Browsing by study line "Farmakologi"
Now showing items 21-40 of 51
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(2021)Dilated cardiomyopathy is a non-ischemic cardiac disorder predisposing to heart failure, and the characteristics of dilated cardiomyopathy emerge under normal loading conditions. Dilated cardiomyopathy can be consequence of various conditions e.g. genetic mutations, virus infection or toxin exposures. One of the significant causes of familial dilated cardiomyopathy in Finland is mutation S143P in LMNA-gene, coding for A type lamins. Current drug therapy for dilated cardiomyopathy aims to alleviation of symptoms, prevention of complications and progression of the disease, however, efficacy of current therapy is insufficient, and novel therapy strategies are urgently required. Transcription factors are fundamental regulators of gene expression, and GATA4 is a crucial transcription factor both in embryonic and in adult heart and thus an intriguing target for therapeutic manipulation. Compounds targeting GATA4 have shown anti-hypertrophic and cardioprotective effects. Here, effects of two different hypertrophic stimuli, endothelin-1 and mechanical stretch, on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were examined with high-content analysis and quantitative reverse transcription PCR (qRT-PCR), respectively. One hiPSC-CM line was used as a healthy control, whereas the other carried the S143P mutation in LMNA-gene (DCM-CMs). Additionally, effects of GATA4-targeting compound C-2021 on cardiomyocytes were investigated. In summary, according to proBNP staining, DCM-CMs are more hypertrophied at baseline. DCM-CMs seemed to be less susceptible to mechanical stretch-induced enhancement in BNP gene expression. In addition, compound C 2021 may have anti-hypertrophic properties suggesting it to be a potential drug candidate in cardiac diseases. Finally, lamin A seemed to mislocalize to nucleoplasm instead of nuclear lamina in DCM-CMs.
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(2022)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.
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(2023)Alcohol use disorder (AUD) is a chronic relapsing brain disorder causing a high burden of disease and significant social and economic consequences to both individuals and society. Alcohol addiction, the most severe form of AUD, is characterized by compulsive seeking and use of alcohol, loss of control over limiting alcohol consumption despite negative consequences, emergence of negative emotional states, and long-lasting vulnerability to relapse related to alcohol abstinence. Powerful craving for alcohol and the chronic, relapsing nature of the disease are major problems complicating recovery from alcohol addiction and predicting poor clinical outcome. Relapse to alcohol intake can occur even after an extended period of abstinence in humans, relapse rates being highest during the first three months of alcohol withdrawal. Associative learning is a critical factor in alcohol craving when alcohol consumption is accompanied by conditioned stimulus. Cues associated with alcohol are known to induce craving and alcohol-seeking behavior increasing the risk of relapse, and this craving can be triggered by alcohol itself, alcohol-associated stimulus, or stress. Chronic alcohol exposure has been linked to changes in synaptic plasticity, neurogenesis and cell-signaling. Thus, elucidating the neural mechanisms that underlie alcohol craving and relapse would help to understand the pathology of alcohol addiction and facilitate the development of efficient treatments. In this experiment, the effects of subanesthetic-dose 10 mg/kg ketamine, an NMDAR antagonist and a major inducer of synaptic plasticity, on cue-induced alcohol-seeking behavior after withdrawal were investigated in social context in female mice. Mice were trained to voluntarily drink alcohol, and a novel methodology to study alcohol-seeking behavior after withdrawal allowed to perform the experiment with a minimum of human interference in totally automated social home cage environment. The analyses of behavioral data showed that pairing sweetened alcohol with conditioned stimulus resulted in cue-induced alcohol-seeking behavior, and no differences in alcohol conditioning were observed between treatment groups. However, the behavioral activity in extinction tests after withdrawal showed that alcohol-seeking behavior was not altered by ketamine treatments. In biochemical analyses, the effects of subanesthetic-dose ketamine on ΔFosB and BDNF protein levels in the brain areas important for alcohol addiction were studied. ΔFosB expression levels in the mouse nucleus accumbens were analyzed with western blot and BDNF protein levels in the mouse prefrontal cortex were determined using enzyme-linked immunosorbent assay (ELISA). The results from biochemical analyses showed that levels of ΔFosB and BDNF were unaltered by ketamine treatments. Anyhow, the experiment provided important insights into the interactions of ketamine and alcohol craving and relapse, a topic that has been insufficiently studied in novel preclinical models.
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(2024)Parkinson’s disease (PD) is a progressive neurodegenerative disorder in which dopaminergic neurons in the substantia nigra (SN) degenerate and die. This causes multiple motor symptoms such as rigidity, bradykinesia and tremor and non-motor symptoms such as depression, hallucinations, and cognitive impairment. At the time of the diagnosis, approximately 60% of the dopaminergic cells can already be lost, which underlines the importance of neurorestorative treatments for PD. Currently used treatments are only symptomatic and mostly based on levodopa, which can lose its effectiveness as the disease progresses and additionally cause significant side effects such as dyskinesia. Neurotrophic factors (NTF) such as glial cell-line derived neurotrophic factor (GDNF) and neurturin (NRTN) have been studied in clinical trials with PD patients but have shown only modest effects on motor function. Additionally, they have been administered with invasive techniques such as intraputamenal or intracerebroventricular injections which includes many risks. Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to unconventional NTF’s with unique molecular structure and mode of action. MANF has shown both neuroprotective and neurorestorative properties for nigrostriatal dopamine system in in vivo study in rat model of PD. To enable systemic administration of MANF, the molecule has been modified by retaining only the C-terminal domain to form C-terminal MANF fragment (C-MANF). C-MANF has shown neurorestorative effects when administered intrastriatally in 6-OHDA lesioned rats and when injected subcutaneously (s.c.) in an ALS animal model. The aim of this study was to assess the effect of daily and weekly s.c administered C-MANF in 6-OHDA mouse model of PD. Cylinder and amphetamine-induced rotation tests were performed as behavioural tests and thereafter morphological studies were done by analyzing tyrosine hydroxylase (TH)+ cells in the substantia nigra pars compacta (SNpc) and optical density from TH+ axons in the striatum. Interestingly, weekly C-MANF treatment decreased the number of TH+ cells in SNpc and the density of TH+ fibers in the striatum compared to PBS. However, it decreased ipsilateral rotations and showed some positive effects in the cylinder test. On the other hand, daily C-MANF treatment increased the number of TH+ cells in SNpc and the density of TH+ fibers in the striatum but had a modest effect on ipsilateral rotations and the cylinder test compared to PBS. Although no statistically significant effects were observed in behavioral and morphological studies, s.c administered C-MANF presents a promising treatment option for PD. Particularly, daily administration of C-MANF showed neurorestorative effects in morphological studies; however, further research is required for validation. Additionally, the investigation of higher doses of C-MANF should be considered.
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(2023)Parkinson’s disease is a progressive neurodegenerative disorder which is commonly treated with Levodopa (L-dopa) and Dopa Decarboxylase (DDC)/ Catechol-O-methyltransferase (COMT) inhibitors. The main problem with this treatment is the intestinal conversion of L-dopa to dopamine despite DDC and COMT inhibition which probably occurs by the Tyrosine Decarboxylase (TyrDC) of intestinal bacteria. This study aims to find new inhibitor molecules that would have dual inhibitory effects towards both DDC and TyrDC enzymes. Currently, available DDC inhibitors cannot inhibit the bacterial TyrDC enzyme. A recently found TyrDC inhibitor (S)-α-Fluoromethyltyrosine (AFMT) is not able to inhibit the human DDC enzyme, respectively. The dual inhibition of both decarboxylases could reduce the dosing frequency and side effects related to L-dopa. In addition, the object of this study is to produce the human DDC enzyme by DNA recombinant technique as well as develop and optimize a biochemical DDC inhibition assay to study the effect of selected small molecule compounds towards inhibition of DDC and L-dopa conversion in E. faecalis model by previously developed cell-based assay. The human DDC was successfully produced in a TB medium with a yield of 1.8 mg/mL. The Km value of DDC for L-dopa was found to be 34 μM which indicates a high affinity for L-dopa. In the optimization of the DDC inhibition assay, the sample volume of 80 μL and incubation time of 3 h with detection reagent was found to give the highest fluorometric signal with sufficient robustness. In the initial screening of test compounds, 14 % of the compounds (n=59) were classified as active towards human DDC, while 31 % of the compounds were active towards L-dopa conversion in the E. faecalis model. Of those compounds, five were having dose-dependent dual inhibitory effects, but the IC50 values of them were higher compared to either carbidopa or AFMT. The most effective compounds were 8009-2501 (IC50 37 μM in E. faecalis model and 19 % inhibition at 1000 μM towards DDC enzyme) and 8012-3386 (IC50 248 μM in E. faecalis model and 37 % inhibition at 1000 μM towards DDC enzyme). However, this study confirms the possibility to find dual decarboxylase inhibitors. By optimizing the structures as well as investigating the mechanism of action, selectivity, and structure-activity relationships of the most active compounds, it is possible to find more effective dual inhibitors in the future.
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(2021)Parkinson’s disease (PD) is a progressive chronic neurodegenerative disorder, which results in the selective loss of dopaminergic neurons in the substantia nigra (SN). The loss of these neurons results in the dysfunction of the nigrostriatal pathway bringing forth the characteristic motor symptoms seen in PD: postural instability, rigidity, slowness of movement and resting tremors. Non-motor symptoms, such as cognitive deficits, depression and impaired olfaction, typically emerge before motor symptoms. Currently available treatments only provide symptomatic relief with diminishing returns over time and no improvements on the overall outcome of the disease. Neurotrophic factors (NTF) have been of particular interest as a possible curative treatment for PD due to their potential for neuroprotection and neurorestoration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an NTF that has shown promising results in numerous in vitro and in vivo studies of PD. However, therapy with MANF and other NTFs involves surgical intervention for local administration, as NTFs cannot cross the blood-brain barrier (BBB). Therefore, the therapeutic potential of a systemically administered NTF would be tremendous, as it would lead to a significantly more favorable risk-benefit ratio for the patient. The aim of the current investigation is to evaluate the efficacy of a next generation variant of MANF in the 6-hydroxydopamine toxin-induced unilateral lesion rat model of PD. Prior in vivo results suggested that subcutaneously injected MANF variant is able to penetrate the BBB. Amphetamine-induced rotational behavior (AMPH-ROTO) was used to evaluate the severity of the unilateral lesions during the experiment every other week until the end of the experiment at week eight. Animals were divided into treatment groups during week two based on their AMPH-ROTO results. Animals received MANF variant either subcutaneously through an implanted osmotic minipump at two different dosages or as a single dose divided into three separate intrastriatal injections. Tyrosine hydroxylase (TH) immunohistochemical staining was performed on brain sections collected from the striatum and SN for data analysis. In addition to AMPH-ROTO results, the efficacy of treatment was determined via the optical density of TH-positive striatal fibers and the number of TH-positive cells in the SN. Statistically significant differences (defined by p < 0.05 and a non-zero mean difference at a 95 % confidence interval) were observed only in the number of TH-positive cells in the SN favoring intrastriatal MANF variant treatment over both intrastriatal MANF and the vehicle treatment. The main concern regarding the validity of the results was related to the heterogeneous lesion sizes in different treatment groups possibly resulting in unsuccessful randomization due to excessive baseline differences. The inadvertent negative effects of this was further exacerbated by low a priori statistical power, which in the end had likely caused inflated effect sizes. Thus, assessment of the definitions of the used statistical parameters and the limitations of the experimental design suggest that presently, the efficacy of the MANF variant could not be evaluated reliably, in spite of the statistically significant result.
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(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.
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(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.
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(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.
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(2024)Current therapies for depression have limitations in efficacy and delayed onset of action. Rapid-acting antidepressants like ketamine, an N-methyl-D-aspartate receptor (NMDA-R) antagonist, have gathered attention as an improved treatment option. However, the neurobiological mechanism underlying their antidepressant effect remains uncertain. Integral mechanisms of action seem to be alterations in synaptic plasticity, global cortical excitation, and repair of neuronal dysfunctions prevalent in the pathophysiology of depression. Emerging evidence does suggest that antidepressant drugs act by facilitating brain derived neurotrophic factor (BDNF) mediated tropomyosin receptor kinase B (TrkB) signaling. Interestingly, rapid-acting antidepressants seem to increase TrkB-associated signaling after their acute pharmacological effect has dissipated, and when animals become sedated and show various physiological changes associated with deep sleep (e.g., slow wave EEG activity, SWA). Indeed, recently a close relationship between sedation and molecular signaling implicated in antidepressant effects has been discovered. The aim of this study was to explore the relationship between sedation and molecular signaling associated with antidepressant effect. This was carried out by assessing the localization of TrkB-associated phosphorylation signaling in the adult male mice medial prefrontal cortex (mPFC) using dexmedetomidine, a sedative. Key signaling molecules such as ribosomal protein S6 kinase (p70S6K), ribosomal protein S6 (rpS6), glycogen synthase kinase 3 (GSK3), mitogen activated protein kinases (MAPKs) and immediate early gene c-Fos, were examined through immunohistochemical (IHC) analysis. Two separate experiments were conducted using naïve adult 8-13-week-old (n=8 and n=10) male C57BL/6JRccHs mice. In the experiments mice were injected intraperitoneally with either dexmedetomidine (0,05 mg/kg, Dexdomitor®), or saline followed by a 30-minute recovery period whereafter mice were euthanized. In the first experiment, medial prefrontal cortex samples were collected immediately post decapitation for western blot (WB) analysis. The results showed that dexmedetomidine significantly activated TrkB-associated signaling in brain homogenates, consistent with expectations. In the second experiment, mice were perfused with 4% paraformaldehyde (PFA) before brain collection for IHC analysis. However in this experimental setting, no significant difference in the localization of TrkB-associated signaling induced by dexmedetomidine was observed compared to saline. Although, no significant results for signal localization were observed, the results provide insights into the neurobiological effect of sedation induced TrkB-signaling. Further research factoring in limitations is needed to uncover the involvement of physiological states in antidepressant mechanisms.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(2024)Ischemic heart disease, which often progresses to heart failure, is one of the leading causes of death worldwide. Ischemic conditions result in the death of heart muscle cells i.e. cardiomyocytes. Due to their poor regenerative ability, lost cardiomyocytes are replaced with a fibrotic scar. The loss of cardiomyocytes further leads to compensatory mechanisms, including cardiomyocyte hypertrophy and fibrosis. When prolonged, these responses turn maladaptive leading to pathological cardiac remodeling and alterations in cardiac function. In order to achieve better clinical results, discovery of new drug treatments that promote cardiomyocyte regeneration and decrease pathological cardiac remodeling would be invaluable. One potential target is serine/threonine protein kinase AKT (also known as protein kinase B), a key component of the PI3K/AKT signal pathway, which has been shown to be one of the mechanisms regulating heart regeneration and remodeling post-ischemia through its several downstream targets. The aim of this study was to investigate the effects of AKT-targeted compounds with and without endothelin-1-induced hypertrophy on the phenotype of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The compounds were either commercially available substances linked to AKT regulation, or new experimental compounds synthesized at the Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki. Prior to the hiPSC-CM phenotypic studies, the toxicity of the compounds was investigated using the lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in three different cardiac cell models: human cardiac fibroblasts (HCFs), H9c2 cells derived from embryonic rat myocardium, and hiPSC-CMs. Compound-induced proliferative and hypertrophic responses of hiPSC-CMs were studied using immunofluorescence staining and high-content analysis. Toxicity screening of the compounds showed that only AKT045 was concentration-dependently toxic in all three cell types after 24-hour treatment. Based on the toxicity screening, several compounds caused more pronounced concentration-dependent effects in H9c2 cells as compared to hiPSC-CMs and HCFs. The most considerable effects were observed with AKT042 and AKT048, as they decreased the viability of H9c2 myoblasts 46% and 55% at 30 μM concentration, respectively. In phenotypic studies, AKT050 decreased hiPSC-CM proliferation significantly. This result indicated inhibition of AKT and was consistent with previous studies. Commercially available AKT activator SC79 did not induce expected effects, as it tended to attenuate both proliferative and hypertrophic response in hiPSC-CMs. However, AKT activation has been shown to increase both proliferation and hypertrophy in previous studies. Other compounds induced a prohypertrophic rather than an antihypertrophic effect in hiPSC-CMs. Although proliferative responses to other compounds varied slightly, AKT042 and AKT043 seemed to increase the proliferation of hiPSC- CMs. However, the AKT activation or inhibition could not be confirmed in this study and therefore additional studies are needed to assess the full extent of effects and mechanisms of these compounds.
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(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.
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