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Autophagy is a pathway for cells to degrade intracellular components that are no longer needed or are detrimental for the cells. It is essential for cell homeostasis and survival and has been related to various diseases and pathophysiology. Autophagy is a complex process and there are still several unclear und unknown aspects to it. Regulation of autophagy is essential to prevent unwanted and escess activation, and several pathways and molecules, both stimulatory and inhibitory, are included. Different signaling pathways are sensitive to a variety of environmental clues. Two main autophagy pathways are mTOR-dependent pathway and mTOR-independent pathway. Induction of autophagy in the latter pathway is dependent on the interaction of Bcl-2 and Beclin 1. Prolyl oligopeptidase (PREP) is a peptidase enzyme that has several substrates. PREP-inhibition by KYP-2047 can reduce aggregation of α-synuclein in two ways: by increasing rate of autophagy and by decreasing dimerization. The aim of this study was to find out how PREP affects the interaction between Bcl-2 and Beclin 1 and how this affects autophagy. Based on previous studies, PREP-inhibition seems to increase the amount of Beclin 1 and to affect the phosphorylation of Bcl-2 and Beclin 1, leading to dissociation of the complex. Hypothesis was to see differences in colocalization of Bcl-2 and Beclin 1 in cells treated with different PREP-modifications and for PREP-inhibition to decrease the colocalization. Human embryonic kidney cells 293 (HEK-293) and hPREP knockout cell line created from them by using CRISPR/Cas9-silencing were used in the experiments. Two experiments were performed on regular HEK-cells: inhibitor experiment with KYP-2047 (1 or 10 µM) and overexpression experiment (transfection with either active or inactive hPREP plasmid). After immunofluorescence staining, cells were analysed with confocal microscope and colocation analysis of Bcl-2 and Beclin 1 was performed. The intensity of Beclin 1 in the nuclei was stronger than in other parts of the cell in all samples, which could indicate a stronger activity of its nuclear tasks compared to autophagy. However, the antibody used for immunofluorescence has most likely caused this staining pattern. Based on previous knowledge, it was expected to see differences in colocalization of Bcl-2 and Beclin 1 in cells treated with different PREP-modifications. However, there were no significant differences in colocalization of Beclin 1 and Bcl-2 in any of the experiments but it was nearly 100 percent in all treatments. Since rate of autophagy in cells was not detected, it is impossible to determine, if there were changes in autophagy that were not reflected as changes in colocalization of these two proteins. It is possible that even a small change in colocalization can affect the rate of autophagy or there might be subpopulations where the interaction is interrupted and these changes are so small that they are not detectable with the methods used in this experiment. Both Bcl-2 and Beclin 1 also have functions not related to autophagy, which could be one reason behind the results gained in this study.

The main goal of this thesis was to examine the effect of the compaction speed on the compressional behaviour of two excipients, microcrystalline cellulose and starch, using an eccentric and rotary presses. First, the average weights of the tablets have changed due to the increasing speed, as the volume of die kept constant. They were grown, for eccentric press, or were reduced, for rotary press. Second, Compression force, needed to obtain tablets with similar strength, was increased during both tableting methods. The eccentric compaction was more stable regarding to the speed increase. Tablets were formed from all of the blends, with more or less success. Additionally, as a result of force increase, resulted tablets were denser and less porous because of speed expansions during eccentric press. However, the blends containing 80% or more starch were not able to form tablets during the rotary press, because of the very poor die filling. Furthermore, blend containing 60% starch has shown very poor tabletability at speeds over 34 rounds per minute. The elastic recovery of tablets was very sensitive to the speed rises and to the concentrations of excipients during the eccentric press. Tablets have demonstrated an increase in their elastic recovery values in all cases. However, the tablets with a higher concentrations of starch were significantly more sensitive to the increasing compaction velocity. According to these results, it can be concluded that the starch exhibit more elasticity than microcrystalline cellulose. The effect of magnesium stearate on tablets' properties, such as the weight and the porosity, and compaction parameters, such as ejection force have also examined. As it expected from boundary lubricants, magnesium stearate has significantly reduced the ejection force values, required for removing the tablet from the die, compared with unlubricated tablets. Additionally, tablets with lubricants were heavier and more porous. The compression force was adjusted according to the crushing strength values in rotary press. This was due to the fracture variations of such tablets during diametrical compression, which would give unreliable values of tensile strength. Moreover, elastic recovery, porosity, density values were not calculated for scored tablet, due to either the lack of punch displacement data from rotational machine or the relative complexity of measuring the volume of such tablets. If these values had been available for both machines, their comparison with respect to these parameters would be possible and the results of this thesis would have been more appropriate.

Drug transporters and metabolizing enzymes have an important role in drug absorption in the small intestine. Food-drug interactions can affect the function of drug transporters and metabolizing enzymes in the small intestine and hence the bioavailability of drugs may change. Certain beverages have clinically relevant interactions with drugs and drinking of them should be avoided during certain drug treatments. However, possible food-drug interactions need more in vitro and in vivo studies, for example in the case of food additives which are used in the food industry increasingly, to investigate their clinical significance as inhibitors. Overall, investigating food-drug interactions is important as they might be as relevant as drug-drug interactions, especially for drugs that pass the gut wall mainly via transporters or have high presystemic metabolism. In this thesis, the inhibitor potential of 23 food additives was studied toward intestinal transporters and CYP enzymes. The food additives included sweeteners, colorants, and antioxidants. Food additives were tested against four efflux transporters with vesicle transporter assays and in OATP2B1 influx transporter with HEK293 uptake assay. The inhibition of CYP enzymes was tested in human intestinal microsomes. Six food additives were identified as possible inhibitors of BCRP, MRP2, OATP2B1, or P-gp. Two food additives were dual inhibitors. IC50 values were determined in dose-response studies for the potential inhibitors. The IC50 values were compared to the maximum expected concentration in the intestinal lumen to evaluate if the in vivo inhibition of intestinal transporters is possible. Only one food additive had a higher IC50 value than the maximum expected concentration. Eight food additives, specifically six antioxidants and two colorants, inhibited CYP-enzyme metabolism by more than 50%. Based on the results of this thesis, further studies could be performed for the identified inhibitors whose daily consumption is higher than the IC50 value. Certain food additives may inhibit CYP enzymes and the microsome assay used in this thesis is valid and could be used to study the metabolism of intestinal drug-metabolizing enzymes. However, the inhibition of transporters and CYP enzymes could be tested in cell lines, for example Caco-2 cells, to have more realistic intestinal test conditions.

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.

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.

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and it's activity is mediated via metabotropic GABAB - and ionotropic GABAA receptors. Receptors containing αβγ subunit combination are localized in both synaptic and extrasynaptic sites and they mediate fast synaptic phasic activity. αβδ- and α5βγ-containing receptors reside only in extrasynaptic space where they regulate tonic inhibition of the nerve cell. Disruption in tonic inhibition may cause several diseases. Drugs that selectively affect extrasynaptic GABAA receptors are believed to help treatment of diseases like sleep disorder, neuropsychiatry disorders, epilepsy, cognition impairment and recovery from stroke. Drug development of δ- selective GABAA agonists and positive modulators that enhance tonic inhibition as well as α5βγ selective inverse agonists that reduce tonic inhibition and enhance cognition are under investigation right now. Muscimol is a psychoactive molecule which activates all GABAA receptors but has higher affinity to cerebellar granule cell-specific receptor subtypes α6β2γ2 and extra synaptic α6β2δ than to the most common receptor subtype α1β2γ2. However, the binding of [3H]muscimol has produced contradictory results in former studies. Binding to membrane homogenates results to binding levels of the same magnitude in most brain regions, while in receptor autoradiography the high affinity binding is δ-subunit dependent. The affinity of muscimol to GABAAR subtypes has thus far been determined using saturation analysis, i.e. by measuring concentration-dependent binding of [3H]muscimol at equilibrium and by determining KD, the dissociation constant of the ligand. However, this value expresses the affinity of the ligand to the receptor, but does not give any information on the rate of the association and dissociation. In this master's thesis I have investigated association and dissociation rate of [3H]muscimol from recombinant GABAA receptors and from native GABAA receptors present in wild-type (WT) and δ-subunit knock-out (δKO) mice forebrain and cerebellar membranes. We concluded from binding assays that [3H]muscimol dissociates extremely slowly from δ-receptors. This explains further contradictory results: in membrane/filtration assay the washing procedure is generally much faster resulting in only low dissociation from αβγ receptors, while in the long washing procedure of receptor autoradiography only αβδ-binding is retained. The high affinity of muscimol to αβδ receptors is suggested to be due to its extremely slow dissociation from these receptors. Also association of [3H]muscimol to αβδ receptors seems to be slower, but this needs confirming studies. The exceptional binding properties of muscimol make it an interesting leading molecule in development of drugs which act via extrasynaptic GABAA receptors.

Eläinlääkkeiden kehitys kokee nykyaikana huomattavia muutoksia. Perinteisesti 70 % eläinlääkkeistä on ollut tarkoitettu tuotantoeläimille, mutta lemmikkieläinten lääkkeiden kulutus kasvaa jatkuvasti. Transdermaalisen antotavan tärkeitä etuja ovat maksassa tapahtuvan LA:n alkureitin metabolian vähentäminen, noninvasiivisuus ja eläinten omistajan hoitomyöntävyyden parantaminen. Usein kuitenkin eläimille joudutaan käyttämään ihmiselle tarkoitettuja lääkkeitä sopivien eläinlääkkeiden puuttumisen takia Meloksikaami on oksikaamiluokkaan kuuluva, alun perin ihmiskäyttöön kehitetty, tulehduskipulääke jonka indikaatio 1990 luvun lopussa laajennettiin eläinkäyttöön. Tutkimuksen tarkoituksena oli kehittää meloksikaamin preformulaation, jonka in-vitro transdermaalinen virtausnopeus on riittävän korkea meloksikaamin transdermaaliseen annosteluun sialle. Formulointi strategioiksi valittiin keräliuottimien, meloksikaamin liukoisuutta sekä ihon penetraatiota parantavien aineiden käyttö. Formulatioiden perimiaatiokokeita suoritettiin sian ihon preparaattien ja Franz diffusiosolujen avulla, joissa luovuttajakammiossa oli tutkittava formulaatio ja vastaanjottajakammiossa - PB 7,4/saliiniliuos. Sian ihon preparaatti oli valmistettu sian korvan ihosta. Valittujen keräliuottimien ja penetraatiota parantavien aineiden käyttö todettiin tehottomaksi keinoksi nostamaan transdermaalista virtausnopeutta. Tutkimuksen päämäärän saavuttamiseksi päätettiin syntesoida ja kokeilla Enhancer-S:ksi nimitettyä ainetta, joka sekä nostaa meloksikaamin liukoisuutta formulaatiossa että vaikuttaa ihon permeabiliteettiin. Permiaatiokokeiden jälkeen todettiin Enhancer-S apuaineen lisäävän meloksikaamin transdermaalista virtausnopeutta 22 µg/(h·cm2) asti. Todettu meloksikaamin virtausnopeuden taso riittää ylläpitämään 21,5 mg:n vuorokausiannoksen formulaation pinta-alan olevan 41 cm2 (6,4 cm x 6,4 cm).

Despite the long history of skin grafting, there is no standardized treatment for split-thickness skin graft donor sites. These sites cause a notable amount of pain and discomfort to the patients and open wounds also introduce a risk for infection. There is an extensive need for treatment options promoting the fastest and least painful healing possible while also being infection-free. The treatment of split-thickness skin graft donor sites is constantly studied and there is plenty of scientific literature available about this topic. In the theory section of this Master’s thesis, the structure of skin, the process of wound healing, skin grafting surgery and wound care products for split-thickness skin graft donor sites are briefly introduced. Additionally, the method of systematic review is described. In the empirical section, a systematic review is performed to compare animal- and non-animal-based wound care products in the treatment of split skin graft donor sites. The methodological quality of the included studies is reviewed. In the literature search, 3552 references were found. In this systematic review a total of 23 articles were included comprising of 21 comparative clinical studies and two previous literature reviews. Of the original studies, 20 reviewed healing, 14 infection and 17 pain of the split-thickness skin graft donor sites. Based on the results of the systematic review, animal-based wound care products might promote healing and reduce pain experienced by patients in the treatment of split-thickness skin graft donor sites when compared with non-animal-based wound care products. The results concerning infection were inconsistent. Generally, the reporting of the clinical original studies was not comprehensive enough for proper evaluation of methodological quality. Some defects, mostly in the blinding of the patients, study personnel and the assessors of outcomes, were also found. Moreover, the studies were heterogeneous in their definitions and measuring of the reported outcomes. Therefore, there is substantial uncertainty in the results of this systematic review. The systematic and transparent way of conducting the literature search, the review of the methodological quality and the reporting of the outcomes can be considered as a strength of this thesis. The main weakness is, that only one person performed the critical steps of this study, which might increase the risk of bias and reduce the repeatability of the study.

Literature review part: The enteric nervous system (ENS) often called “the second brain” is considered its own autonomic division that can independently regulate gut function. The ENS is derived from enteric neural crest-derived cells (ENCCs), which colonize the gut during development. Development of the ENS is a complex process, and many signalling pathways are required for a properly functioning ENS, especially GDNF/Gfrα1/RET signalling controlling survival, proliferation, migration, and differentiation of ENCCs. Hirschsprung’s disease (HSCR) is the most common congenital disease affecting gut motility. The prevalence of HSCR is 1:5000, and it is characterized by a complete lack of enteric neurons (aganglionosis) in the distal colon. Due to impaired intestinal motility, infants may have constipation, emesis, abdominal pain or distention, and, in some cases, diarrhea. The most life-threatening symptom is HSCR-associated enterocolitis (HAEC), which occurs in 30-50% of patients. Routine treatment for HSCR is a surgical operation called “pull through” in which the aganglionic segment is removed, and the remaining ganglionic segment is joined to the anus. However, the risk of developing HAEC after successful surgery still exists. Histopathological analysis has revealed that HAEC is accompanied by various changes in the gut epithelium, especially in mucin-producing goblet cells. These changes include hyperplasia of the goblet cells, altered mucin profile, retention of mucin, damaged and disorganized epithelium structure, inflammation, and bacterial adherence to the epithelium. However, a lack of suitable postnatal HSCR mouse models has partially hindered the progress of pinpointing the exact order of these events. A RET mutation found in half of the patients is overwhelmingly the biggest risk factor for HSCR. RET is a receptor on the cell membrane that mediates the effects in GDNF/Gfrα1/RET signaling pathway. of Knock-out mice of Gdnf, Gfra1 and Ret all have intestinal aganglionosis, resembling HSCR. However, to date, no mouse models of HSCR affecting GDNF/Gfrα1/RET signalling exist because pups are born without kidneys and die soon after birth. Experimental part: The GFRa1 hypomorphic mouse line (Gfra1hypo/hypo) created by Dr. Jaan-Olle Andressoo is the first successful model that survives past birth while manipulating GDNF-Gfrα1-RET signalling and phenocopying HSCR. These mice have 70-80% reduction in the expression of Gfrα1 in the developing gut and kidneys, which is sufficient to cause aganglionosis in the distal colon, yet not enough to impair kidney development.These mice are sacrificed between P7-P25 because of welfare problems yet giving a time window for analysis of the development of HAEC. Histological analyses revealed that Gfra1hypo/hypo mice had goblet cell hyperplasia and a shift away from acidic mucin production in the distal colon. Goblet cell hyperplasia was first observed at P10, but the shift in mucin profile already appeared at P5. It is not known what causes goblet cells to change their mucin production, but it seems to be the earliest histopathological change in HAEC preceding goblet cell hyperplasia. qPCR-analysis revealed that Muc2, the main secreted mucin that protects epithelium from invading pathogens, was upregulated at both P5 and P10. mRNA levels of Tnfa were also upregulated at P10. The aforementioned changes were not observed in the duodenum where the ENS had developed normally despite the reduction in Gfra1 expression. This indicates that the changes observed in the colon are likely due to the lack of ENS innervation, rather than a direct effect from GDNF-GFRa1-RET signalling itself. Finally, serum analysis indicated that systemic inflammation did not occur from P10-P16, although one Gfra1hypo/hypo animal had high levels of IL6 and TNFa at P14-16. This indicates that inflammation is not an early stage event and it is preceded by goblet cells related changes. In conclusion, changes in goblet cells seems to be earliest histopathological findings preceeding HAEC.

Lääkevaihto ja sitä täydentävä viitehintajärjestelmä ovat laskeneet lääkekustannuksia Suomessa. Epilepsialääkkeet eivät ole aiemmin kuuluneet lääkevaihdon piiriin, sillä epilepsian hoidossa eri valmisteet eivät välttämättä ole terapeuttisesti tarpeeksi samanarvoisia, ja pienikin muutos hoitotasapainossa voi altistaa epilepsiakohtauksille. Nykyisin epilepsialääkkeitä käytetään kuitenkin usein muihinkin käyttöaiheisiin, kuten psykiatrisiin sairauksiin ja kivun hoitoon. Vuonna 2017 lääkekorvausjärjestelmään tehtiin säästötoimenpiteitä, joiden yhteydessä epilepsialääkkeet sisällytettiin lääkevaihdon piiriin muissa käyttöaiheissa kuin epilepsian hoidossa. Lisäksi otettiin käyttöön poikkeava viitehintaryhmä, joka koski epilepsialääkkeistä pregabaliinia neuropaattisen kivun käyttöaiheessa. Tutkimuksen tavoitteena oli tarkastella epilepsialääkkeiden (pregabaliinin, gabapentiinin, topiramaatin, lamotrigiinin ja valproiinihapon) vaihtamista sekä hintojen kehitystä lääkevaihtoon ja viitehintajärjestelmään sisällyttämisen jälkeen vuoden 2017 alusta vuoden 2019 puoliväliin. Lisäksi tarkasteltiin näiden lääkeaineiden kustannusten, korvausmenojen sekä käyttäjä- ja reseptimäärien kehitystä. Aineistona käytettiin Kansaneläkelaitoksen reseptirekisteriin pohjautuvia tilastoja epilepsialääkkeiden lääkeostoista sekä lääkkeiden hintalautakunnan päätöksiä epilepsialääkkeiden viitehintaryhmistä ja viitehinnoista. Epilepsialääkkeiden vaihtaminen yleistyi tarkastelujakson aikana kaikilla lääkevaihdon piirissä olleilla viidellä lääkeaineella, ja vaihtokieltojen osuus resepteistä laski useimmilla lääkeaineista. Viitehinnat laskivat useimmissa tarkastelluista viitehintaryhmistä, mutta lähes yhtä usein viitehinta ei muuttunut. Viitehinnat laskivat enemmän viitehintaryhmissä, joissa oli useampia vaihtokelpoisia valmisteita. Lääkevaihdon ensimmäisenä vuonna 2017 lääkevaihtoon kuuluvien epilepsialääkkeiden kustannukset ja korvausmenot pääosin laskivat, vaikka lääkkeiden käyttö ei vähentynyt. Lääkevaihdon toisena vuonna kustannukset eivät juuri laskeneet. Pregabaliinin poikkeavan viitehintaryhmän vuoksi vaihtamatta jääneet reseptit aiheuttivat merkittävän osan lääkevaihtoon kuuluvien epilepsialääkkeiden kustannuksista. Pregabaliinille jäi siten todennäköisesti yhä säästöpotentiaalia poikkeavan viitehintaryhmän voimassaolon päätyttyä vuoden 2019 heinäkuussa, mitä on syytä tarkastella jatkotutkimuksissa.