    Obesity has increased in our society for decades and is still increasing. It is a burden for individuals and societies. The healthcare costs, disability, illnesses, and deaths caused by it are unfortunately a big burden on global scale. Binge eating disorder is an eating disorder in which a person uncontrollably devours an excessive amount of food due to a lack of self-control. Binge eating disorder is strongly linked to obesity and it further increases the weight of both normal weight and obese people. Many mechanisms influence the regulation of eating. A long-term research subject and affecting the regulation of eating, serotonergic and serotonin receptors, affect the amount of food eaten and the reward system, and disturbances in serotonin signaling have been linked to obesity. Aim of this study was to exam binge-like eating modelled C57Bl/6J mice and their food consumption, while affecting serotonergic signaling. I studied psychoactive LSD and antipsychotic MDL 100907 effects on serotonergic signaling in a binge-like eating model, using drugs both separately and simultaneously. Mice were induced into a stress-free model of binge-like eating by providing high-energy food once a week for 24 hours. When the binge eating model was runnig, once a week the mice were dosed with a drug or substances and given energy-dense food to binge on. In the study, consumed food and water were measured. The mice were also subjected to locomotor tests to ensure that they were able to eat motorically. Induction of the binge-like eating model was successful and a reduction in binge eating was observed in mice under LSD alone at significant time points. MDL reduced binge-like eating at the first time point. No significant changes were observed in the water intake. The locomotor tests ensured a sufficient amount of movement to enable eating. Even though the drugs individually reduced binge-like eating, it should be noted that the properties of the drugs, and especially the trials of their combined use, which did not show significant results, do not promise significant discoveries in terms of similar research.
  • Qvist, Teo (2022)
    Ahmintahäiriö on epätyypillinen syömishäiriö, johon liittyy toistuvia ahmintakohtauksia, joiden aikana syödään hallitsemattomasti suuria määriä ruokaa, vaikka olo olisi jo epämiellyttävän täysi. Mantelitumake on ohimolohkon pohjukassa sijaitseva pelon ja tunteiden, mutta myös ruokahalun ei-homeostaattisen säätelyn, kannalta tärkeä rakenne. Mantelitumakkeen sentraalisen tumakkeen lateraalisen osan (CeL) solut säätelevät muun muassa ruoan palkitsevuutta ja tyydyttyneisyyden tunteen muodostumista ruokailun aikana. Suurin osa CeL:n kolinergisista hermoyhteyksistä on aivorungon pedunculopontisesta tegmentaalisesta tumakkeesta (PPT) projisoituvia hermoratoja. PPT:een kolinergisten hermopäätteiden in vivo optisen aktivaation on aikaisemmissa tutkimuksissa havaittu säätelevän opittua välttämiskäyttäytymistä, mutta optisen aktivaation vaikutusta ruoankulutukseen koe-eläimillä ei ole vielä tutkittu. Tämän pro gradu -tutkielman kokeellisen osan tavoite oli tutkia PPT:sta CeL:aan projisoituvan kolinergisen hermoradan in vivo optisen aktivaation vaikutusta ruoankulutukseen C57BL/6N -hiirillä. Optisen aktivaation vaikutusta tutkittiin sekä homeostaattiseen että ei-homeostaattiseen ruoankulutukseen. Ei-homeostaattisen ruoankulutuksen tutkimiseksi hiirille indusoitiin ahminnan kaltaista syömiskäyttäytymistä tarjoamalla hiirille rasvapitoista ruokaa viikoittain 24 tunnin jaksoissa. Tämän lisäksi tutkittiin ovatko optisen aktivaation vaikutukset kumottavissa asetyylikoliinireseptorien antagonistien mekamyyliamiinin tai skopolamiinin intraperitoneaalisella annostelulla. In vivo optisella aktivaatiolla ei havaittu olevan tilastollisesti merkitsevää vaikutusta ruoankulutukseen C57BL/6N -hiirillä. Tästä johtuen myöskään asetyylikoliinin antagonistien annostelun vaikutusta optisen aktivaation vaikutuksiin ei voitu arvioida. Tulokset viittaavat siihen, että PPT:een ja CeL:n välinen kolinerginen hermorata säätelee koe-eläimillä opittua välttämiskäyttäytymistä, mutta ei ruokahalua. Näin ollen on myös epätodennäköistä, että optisen aktivaation vaikutukset edellyttävät CeL:n proteiinikinaasi C deltaa ilmentäviä soluja. PPT:een kolinergisten hermosolujen vaikutus CeL:n toimintaa hermosolutasolla tulee kuitenkin varmistaa tarkemmissa jatkotutkimuksissa. Vaikka tutkimuksessa ei havaittu tilastollisesti merkitsevää vaikutusta, havaintoihin on kuitenkin suhtauduttava varauksella, sillä tutkimuksen toteuttamiseen liittyneet haasteet voivat rajoittaa havaittujen tuloksien luotettavuutta.
  • Mandelin, Ronja (2024)
    MDMA is an illegal stimulant known for its empathy-enhancing effects. Its positive effects are mainly based on increasing the concentrations of monoamines such as serotonin (5-HT), dopamine (DA) and norepinephrine (NE). In addition to its positive effects, MDMA can cause adverse effects such as hyperthermia and neurotoxicity. Especially with long-term use, MDMA can cause serotonergic and dopaminergic neurotoxicity. In addition, there are also indications of MDMA-induced neurotoxicity in systems where gamma-aminobutyric acid (GABA) functions as the main neurotransmitter. Glutamate decarboxylase (GAD) 67 is an enzyme that synthesizes GABA from glutamate and is a specific marker for GABAergic cells. The amygdala is a nucleus in the brain that regulates anxiety and fear response. In addition to GABAergic interneurons, there are also glutamatergic cells in the basolateral nucleus (BLA) of the amygdala, however in the central nucleus (CeA) there are only GABAergic cells. Disturbances in the GABAergic system can predispose to psychiatric diseases such as anxiety. The aim of thisstudy was to investigate the effects of MDMA (20 mg/kg) on the number of GAD67-positive cells in two nuclei of the mouse amygdala, BLA and CeA. In addition, this study aimed to examine the importance of the dose (4 or 16 injections) for neurotoxicity and the duration of the effects (2, 7 or 30 days). Adolescent wild type mice were divided into 12 groups according to the treatment (MDMA or saline), dose and timepoint. After euthanasia, the brain sections at the level of the amygdala were collected and stained with an immunohistochemical method and imaged using a confocal microscope. This study showed that MDMA reduced the number of GAD67-positive cells in the BLA when mice were given a total of 4 injections. This effect lasted up to 30 days. In contrast, MDMA did not reduce the number of GAD67-positive cells in the BLA in mice that were given 16 injections. Also, MDMA did not decrease the number of GAD67-positive cells in the CeA, regardless of dose. Statistical significance could have been improved, for example, by using more mice or analysing more sections from each individual animal. It is important to continue studying the effects of MDMA to better treat and prevent its adverse effects. In addition, increased understanding would urge users to exercise caution when using MDMA.
  • Mankila, Anja; Mankila, Anja (2022)
    Cardiovascular diseases are the most common causes of mortality worldwide. More adequate human-based models would be needed for the purposes of disease modeling and drug development. One of the most promising fields of in vitro modeling is the use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). A central problem of hPSC-CMs is their immature or fetal-like phenotype compared to adult human cardiomyocytes regarding many structural, functional, and metabolic properties. The development of metabolic properties is considered to be a central driver of cardiomyocyte maturation. One practicable way to promote the metabolic maturation of hPSC-CMs in vitro is the use of various biochemical cues in the cell culturing media. The topic of this study was the metabolic maturation of hPSC-CMs. The research questions were: What biochemical cues have been suggested to be involved in the hPSC-CM maturation in vitro? What signaling pathways connected to the biochemical cues have been explored in the context of the maturation of hPSC-CM? What experimental results have been achieved on the effects of the biochemical cues and the involvement of the signaling pathways? The study was conducted as a systematic review with the database Scopus (Elsevier). The final set of materials consisted of 46 original research articles published in peer-reviewed journals in English in the years 2013–2022. Out of the materials, 11 articles (24%) were characteristically longitudinal studies. They indicated that the pathways leading to metabolic changes such as PPARs (peroxisome proliferator-activated receptors) and PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) are activated already in early stages. In 12 articles (26%), pharmacological agents were used to target the metabolic pathways, and in 8 articles (17%) techniques affecting the gene expression were utilized. The most recent studies involved ever more frequently combinations of different techniques. Considering the use of biochemical cues, the trend has been to favor fatty acids, thyroid hormone and dexamethasone over glucose, insulin and insulin-like growth factor. Some cues such as retinoic acid and neuregulin 1 have been tested only in single experiments. In addition to the nuclear receptor mediated pathways, the energy sensors AMPK (AMP-activated protein kinase) and mTOR (mechanistic target of rapamycin), the oxygen sensor HIF-1α (hypoxia-inducible factor 1α), and the microRNAs turned out to be central.
  • 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.
  • 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.
  • Urpelainen, Katja (2021)
    Cardiomyocyte oxygen deprivation followed by apoptosis and cardiomyocytes being replaced with fibrotic tissue can lead to heart failure. Cardiovascular diseases are the most common cause of death world-wide, contributing to 17.8 million deaths in 2017. Treatments currently available aim to maintain cardiac function but are unable to repair the damage, resulting in a poor prognosis for heart failure. Cardiomyocytes are able to proliferate but the endogenous mechanisms of cardiac repair are insufficient to replace the damaged cardiomyocytes, as only an estimated 0.3-1 % of adult cardiomyocytes are regenerated annually. It is known that before birth and up to seven days after birth mice can maintain ability to regenerate cardiomyocytes even after large damage, but this capability is lost within seven days following birth. After this, cardiomyocytes exit cell cycle and will not re-enter it sufficiently to enable cardiac repair. In adults the growth of heart muscle results mainly from hypertrophic growth meaning that the cells grow in width and length. Cardiomyocyte regeneration is an important therapeutic target to which there are no effective pharmacological therapies available yet. The aim of this study was to investigate the effect of 14 novel compounds on cardiomyocyte viability, phenotype and cell cycle activation. Novel compounds were synthesized at the Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, University of Helsinki in Finland. Initial toxicity and cell viability screening was conducted with lactate dehydrogenase assay (LDH assay) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) using COS-1 cells. Based on these assays tolerable concentrations of compounds were determined. Activity analysis was conducted using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and immunocytochemistry staining in conjunction with high-content analysis (HCA). Stress response was measured by imaging and analyzing expression of pro-B-type natriuretic peptide (proBNP) and cell cycle activation was imaged and analyzed by using 5- bromo-2’-deoxyuridine (BrdU) as a marker of active cell cycle. In addition, the DNA content of the cardiomyocytes was measured using 4’,6-diamidino-2-phenylindole (DAPI) staining as well as cardiomyocyte morphology investigated with cardiac troponin T (cTnT) staining. One of the compounds, K6, decreased proBNP expression, which can be considered as a sign of decreased stress response. However, K6 also decreased the number of BrdU positive cardiomyocytes that can be considered as a sign of decreased cell cycle activity. Together these markers indicate that the decreased activity may not be due to a stress response caused by the compound. Another compound, K12, increased proBNP expression in all tested concentrations and it also decreased the number of BrdU positive cardiomyocytes. Together these could be considered as an indication of cardiotoxicity. The rest of the compounds did not exhibit remarkable biological activity or there was too great variance between the results of the independent experiments (n=3) to draw definite conclusions. Compounds for this study were chosen for the sole reason of not been tested for biological effects before. Using a defined compound library or screening a larger number of compounds could deliver more predictable results. Early toxicity and viability screenings were a good approach allowing to define toxic compounds and concentrations before continuing with further studies. Pharmacological therapies to induce cardiac regeneration will continue to be an important area of interest in cardiovascular drug research. Phenotypic screening in conjunction with high-content analysis offers variable and statistically significant data on cardiomyocyte proliferation and stress response. The results of the screening could be improved with careful selections of test molecules based on their structure and biological activity. Early toxicity and viability screening further improve the predictability of results. As a result of this study a compound that would induce cardiomyocyte proliferation was not found, however, one compound that seemed to decrease cardiomyocyte stress response was detected and this compound could be of interest for further studies.
  • Iljin, Arto (2023)
    Changes in environmental and psychological factors have greatly influenced human eating behavior, leading to increasing rates of overeating. In the long-term continuous overeating causes the body to accumulate an excess amount of body fat tissue, which is a leading factor in the development of obesity and obesity related diseases that reduce the quality of life. Despite the available treatment options, the prevalence of obesity has been increasing worldwide. In avoiding obesity and obesity-related diseases, it is crucial to understand the outside factors which lead to obesity and the mechanisms which regulate body energy balance. The brain's serotonin system seems to play a significant role in regulating both energy balance and the tendency that promotes an overeating type of eating behavior. The properties of psychedelics to affect human's emotional state and thus modulate behavioral patterns through the brain's serotonin system has aroused interest in psychedelics for their possibilities in treating eating behaviors that promote overeating which results in obesity. This study aimed to examine the dose-dependent effect of three different psychedelics on eating behavior in mice and evaluate their potential drug therapy properties in overeating indulged obesity. In total, 16 female mice were trained to perform in an operant setting used in behavioral experiments measuring appetite and motivation for food reinforcers. The psychedelic-derived changes were observed in mice's eating behaviors by reward deliveries received during the trials. The examined psychedelics (lysergic acid diethylamide (LSD), psilocybin (PSI), and 25CN-NBOH) showed no statistically significant changes in the mice's eating behavior in terms of appetite and motivation. However, while statistically non-significant, some changes in hunger and motivation were observed in the mice, for example, in days followed by the dosage of the psychedelics. These results indicate no effect on appetite and food motivation by the studied psychedelics.
  • Kulmala, Veera (2022)
    Parkinson’s disease (PD) is a progressive neurodegenerative disorder with the neuropathological hallmark of intraneuronal inclusions called Lewy bodies (LB). Accumulation of α-synuclein (α-syn) and cellular components into LBs coincides with degeneration of dopaminergic neurons in the midbrain, substantia nigra. Degeneration of dopaminergic neurons eventually leads to motor dysfunctions. Currently, the treatments for PD are symptomatic. For this reason, new disease-modifying treatments are needed to slow down or prevent the disease progression. Neurotrophic factors (NTFs) have been an interest of research for a couple of decades because of their neuroprotective properties. The main aim of this study was to investigate if brain-derived neurotrophic factor (BDNF) reduces pre-formed fibril (PFF) induced aggregation of α-syn in dopaminergic neurons. PFF-model was used to mimic the accumulation of LBs in neurons, as PFFs induce aggregation of endogenous α-syn in neurons. Additionally, the dose dependence of BDNF was tested. The secondary objective was to investigate the interaction of tropomyosin receptor kinase B (TrkB) signaling pathway and α-syn aggregation using TrkB agonists and antagonists. The cultured dopaminergic neurons isolated from the midbrain of mouse embryos were treated with PFFs on the day in vitro (DIV) 8. BDNF or control treatments were added either 1 hour after the PFF-treatment or on DIV 12. Neurons were fixed on DIV 15 and fluorescent immunohistochemistry was performed. After the detection of fluorescence with automated, high-content imaging, image analysis was done for quantifying dopaminergic neurons, and dopaminergic neurons positive for LB-like aggregates by using unbiased image analysis CellProfilerTM software. Both BDNF and positive control glial cell line-derived neurotrophic factor (GDNF) significantly reduced LB-like aggregates in dopaminergic neurons at both timepoints. GDNF was more effective at both timepoints than BDNF. Both tested doses of BDNF lowered the number of LB-like aggregates, but a more robust effect was seen with the higher dose. The highest tested dose for the TrkB agonists was toxic to the cultured dopaminergic neurons, whereas the lower doses did not affect either the survival or the number of LB-like aggregates. BDNF promoted the survival of the dopaminergic neurons despite the survival-reducing adverse effect of TrkB antagonist K252a. This study provided new information on the effects of exogenously added BDNF on PFF-model with primary neuronal culture. Research on the underlying mechanisms of α-syn aggregation and the protective effects of NTFs can forward the development of new therapies against PD.
  • Ignatius, Adele (2021)
    Misfolding and aggregation of alpha-synuclein (α-syn) protein, leading to dysfunctional proteins and toxic protein aggregates, are seen as major factors in the pathogenesis of Parkinson’s disease (PD). Direct protein-protein interactions (PPI) between α-syn and a serine endopeptidase, prolyl oligopeptidase (PREP), have been shown to increase α-syn aggregation. Small molecular PREP inhibitors, in turn, have been shown to reduce the ɑ-syn aggregation process both in vitro and in vivo. Inhibition of PREP has been shown to have dual effects on ɑ-syn aggregation: first of all, blocking PREP mediated seeding and secondly, inducing the clearance of ɑ-syn aggregates via increased autophagy. Thus, PREP inhibitors should be further studied as a potential treatment for PD and other synucleinopathies. In this study, we evaluated the effect of two different PREP inhibitors, 4-phenylbutanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047) and HUP-115 in a virus vector-based unilateral A53T-ɑ-syn overexpression mouse model. AAV-A53T-ɑ-syn injections used in this study caused a significant increase in oligomer-specific alpha-synuclein (ɑ-synO5) immunoreactivity and a mild dopaminergic neuron loss, together with mild motor deficits. Neither 2-week PREP inhibition with KYP-2047 or 4-week PREP inhibition with HUP-115 reduced ɑ-synO5 immunoreactivity or protected dopaminergic neurons in the substantia nigra (SN). Concordant to this, the treatments did not restore the slight behavioral deficit AAV-A53T-ɑ-syn injections caused in the cylinder test. In previous studies, PREP inhibition with KYP-2047 decreased ɑ-synO5 immunoreactivity, attenuated dopaminergic neuron loss and restored behavioral deficits in other α-syn overexpression mouse models. It is suggested that PREP inhibitors mainly have an effect on soluble ɑ-syn oligomers, rather than insoluble fibrils. In case A53T-ɑ-syn forms insoluble fibrils too rapidly in mice, overexpression of A53T-ɑ-syn might not be a suitable option when studying the effects of PREP inhibitors. Our results suggest that further characterization of this model in mice is much needed before drawing any conclusions about the effect of these PREP inhibitors.
  • Jaskari, Iida (2022)
    Multiple sclerosis is a progressive inflammatory disease of the central nervous system that affects young adults. The pathological hallmark of MS is the degradation and loss of oligodendrocytes resulting in demyelination. Damage to axons caused by demyelination severely impairs physical function. Currently there is no cure for MS, but current drugs aim to modify the course of the disease and relieve symptoms. However, they are unable to promote the repair of damaged myelin sheaths, and thus new therapies are needed. In this study, the effect of V-MANF on remyelination was investigated in two commonly used experimental toxin models. V-MANF is a modification of the endoplasmic reticulum located protein MANF, which has been found to have neuroprotective and regenerative properties. Additionally, MANF can regulate ER stress, which contributes to demyelination in MS. The effect of V-MANF on lysolecithin-induced demyelination was examined in organotypic cerebellar brain sections from C57B/6 mice. The study was conducted exceptionally using the brains of adult mice because they are a better model for neurodegenerative diseases. However, when analyzing the results, it was found that there was no demyelination in the tissue cultures, so the effect of V-MANF could not be analyzed. In the other study, C57B/6 mice were given dietary cuprizone for six weeks, followed by daily intranasal administration of either V-MANF or vehicle for seven days. Mice were subjected to behavioral experiments, in which a light/dark box test showed that V-MANFs had a potential anxiolytic effect in mice receiving cuprizone. No significant demyelination was observed by immunohistochemical analysis and therefore the effect of V-MANF on remyelination could not be assessed. However, the results of the study can be utilized in the design of further studies.
  • Turunen, Iida (2023)
    Metastatic prostate cancer is often fatal disease stage. Mechanism causing prostate cancer remains unknown, but possible mechanism relies on hormones. Testosterone may activate spontaneous cell division of oncogenes. Prostate cancer cells require androgen cell stimulation of AR to grow in early stages of prostate cancer, approximately 80-90% of prostate cancer cases are androgen dependent. 3bHSD1, encoded by HSD3B1, catalyzes the conversion of dehydroepiandrosterone to androstenedione and further to T and DHT. SNP (1245A to C) in HSD3B1 changes asparagine to threonine in position 367 resulting the enzyme accumulation and increased function. With androgen deprivation therapy castrate levels of testosterone are often achieved and it induces positive response in most PCa patients, but the polymorphism of 1245C is related with lower survival rate and higher probability for PCa to develop into CRPC. The aim of this study was to find out the effect of SNP in 3bHSD1 to androgen levels in patients treated with ADT. 32 patients were first genotyped based on SNP in the HSD3B1 gene (rs1047303) with 96.9 % success rate. 21 patients represented genotype AA, 9 AC and 1 patient CC. Other mutation in rs6203 was also detected. Genotyping was done by isolating DNA from blood samples and preparing it further for Sanger sequencing. Steroid analysis was performed by using LC/MS, using liquid-liquid extraction as sample preparation method. Altogether 21 steroids were analyzed from serum samples. Samples were taken every 3 months, during 33 months period for longest. The concentrations of T and DHT were reduced in AA genotype group after ADT as was expected to happen in all of the groups. In fact, the only significant changes were seen in AA genotype with for example the concentrations of previously mentioned T, DHEA and also A4. The changes in measured androgen levels cannot be generalized to concern especially the CC genotype, as there was only one patient homozygote with the mutation. Even though these results gave promising data of possible androgen synthesis pathways, a similar study must be rerun with larger patient data to be sure of the characteristics of different genotypes. Also, the effect of SNP in rs6203 remains still unknown.
  • Niittymäki, Erika (2021)
    Since the discovery of ketamine’s antidepressant response, numerous of studies have been observed it to alleviate depressive symptoms rapidly and effectively within hours. This is a significant advantage compared to traditional antidepressants, which take weeks to show treatment efficacy. Ketamine is a N- methyl-D-aspartate receptor (NMDA) antagonist and its underlying mechanism of is proposed to be in its ability to increase synaptic plasticity and this is ultimately believed to improve mood. On a molecular level, the antidepressant effects have been observed to be dependent on the activation of tropomyosin receptor kinase B (TrkB) signalling pathway. However, the antidepressant mechanism of ketamine remains still poorly understood as no new NMDA-antagonist or other rapid-acting antidepressants have been successfully developed for clinical use despite many years of effort. Therefore, some have proposed that the missing pieces of understanding its antidepressant effects might be linked to ketamine’s ability to modify sleep patterns and circadian-related molecules. Ketamine has especially been demonstrated to increase slow-wave activity during the following night of treatment and these changes have been shown to predict the clinical outcome in patients with major depressive disorder (MDD). Slow-wave activity is a low-frequency and high-amplitude wave seen in electroencephalography, which is highly expressed during the deepest stage of sleep, and this has been prominently found to be reduced in MDD patients. Even more intriguing, there are indications that ketamine might increase slow-wave activity also immediately after its administration. During this time, TrkB signalling is observed to became active. Following these molecular findings, we sought to investigate the link between the TrkB signalling pathway and two prominent processes occurring during slow-wave sleep. During slow-wave sleep processes such as (1) reduction of brain’s energy expenditure and (2) the activation of glymphatic system is known to occur. The glymphatic system is as lymphatic-perivascular network, which is responsible for clearing the brain from the metabolic waste. Thus, in this study, our objective was to investigate whether by causing an acute decline in adenosine-triphosphate (ATP) production or by stimulating the glymphatic network, we could activate the same plasticity-related pathways as ketamine is capable of activating in mice prefrontal cortex. The results of this study suggest that acute metabolic reduction can trigger pathways regarding synaptic plasticity. The metabolic inhibitor, 2-deoxy-D-glucose and mercaptoacetate (2DG+MA), was found to phosphorylate the TrkB receptor and its downstream signalling molecules GSK3β and p70S6K, while MAPK was dephosphorylated. These results correlate with the previous findings of ketamine’s effect after its administration. We also found a plasticity-related marker, MAP2, to be heavily phosphorylated by 2DG+MA, indicating 2DG+MA having a surprising role on neuroplasticity. These results are promising indication of understanding the rapid effects of ketamine and might even give important insight to developing novel antidepressants. However, these findings are only preliminary, and more research is needed to directly link antidepressant effects and energy metabolic inhibition together, as our study did not directly investigate antidepressants and depression-like behaviour in mice.
  • Annala, Iina (2021)
    Subanesthetic-dose ketamine, an N-methyl-D-aspartate receptor (NMDAR) blocker, exerts rapid antidepressant effects that sustain long after its elimination from the body. The precise mechanism remains unknown, but regulation of TrkB (tropomyosin receptor kinase B), ERK (extracellular-regulated kinase 1 and 2), GSK3β (glycogen synthase kinase 3β) and mTOR (mammalian target of rapamycin) signaling within the prefrontal cortex (PFC) have been deemed important for its antidepressant-like effects in rodents. In addition, activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is thought to be an important step in its mechanism. Nitrous oxide (N2O), another NMDAR antagonist and a putative rapid-acting antidepressant, regulates the same molecular pathways as ketamine in the rodent PFC. The fast pharmacokinetics of N2O have been exploited to show that markers of neuronal excitation, including phosphorylation of ERK, are upregulated in the PFC during its acute pharmacological effects (NMDAR blockade), while regulation of TrkB, GSK3β and P70S6K emerges only upon N2O withdrawal. In the first part of this study, we investigated the N2O-induced biochemical changes associated with neuronal excitation and BDNF-TrkB signaling in the PFC and further, the requirement for AMPAR activation in inducing them. We focused on the effects seen after the acute pharmacological effects of N2O. N2O (65% for 20 min) was administered to adult male C57BL/6 mice with or without pretreatment with AMPAR antagonist (NBQX, 10 mg/kg) and PFC samples were collected 15 minutes after stopping N2O delivery. Within this time N2O is expected to be completely eliminated. The brain samples were analyzed using western blot, enzyme-linked immunosorbent assay and quantitative reverse transcription PCR. We observed that N2O increased levels of phosphorylated TrkB, GSK3β and P70S6K, and these effects were not attenuated by NBQX pretreatment. At the same time, we observed a decrease in the levels of phosphorylated ERK, which was attenuated in mice that received NBQX prior to N2O. Tissue levels of BDNF protein or messenger RNA (exon IV) were not different between control and experimental groups. These results indicate that the mechanism of N2O is associated with TrkB and ERK signaling that are regulated independently of each other. It appears that AMPAR activation is not required for TrkB signaling, although it might play a role in ERK signaling. Further, N2O-induced TrkB phosphorylation in the PFC is not associated with changes in total levels of BDNF. In the second part of the study, we aimed to search for new ketamine-like NMDAR blockers with antidepressant potential. Ketamine was used as a query compound for in silico substructure search to find commercial ketamine analogs. The retrieved ketamine analogs were filtered by their computed ADMET properties and then further screened virtually by docking them to the pore region of NMDAR complex (protein data bank code: 4TLM), around the predicted binding site of ketamine. Finally, we sought to study if selected ketamine analogs could elicit ketamine-like effects on TrkB and ERK signaling in mouse primary cortical neurons. However, we did not proceed to test the analogs since ketamine (positive control) did not show any effects on TrkB or ERK phosphorylation in our culture. Overall, this study advances the understanding of the mechanism of N2O, possibly giving new insight of the antidepressant mechanisms of NMDAR-blocking agents more generally. Additionally, we found promising ketamine analogs that await experimental testing.
  • Halinen, Sara (2023)
    Current pharmacological treatments for major depressive disorder leave many patients unresponsive to treatment or treatment response is delayed by weeks. More effective treatments with quicker effect onset are therefore needed. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonists has demonstrated sustained rapid antidepressant activity after single dose. Precise mechanisms behind this effect are unknown, however some crucial contributors to ketamine-induced behavioural effects in rodents include phosphorylation of Tropomyosin receptor kinase B (TrkB), ribosomal protein s6 kinase (p70s6k), glycogen synthase kinase 3 (GSK3), mitogen activated protein kinases (MAPKs), and activation of α-amino-3-hydroxy-5- methyl-4- isoxazolepropionic acid receptors (AMPAR). Similar TrkB related signaling cascades are also activated with another NMDA receptor antagonist and a putative rapid-acting antidepressant, nitrous oxide (N2O). During acute effects of N2O, cortical excitation increases MAPK phosphorylation and upregulates expression of activity dependent immediate early genes (IEG; c-Fos and Bdnf IV). Phosphorylation of TrkB, GSK3 and p70s6k appearing only after N2O has been eliminated suggest that TrkB signaling is induced as an adaptive response to treatment. The first objective of this study was to corroborate previous results from our group to validate our gas administration set up and protein analysis protocol. To analyze N2O-induced phosphorylation of proteins implicated in ketamine’s behavioral effects in mice, we treated C57BL/6J male mice with either room air (control) or 65% nitrous oxide for 20 minutes. After gas exposure and 15-minute washout period, medial prefrontal cortex samples were dissected to be analyzed with western blotting. In this study nitrous oxide exposure did not induce increased TrkB signaling in nitrous oxide withdrawal. Another aim of this study was to investigate the involvement of AMPARs in inducing cortical excitation with N2O. Pretreatment of AMPAR antagonist (10 mg/kg, NBQX) or saline was given to C57BL/6J male mice 10 minutes prior to 1 hour exposure to 50 % O2 or 50 % N2O, a N2O dose previously shown to induce IEG expression. One hour after gas exposure mice were euthanized and mPFCs were dissected and analyzed with reverse transcriptase quantitative PCR (RT-qPCR). No regulation in IEG expression was induced with nitrous oxide, NBQX pretreatment or combination compared to control. Additional studies factoring in limitations of this study are needed to uncover the involvement of AMPAR in inducing cortical excitation and antidepressant-like behavioral effects of N2O in preclinical models of depression.