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  • Nousiainen, Sini (2016)
    Parkinson's disease is a progressive neurodegenerative disease where dopaminergic neurons die in the substantia nigra pars compacta. Dopamine depletion induces typical parkinsonian motor symptoms which are treated by the golden standard medication levodopa and compounds enhancing the effect of levodopa. However in 4-6 years after the initiation of the chronic levodopa therapy abnormal involuntary movements (AIMs, also called levodopa-induced dyskinesia, LID) often develop and can notably worsen the quality of life. The most effective treatment for LID is deep brain stimulation (DBS), but as an invasive method its use is rare and not suitable for all patients. To date the only effective therapy for LID with marketing authorisation is amantadine. The disadvantage of amantadine is loss of efficacy which might appear less than a year after the initiation of medication. The pathophysiology of LID is a diverse phenomenon and includes dysfunctions in several different neurotransmitter systems both in the basal ganglia and in surrounding brain areas. The role of nicotinic acetylcholine receptors (nAChRs) in the pathophysiology of LID has been studied recently. Both nicotine and several nicotine-like agents have been shown to alleviate LID in preclinical studies and nicotine itself has been tested in a clinical phase II study as a potential LID medication. Of various different nAChR subtypes, the α7 receptor seems to be a potential option for future therapy of LID. It has been shown that α7 nAChR knock out mice display an increase in LID suggesting that this nicotinic receptor subtype has an inhibitory impact on the development of LID. Other studies have confirmed this view by showing that a selective α7 nAChR agonist (ABT-107) alleviates LID in primates and is neuroprotective for dopaminergic neurons in rats. Based on these observations, the aim of this study was to examine the effect of a novel α7 nAChR agonist (AZD0328) on LID in a 6-OHDA mouse model of Parkinson's disease. C57BL/6J female mice (n=17) were injected unilaterally 6-OHDA solution (3 µg) into the right medial forebrain bundle (MFB). Degeneration of dopaminergic neurons was detected two weeks after the 6-OHDA injection by measuring the motor performance in rotating rod with accelerated speed and with amphetamine-induced rotametry (2.5 mg/kg, i.p.). In the beginning of the chronic treatment, levodopa (4.5 mg/kg, s.c.) was administered twice daily for four days and then continued once daily (from Mon to Sun) to the end of the experiments. Levodopa treatment had been ongoing for 10 days before the first testing of drug effects. The pretreatment (AZD0328 0.06, 0.19, 1.9 mg/kg or 0.9 % saline, s.c.) was given 30 minutes before levodopa. The study was conducted using a within subject design so that each mouse received all four treatments on four test days during three weeks. Mice were videorecorded for 1 minute 20, 40, 60, 80 and 100 minutes after the levodopa injection was given. After the last recording day mice were killed under anesthesia via perfusion fixation and brains were collected for immunohistochemical staining to measure the extent of degeneration of dopaminergic neurons. 54 % of mice who survived from surgery (13/17) were dyskinetic (n=7). AZD0328 alleviated axial dyskinesia statistically significantly 40 minutes after levodopa injection but the statistical analysis did not reveal which of the doses was the most effective. The pretreatment with AZD0328 did not affect orolingual or forepaw dyskinesia. A potential mechanism of AZD0328 in alleviating LID might be the desensitization of α7 nAChRs which would happen only at very low doses. This means that LID are only attenuated when receptors are temporarily activated and then immediately gradually inactivated. The doses used in this study might have only activated the α7 nAChRs which might explain why no clear alleviation of LID was observed. On the other hand, the acute treatment may also be insufficient to develop desensitization. Additional studies are needed to investigate the effects of chronic administration of AZD0328 on LID in mice.
  • Korpi, Anna (2011)
    Heart failure is a complex and severe syndrome caused by different kinds of cardiovascular diseases. Pathophysiology of heart failure involves, for example, activation of sympathetic nervous system and the renin-angiotensin-aldosterone system (RAAS), insufficiently contracting left ventricle, cardiac remodeling, myocyte mishandling of Ca2+ and myocyte loss owing to apoptosis. Despite advances in the management of patients with heart failure, the mortality of patients with heart failure remains high. The use of classic inotropic agents is hampered by poor prognosis due to increase in [Ca2+]i, induction of arrhytmias and increase in the myocardial oxygen consumption. Levosimendan is an inotropic agent that has positive inotropic and anti-stunning effects mediated by the calcium sensitization of the contractile proteins and vasodilatory, anti-ischemic and cardioprotective effects mediated by opening of sarcolemmal and mitochondrial KATP channels. Levosimendan also inhibits cardiac PDE3 predominately at higher concentrations. Levosimendan is currently used only as 24-hour infusion to improve symptoms of acute decompensated heart failure. However, other promising indications have also been discovered. For example, chronic use of oral levosimendan improves survival and protects cardiovascular system in vivo. In the present study, the effects of oral levosimendan, valsartan and their combination use on survival, blood pressure and cardiac remodeling were examined in Dahl/Rapp rats on a high salt diet (8 %). Levosimendan improved the survival in Dahl/Rapp rats on a high-salt diet, although not statistically significantly when compared to control group. The drug combination prevented completely salt-induced cardiovascular mortality. The combination therapy also produced a blood pressure-dependent protection against hypertension-induced hypertrophy measured by heart weight-to-body weight ratio (HW/BW) and echocardiographic parameters. Interestingly, the combination use of levosimendan and valsartan had an additive antihypertensive effect in Dahl/Rapp rats. Levosimendan slightly improved systolic function. However, echocardiography revealed increased IVRT in Dahl/Rapp control rats when compared to control group on low salt diet (0,2 %) indicating impaired diastolic relaxation in Dahl/Rapp rats. In the present study, levosimendan, alone and in combination with valsartan, also corrected hypertension-induced diastolic dysfunction.
  • 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.
  • Karumo, Suvi (2022)
    Liposomes are biocompatible spherical nanosized vesicles consisting of hydrophobic phospholipid bilayer encasing an aqueous core. They can be utilized as drug carriers by either encapsulating molecules inside the core or embedding them in the bilayer accordingly to achieve numerous advantages such as prevention of rapid clearance and reduction of adverse effects as systemic exposure is reduced. Despite the marked efforts in designing the liposomes to improve therapeutic outcomes, only limited drug concentrations are achieved at the target sites such as in solid tumors. Stimuli-responsive liposomes could be applied as potential delivery systems to achieve spatiotemporally controlled drug delivery, i.e., the drug release could be pinpointed and restrained to the target site. In this thesis, the objective was to study the light-activated indocyanine green (ICG) liposomes as nanocarriers for peptide-based anti-tumor agents. The physicochemical characteristics, stability and functionality of the prepared liposomes were determined alongside optimizing the formulation as needed and utilizing different model peptides as encapsulated compounds. Additionally, the peptide stability during near-infrared (NIR) light illumination and the effects of the anti-angiogenic model peptides in vitro were investigated. The stability of the liposomes was assessed by monitoring the size of the liposomes, intactness of ICG, and passive leakage of the peptides over time, and by determining the phase transition temperatures of the different formulations. The liposomes remained adequately stable in different relevant conditions, and the observed phase transition temperatures did not indicate the lipid bilayer becoming permeable in physiological temperatures. However, the rate of passive leakage was rather high in all formulations, although with stiffer lipid bilayer in the “rigid” formulation, the unintended release was able to be decreased slightly in comparison to the other formulations. On the other hand, light-triggered release upon illuminating the liposomes remained considerably low in all formulations. The intactness of peptides seemed to not be impacted by the illumination. Also, no cytotoxic effects were observed after exposing human umbilical vein endothelial cells (HUVEC) to the peptides. The final “rigid” formulation showed the best functionality out of those included in the studies. It remains to be investigated whether the formulation could be improved further for optimal functionality and stability, and to what degree do the properties of the cargo molecule affect the performance of the liposomes.
  • Mäki-Mikola, Eija (2020)
    Liposomes are nano-sized vesicles, that are composed of a phospholipid bilayer structure. They can be utilized as drug carriers, in which case the drug is incorporated either to their hydrophilic internal cavity, or into their hydrophobic bilayer structure. For anticancer drugs, liposomal formulations have exhibited their capability in reducing adverse effects of anticancer drugs. This is achieved mainly by the enhanced permeability and retention (EPR) effect, in which liposomes accumulate into tumour tissue. However, the conventional liposomes release their drug content passively, and a proportion of drug is distributed to off-target tissues. Therefore, there is a demand to develop liposomes from which the content can be released in a controlled manner, by an external stimulus. The objectives of this master’s thesis project were to determine the potential of light-activated paclitaxel (PTX) liposomes for the treatment of lung cancer, and to optimize a dynamic cell culture system, QuasiVivo® (QV), to study the off-target effects of light-activated PTX liposomes. The hypothesis was that the induction of the light-activated PTX liposomes would increase the efficiency of paclitaxel treatment. For QV experiments, it was expected that the presence of flow would improve the viability of the cells. The encapsulation efficiency of PTX into the liposomes and the effect of the PTX incorporation into the phase transition temperature of the liposomes were determined. The stability of liposomes was determined by monitoring the liposomal size and light sensitizer absorbance during a storage period. The cells of lung cancer cell line A549 were cultured inside QV system, and their viability was monitored with two commercial cell viability assays. Incorporation of PTX decreased the phase transition temperature, but the liposomes remained stable in the studied conditions. The PTX liposome treatments with and without light activation resulted in the similar efficacy as free PTX treatment did. A549 cells failed to display superior viability inside the QV compared to static conditions. Cells cultured under lower flow rate portrayed modestly higher viability. The light-activated PTX liposomes did not improve the efficacy of PTX treatment. Neither of the flow rates were optimal for A549 cells, as the variation between experiments was high. The EPR effect is the main reason for the improved effects of liposomal anticancer drugs, therefore, it is likely that in vivo experiments would elicit the differences between the efficacy of the liposomal and free PTX. The non-existent effects of light activation on the viability are likely caused by the low total concentration of the light sensitizer in the treatment solution.
  • Kallio, Varpu (2014)
    The purpose of this study is to evaluate patients' quality of life and healthcare use before and after bariatric surgery and produce new, clinical data-based information on the cost-effectiveness of bariatric surgery. Healthcare resources are limited and expenditures have grown from year to year. Therefore it is important to make cost-effectiveness evaluations so that financial resources could be allocated properly. The research population consists of patients who have undergone gastric bypass or sleeve gastrectomy in the Hospital District of Helsinki and Uusimaa, during the years 2007-2009. The study population consists of 147 gastric bypass patients and 79 sleeve gastrectomy patients. In this study the decision analytic model, used in the Finohta study "Sairaalloisen lihavuuden leikkaushoito" was updated using actual, up-to-date information. The analysis was done using a decision tree and a Markov model with a time horizon of 10 years. The cost data in this study was based on actual data for the first two years after surgery. A forecast model was used to predict the costs for the years 3-10 after surgery. Patients' quality of life scores were based on real data for the years 1 (the year of operation) to 4. Quality of life scores for the other years were predicted. In the literature review section, international studies on the cost-effectiveness of bariatric surgery and its impacts on drug therapy were evaluated. The studies showed that the use of medicines, which were used to treat obesity-related diseases were lower in the surgery group. However, drugs used to treat vitamin deficiencies, depression and gastrointestinal diseases were higher in the surgery group. Most studies found that surgery is the most cost-effective way to treat morbid obesity. This study confirms the role of the bariatric surgery in the treatment of morbid obesity in Finland. Even though the healthcare costs were increased in the first two years after the operation, the conclusions of the Finohta study didn't change. The bariatric surgery is cheaper and more effective than ordinary treatment and the most cost-effective way to treat morbid obesity. The mean costs were 30 309 € for the gastric bypass, 31 838 € for the sleeve gastectomy and 36 482 € for ordinary treatment. The mean numbers of quality-adjusted life-years were 6.919 for the gastric bypass, 6.920 for the sleeve gastrectomy and 6.661 for ordinary treatment. However, there is demand for more information for the long-term effects, benefits and risks of the surgery. How much the surgery will actually save money, will be hopefully clarified in the long-term follow-up study, which should also include an actual control group.
  • Rosenholm, Marko (2016)
    Pharmacologically induced neuronal plasticity holds unprecedented potential in treatment of several neurological disorders, such as depression. Several antidepressant drugs have been shown to induce neuronal plasticity by stimulating BDNF (brain-derived neurotrophic factor) receptor TrkB (tropomyosin receptor kinase B). Studies with rapid-acting antidepressant treatments suggest delta range slow wave EEG (electroencephalography) activity to function as a potential non-invasive biomarker for activation of TrkB-related neuroplastic signaling responses. A sedative GABAA-agonist THIP (gaboxadol) has been shown to induce slow wave EEG activity (SWA) and preliminary studies suggest it to activate TrkB signaling as well. The aim of the present study was to examine the potential connection between SWA, neuroplastic signaling responses and neuronal inhibition by utilizing EEG measurements and THIP administration in genetic and developmental mouse models. The pharmaco-EEG experiments showed acute THIP administration (6 mg/kg, i.p.) to increase SWA in wild-type but not in GABAA δ-subunit knockout mice. TrkB signaling responses from similar treatment groups showed a trend of increased TrkB-related protein phosphorylation in wild-type but not in GABAA δ-subunit knockout mice indicating a positive connection between SWA, neuronal inhibition and TrkB-related signaling response. Autophosphorylation response of TrkB and related proteins in mice of different age showed most TrkB phosphorylation in postnatal day 16 (P16) mouse pups, whereas phosphorylation response of CREB and p70S6k was the highest in postnatal day 8 (P8) mouse pups. Since SWA emerges during the second postnatal week in mice, the obtained result further supports the connection between SWA and TrkB signaling. Acute THIP administration caused no significant phosphorylation changes in P8 or P16 mouse pups. The results support the hypothesis of a positive connection between SWA, neuronal inhibition and TrkB-related signaling response. Further studies with different excitatory and inhibitory interventions are required to better understand the role of neuronal excitation and inhibition in TrkB signaling responses and corresponding EEG signatures.
  • Uimari, Elisa (2023)
    DNA-origamit ovat pitkästä DNA-juosteesta laskostettuja nanorakenteita, jotka voidaan suunnitella monen muotoisiksi ja kokoisiksi. DNA-origamit ovat muodostuneet luontaisesti biohajoavasta materiaalista, joten ne sopivat hyvin biolääketieteellisiin tarkoituksiin, kuten lääkekantajiksi. Aiemmissa tutkimuksissa on kuitenkin huomattu, että ihmiskehon entsyymit voivat hajottaa DNA-origamin rakenteen. DNA-origamien lipidipäällystäminen on yksi keino suojata rakennetta tällaiselta entsymaattiselta hajoamiselta. Tässä tutkimuksessa DNA-origameja päällystettiin kationisilla DOTAP (1,2-dioleyyli-3-trimetyyliammonium-propaani) lipideillä. Tutkimuksen tavoitteena oli tutkia ja optimoida päällystämisprosessia. Tutkimuksessa testattiin erilaisten inkubaatioaikojen ja -lämpötilojen sekä ionisten olosuhteiden vaikutusta muodostuvaan lipidipäällystykseen. Lipidipäällystettyjä DNA-origameja altisteettiin sonikaatiolle ja ekstruusiolle, jotta havaittaisiin, voisivatko nämä menetelmät häiritä muodostunutta lipidipäällystettä. Neutraalia DOPC (1,2-dioleoyyli-sn-glysero-3-fosfokoliini) lipidiä käytettiin tutkimaan, kuinka erilaiset lipidikombinaatiot vaikututtavat päällystyksen muodostumiseen. Työssä käytettyjä tutkimusmenetelmiä olivat dynaaminen valonsirontamenetelmä, agaroosigeelielektroforeesi ja transmissio-elektronimikroskopia. Kokeissa lipidipäällystäminen onnistui ionittomissa olosuhteissa ja 30 minuutin inkubaatioaika huoneenlämmössä oli riittävä päällystyksen muodostumiselle. DNA-origami-partikkelit olivat täysin päällystettyjä, kun käytettiin stoikiometristä suhdetta nDOTAP/nORIGAMI ≈ 20 000. Tällöin lipidipäällystetyt DNA-origamit muodostivat mikrometrien kokoisia rykelmiä. Kun käytettiin stoikiometristä suhdetta nDOTAP/nORIGAMI ≈ 10 000, TEM kuvissa oli havaittavissa päällystämättömiä ja satojen nanometrien kokoisia päällystettyjä DNA-origameja. Kylpysonikaation havaittiin häiritsevän aggregoituneita lipidi-DNA-partikkeleita muodostaen pienempiä partikkelirykelmiä. Kun käytettiin DOPC lipidejä, huomattiin, että ne eivät muodostaneet yhtenevää lipidipäällystettä DNA-origamien ympärille. Kokeissa ionisten olosuhteiden havaittiin epästabiloivat DNA-origamien päällystysprosessia, joka johti partikkelien kokojakaantuman lisääntymiseen. Tässä työssä DOTAP lipidejä käytettiin päällystämään DNA-origameja ja lipidimäärää kasvattamalla saatiin muodostettua monesta lipidikerroksesta muodostuneita lipidi-DNA-komplekseja. Sonikaation havaittiin olevan potentiaalinen menetelmä partikkelikoon pienentämiseen. Muita menetelmiä, kuten NTA-analyysia (engl. Nanoparticle Tracking Analysis), tarvitaan kuitenkin vahvistamaan muodostuneiden lipidi-DNA-partikkeleiden koosta saatuja tuloksia.
  • Huynh, Thi Le Hang (2010)
    In the written part of my master -thesis I discuss about GDNF signalling and more specifically how the changes in the GDNF/GFRα1/Ret signaling affect the nigrostriatal dopaminergic neurons in different mutant mice. In the animal models of Parkinson's disease the neuroprotective and neurorestorative effects of exogenous GDNF are very clear which raises hope for use of GDNF in treatment of Parkinson's disease. In intact animals GDNF stimulates the function of nigrostriatal dopaminergic system. Revealing the role of GDNF/GFRα1/Ret signaling in development, maintenance and protection of nigrostriatal dopaminergic system will certainly help in search for treatment of neurodegeneration in Parkinson's disease. In knockout mouse models GDNF/GFRα1/Ret signaling is not crucial for prenatal nigrostriatal dopaminergic neuron development, but it has been shown that it plays an important role in the early postnatal development. Also, it was shown that reduced GDNF/GFRα1/Ret signaling compromises nigrotriatal dopaminergic system in heterozygous GDNF/GFRα1/Ret knockout mice. However the physiological roles of endogenous GDNF and its signalling in the nigrostriatal dopaminergic neurons are not very well understood. In the experimental part of my master -thesis I studied how reduced endogenous GDNF signaling affects the dopaminergic system after 6-OHDA induced neurotoxicity in the conventional heterozygous GDNF mice. Besides that I examined the effects of elevated endogenous GDNF on dopaminergic system of 7 days old so-called GDNF hypermorphs mice. The effects of reduced endogenous GFRα1 levels on dopaminergic system of 20 days old GFRα1 hypomorphs have also been studied. The obtained date showed that mice with the reduced levels of endogenous GDNF are not more susceptible to the 6-OHDA induced neurotoxicity than the wild type littermates. Elevated endogenous GDNF levels did not affect early postnatal development of the nigrostriatal dopaminergic system in GDNF hypermorphs mice as revealed by normal intensity of TH staining in striatum and normal number of TH-positive cells in the substantia nigra pars compacta. Reduced levels of endogenous GFRα1 levels did not affect monoamine levels in the striatum of GFRα1 hypomorph mice.
  • Kinnari, Päivi (2010)
    Most new drug molecules discovered today suffer from poor bioavailability. Poor oral bioavailability results mainly from poor dissolution properties of hydrophobic drug molecules, because the drug dissolution is often the rate-limiting event of the drug's absorption through the intestinal wall into the systemic circulation. During the last few years, the use of mesoporous silica and silicon particles as oral drug delivery vehicles has been widely studied, and there have been promising results of their suitability to enhance the physicochemical properties of poorly soluble drug molecules. Mesoporous silica and silicon particles can be used to enhance the solubility and dissolution rate of a drug by incorporating the drug inside the pores, which are only a few times larger than the drug molecules, and thus, breaking the crystalline structure into a disordered, amorphous form with better dissolution properties. Also, the high surface area of the mesoporous particles improves the dissolution rate of the incorporated drug. In addition, the mesoporous materials can also enhance the permeability of large, hydrophilic drug substances across biological barriers. T he loading process of drugs into silica and silicon mesopores is mainly based on the adsorption of drug molecules from a loading solution into the silica or silicon pore walls. There are several factors that affect the loading process: the surface area, the pore size, the total pore volume, the pore geometry and surface chemistry of the mesoporous material, as well as the chemical nature of the drugs and the solvents. Furthermore, both the pore and the surface structure of the particles also affect the drug release kinetics. In this study, the loading of itraconazole into mesoporous silica (Syloid AL-1 and Syloid 244) and silicon (TOPSi and TCPSi) microparticles was studied, as well as the release of itraconazole from the microparticles and its stability after loading. Itraconazole was selected for this study because of its highly hydrophobic and poorly soluble nature. Different mesoporous materials with different surface structures, pore volumes and surface areas were selected in order to evaluate the structural effect of the particles on the loading degree and dissolution behaviour of the drug using different loading parameters. The loaded particles were characterized with various analytical methods, and the drug release from the particles was assessed by in vitro dissolution tests. The results showed that the loaded drug was apparently in amorphous form after loading, and that the loading process did not alter the chemical structure of the silica or silicon surface. Both the mesoporous silica and silicon microparticles enhanced the solubility and dissolution rate of itraconazole. Moreover, the physicochemical properties of the particles and the loading procedure were shown to have an effect on the drug loading efficiency and drug release kinetics. Finally, the mesoporous silicon particles loaded with itraconazole were found to be unstable under stressed conditions (at 38 qC and 70 % relative humidity).
  • 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.
  • Kenttä, Laura (2015)
    Susceptibility to antibiotics is constantly developing in bacteria due to selection pressure caused by use of antibiotics. For this reason, finding new antimicrobial substances is imperative. High-throughput screening (HTS) is an important tool to find new active substances. The need to analyse as many substances in as small time as possible is emphasised in modern drug development. Robust methods, suitable for fast throughput of substances, miniaturisation and automation, are particularly useful. In the context of antimicrobial screening, methods utilising bioluminescence can correspond this need, and genetic engineering can help in developing bacterial strains with beneficial features for screening. In this work, two screening methods were developed and optimised using genetically engineered Escherichia coli strains. The screening methods make use of the bioluminescent properties of the strains, and the methods can be used to screen compound libraries for antimicrobials rapidly enough to approach HTS. The strain E. coli WZM120/pCGLS 11 is constitutively luminescent, so weakening of luminescence means the cell viability weakens. The strain E. coli K12/pCSS305, where luminescence is produced by a heat-inducible runaway plasmid, can be used to especially detect compounds inhibiting DNA replication. In developing the method, workflow was optimised and conditions were validated so as to enable possible HTS campaigns. The target was to create as simple, fast and reproducible a method as possible. The Z' values calculated in assessing the performance are excellent for a cell-based method. The signal is readily distinguishable, the bacterial strains are in a stable manner, and the method is well reproducible. It is possible to continue assay development from 96-well format to 384-well format.
  • Tuhkalainen, Juho (2012)
    Natural products have been used as medicines for thousands of years. Of the drugs on the market today a significant proportion are natural products or natural product derivatives. Natural products can be enhanced by the means of chemical modification. Modification of a natural product may result in lesser toxicity, greater efficacy or better chemical stability. Different ways to modify a natural product are represented in the literature review using approved drugs as examples. Biological screening is an important part of a modern drug discovery process. Libraries containing synthetic molecules or natural products can be screened. The literature review discusses different types of natural product libraries and how they differ from synthetic libraries. Natural product libraries are smaller and more laborious to screen compared with synthetic libraries. Natural product libraries contain more hits in proportion of total compounds because natural products have activity in biological systems more often than synthetic molecules. A remarkable part of antibiotics and anti-cancer agents are derived from nature. A need for especially new antibiotics will be notable in the future due to resistant microbial strains and the need can be met with natural product research. The object of the experimental part was to evaluate the bioactivity of eleven synthetic abietic acid derivatives. Antimicrobial activity of the compounds was determined againts six human pathogens which were S.aureus, E.coli, P.aeruginosa, E. aerogenes, E. faecalis and Candida albicans. Cytotoxicity testing on the compounds was performed using mammalian cell lines CaCo-2 and Huh-7. Compounds were tested for albumin binding using bovine serum albumin. The effect of bovine serum albumin on the antimicrobial effect of compounds was studied. Spectrophotometric studies on compound-albumin complexes were carried out using fluorescence and UV absorbance measurement techniques. A primary antimicrobial screening was performed with all the compounds. Minimum inhibitory concentration (MIC) values were determined for compounds that showed antimicrobial activity in the primary screening. Cytotoxicity testing was carried out with all the compounds. Albumin binding was studied only on compounds that showed activity in the antimicrobial screening. Some of the compounds were noticed to have antimicrobial activity against the studied gram-positive bacteria and yeast Candida albicans. Antimicrobially active compounds were noticed to bind to albumin and have cytotoxic effects.
  • Renko-Kaski, Riikka (2014)
    This study explores the research, characteristics, manufacturing processes, safety and applications of graphene from the perspective of pharmacy and medicine. The study also examines how graphene research and commercialization has developed in the last ten years (2004-2013), with an emphasis on biomedical research globally and separately in Finland. The methods employed are an extensive literature review of scientific publications, and a survey of the biomedical research emphases, geographical distribution, and funding of graphene research based on article and patent databases. Graphene holds considerable potential in pharmaceutical use. Clinical trials can commence as soon as the manufacturing processes develop to produce graphene of sufficient quality. The variety of biomedical uses of graphene is vast: antibacterial products and coatings, gene therapy, tissue technology, sensor and imaging technology, as well as utilization in drug delivery. Graphene can be used to enhance therapeutic effectiveness by creating instruments for targeted and controlled drug delivery. In addition to uses in therapeutics, graphene offers possibilities for diagnostics. The biomedical research and commercialization of graphene have accelerated in the recent years, but research and patenting activity has concentrated in Asia, and especially in China. The research has been university driven and primarily publicly funded. In Finland, graphene research has focused on electrical applications, whereas research in the fields of pharmacy and medicine has been limited. As a so-called high-tech country, Finland could increase research into graphene as an innovative pharmaceutical instrument.
  • Nordberg, Antti (2011)
    Nearly one fourth of new medicinal molecules are biopharmaceutical (protein, antibody or nucleic acid derivative) based. However, the administration of these compounds is not always that straightforward due to the fragile nature of aforementioned domains in GI-tract. In addition, these molecules often exhibit poor bioavailability when administered orally. As a result, parenteral administration is commonly preferred. In addition, shelf-life of these molecules in aqueous environments is poor, unless stored in low temperatures. Another approach is to bring these molecules to anhydrous form via lyophilization resulting in enhanced stability during storage. Proteins cannot most commonly be freeze dried by themselves so some kind of excipients are nearly always necessary. Disaccharides are commonly utilized excipients in freeze-dried formulations since they provide a rigid glassy matrix to maintain the native conformation of the protein domain. They also act as "sink"-agents, which basically mean that they can absorb some moisture from the environment and still help to protect the API itself to retain its activity and therefore offer a way to robust formulation. The aim of the present study was to investigate how four amorphous disaccharides (cellobiose, melibiose, sucrose and trehalose) behave when they are brought to different relative humidity levels. At first, solutions of each disaccharide were prepared, filled into scintillation vials and freeze dried. Initial information on how the moisture induced transformations take place, the lyophilized amorphous disaccharide cakes were placed in vacuum desiccators containing different relative humidity levels for defined period, after which selected analyzing methods were utilized to further examine the occurred transformations. Affinity to crystallization, water sorption of the disaccharides, the effect of moisture on glass transition and crystallization temperature were studied. In addition FT-IR microscopy was utilized to map the moisture distribution on a piece of lyophilized cake. Observations made during the experiments backed up the data mentioned in a previous study: melibiose and trehalose were shown to be superior over sucrose and cellobiose what comes to the ability to withstand elevated humidity and temperature, and to avoid crystallization with pharmaceutically relevant moisture contents. The difference was made evident with every utilized analyzing method. In addition, melibiose showed interesting anomalies during DVS runs, which were absent with other amorphous disaccharides. Particularly fascinating was the observation made with polarized light microscope, which revealed a possible small-scale crystallization that cannot be observed with XRPD. As a result, a suggestion can safely be made that a robust formulation is most likely obtained by utilizing either melibiose or trehalose as a stabilizing agent for biopharmaceutical freeze-dried formulations. On the other hand, more experiments should be conducted to obtain more accurate information on why these disaccharides have better tolerance for elevating humidities than others.
  • 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.
  • Viskari, Ansa (2012)
    The purpose of this study was to investigate how the mixing time of the magnesium stearate affects on the compressibility of partially pregelatinized maize starch. Pregelatinized maize starch is used in pharmaceuticals as a filler, binder and as disintegrant. Because pregelatinized maize starch has lubricant characteristics itself, it is known to be sensitive for the amount and the mixing time of magnesium stearate. The aim is that magnesium stearate is not totally homogenously mixed on the powder surfaces so that even, clean powder surfaces exist. Homogeneous mixing means that particles are coated with magnesium stearate, which as a hydrophobic ingredient prevents bond formation between plastically and elastically behaving particles. Too much magnesium stearate and/or too long mixing time may cause weakening of tablet tensile strength, laminating and capping. The weakening of the tensile strength of the tablet increases friability, which causes problems during packaging process and the transportation. Too much magnesium stearate may also lengthen the disintegration time and slow down the dissolution. The aim of this study was to compare four different brands of pregelatinized maize starch. The purpose was to find differences affecting the compressibility behavior. Also the effect of the mixing time of magnesium stearate for compression behavior of masses were studied. The brands investigated were C*PharmGel DC 93000, Lycatab® C, Starch 1500® and SuperStarch 200®. First mentioned was a reference product which is not manufactured any more. There was only one batch of the reference product but three batches from other products to be able to investigate also batch to batch variation. The characteristics studied from pregelatinized starch samples were bulk density, apparent density and true density, flowability, moisture sorption, moisture content, pH value, swelling volume and particle size. Also NIR, FTIR and Raman spectroscopy and X-ray powder diffraction method were used. Weight, tensile strength, dimensions, friability, disintegration time and moisture sorption were studied for tablets. The compressibility of the mass and elastic behavior of tablets was studied. Pictures of the tablets were also taken by scanning electron microscope. When the mixing time of magnesium stearate was increased from 2 minutes to 5 minutes, the compression pressure needed for pressing tablets for 80 N strength increased 200-700 N depending on the brand of pregelatinized maize starch. Based on the results the best alternative to replace C*PharmGel DC 93000 was chosen to be SuperStarch 200®. Scanning electron microscope pictures showed that C*PharmGel DC 93000 deviates from other qualities studied by being roundish and regular in shape. SuperStarch 200® and Starch 1500® reminded remarkably each other. Lycatab® C was the biggest in particle size and very irregular in shape. The differences found in tabletting followed the expectations based on the SEM-pictures. SuperStarch 200® showed to best compressibility in lowest strain strength and after C*PharmGel DC 93000 it was least sensitive for mixing time of the magnesium stearate. It also has least elastic recovery. The differences between SuperStarch 200® and Starch 1500® in compression properties were moderate but clear. Lycatab® C had clearly the weakest compression properties.
  • Tervo, Annukka (2011)
    Alphaviruses are positive-stranded RNA-viruses and they belong to the family of Togaviridae. Alphaviruses are spread by mosquitoes of family Aedes. Alphaviruses have spread on all continents except Antarctica. So far 29 alphaviral species have been identified and they can be divided in two groups, Old and New world viruses, by their geographical distribution and by diseases they cause. Chikungunya virus (CHIKV) is one of the Old World alphaviruses and it has been found in Africa and Asia. However, due to the global warming, Chikungunya is also spreading to southern Europe. In humans, it causes fever, headache, rash and joint pain, which can last for several years and be very painful. In small children, Chikungunya can cause neurological symptoms such as encephalitis. Genome of alphaviruses encodes for four structural proteins and four non-structural proteins (nsP), of which nsP3 contains a macro domain. Macro domains are conserved in most kingdoms of life but their function has not been elucidated. It has been shown that macro domains bind ADP-ribose and its derivatives and it has been shown that nsP3-protein has an important role in alphavirus replication. The aim of the study was to study the use of compounds which bind to macro domain protein as antiviral agents. 45 compounds were chosen for antiviral studies by molecular modeling. These compounds were expected to bind to macro domain proteins. In a competitive binding assay five compounds inhibited more than 50 % poly-ADP-ribose (PAR) binding to MDO1-macro domain protein, which was the protein on which the molecular modeling was performed. When the competitive binding assay was performed with SFV macro domain (nsP3), only one compound inhibited poly-ADP-ribose binding more than 50 %. In SFV-antiviral assay seven compounds had inhibition percentage higher than 50 %. In a CHIKV replicon assay five compounds had more than 50 % inhibition on replicon expression. We also studied possible inhibition mechanism by studying whether the compounds inhibit the virus to enter the cell. Almost all compounds included in this assay inhibited the virus entry to some extent. In general, the inhibition of PAR binding and antiviral activity did not correlate among the studied compounds. Even though compounds which had antiviral potency did not inhibit PAR binding to macro domains, potential antiviral agents were found which deserve further investigation as virus entry inhibitors.
  • Anttila, Jenni (2013)
    Brain-derived neurotrophic factor (BDNF) and the receptor mediating its effects, neurotrophin receptor TrkB, seem to have a role in the pathophysiology and treatment of mood disorders such as depression and mania. BDNF is a neurotrophin that regulates the differentiation and survival of neurons and mediates neuronal plasticity. Lithium and valproate are mood stabilizing agents that are commonly used to treat mania but their mechanism of action is still unclear. However, both acute and chronic lithium treatment have been shown to activate TrkB receptor in the rodent anterior cingulate cortex. It has also been shown that chronic lithium and valproate treatment increase the amount of BDNF in the rodent brain. The aim of the experimental part of this master's thesis was to find out what are the effects of lithium and valproate on TrkB receptor activation and on the amount of intracellular BDNF protein levels in vitro on embryonic day 18 (E18) rat primary cortical neurons. In addition, the possible role of neuronal maturation was investigated by conducting the experiments with neuronal cultures aged 7 and 21 days in vitro. The research methods included two different types of enzyme linked immunosorbent assays (ELISA), phospho-Trk ELISA and BDNF ELISA. Western blot was used to confirm the results. Therapeutically relevant concentration of lithiumchloride and valproate blocked BDNFinduced TrkB receptor phosphorylation in immature neurons aged 7 days in vitro. The effect of valproate was detected only with ELISA. In contrast, therapeutically relevant concentration of valproate increased TrkB receptor phosphorylation in immature neurons after one hour treatment. Lithium and valproate did not regulate TrkB receptor phosphorylation in mature neurons aged 21 days in vitro. However, therapeutically relevant concentration of lithium increased BDNF protein content in mature neurons after 24 hours treatment. Therapeutically relevant concentration of valproate did not alter BDNF protein levels. In conclusion, neuronal maturation does have a role on the effects of lithium and valproate on TrkB receptor activation and regulation of BDNF protein levels. It is possible that lithium and valproate are harmful to immature neurons through blocking BDNF-induced TrkB receptor phosphorylation. Since therapeutically relevant concentration of lithium did not activate TrkB receptor as has been shown previously in vivo it seems that certain developmental processes are essential for lithium-induced TrkB receptor activation.
  • 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.