Skip to main content
Login | Suomeksi | På svenska | In English

Browsing by Title

Sort by: Order: Results:

  • Heiskanen, Vilma (2021)
    Binge eating disorder (BED) is the most common eating disorder characterized by compulsive recurrent binge eating episodes with the sense of a lack of control. During a binge eating episode, one eats a larger amount of food, typically high in fat and/or sugar, than would normally be eaten in a discrete period of time. After the episode, negative emotions, such as shame and self-disgust, are present. However, BED does not include compensatory behavior, such as vomiting or excessive exercise. Due to compulsive and uncontrollable eating behavior, it has been suggested that BED represents a food addiction. Eating energy-dense food activates the dopamine, opioid, and endocannabinoid systems in the brain. This elicits the activation of the reward process. Some drugs and medications affect the same neurotransmitter systems, which may produce neuronal alterations in the reward process, leading to an addiction. Several studies have found that cannabidiol (CBD) reduces the self- administration of cocaine, morphine, alcohol, and sucrose in rodents, suggesting an effect on the reward-response. Some of these effects have been shown to be mediated by cannabinoid receptor 2 and TRPV1 receptor. However, the effects of CBD on bingeing behavior have not been studied up to date. The aim of the study was to investigate the effect of CBD on homeostatic feeding and binge eating behavior in C57BL/6 mice. Five separate experiments were conducted. The first experiment investigated the effect of CBD (15, 50, and 150 mg/kg, i.p.) on locomotor activity in a modified open field test over a 2-hour period. In the second test, the effect of CBD (15, 50, and 150 mg/kg, i.p.) on homeostatic feeding was monitored in non-bingeing mice. Next, a limited intermittent access binge eating model without food deprivation or stressors was inducted. Mice had access to laboratory chow ad libitum, but a high energy diet (high in fat, HED) was presented in 24-hour periods every 5-8 days. Then the effect of CBD (15, 50, and 150 mg/kg, i.p.) on HED and chow intake in bingeing mice was investigated. In the fourth experiment, seven days following the administration, the after effect of CBD was studied by monitoring food intake without CBD treatment. Finally, it was investigated whether the effect of CBD can be inhibited by TRPV1 receptor antagonist AMG9810 (1 mg/kg, i.p). In each test, the food intake was monitored at the time point 0,5, 2,5, and 24 h after CBD treatment. Also, water consumption was measured in each experiment. The results revealed that CBD does not affect locomotor activity or homeostatic feeding at a dose of 15, 50, or 150 mg/kg (i.p). However, the results showed that CBD reduces the intake of HED in a dose-dependent manner (15, 50, or 150 mg/kg; i.p.) and, possibly, increases chow intake. No after effect was observed seven days following the administration. Most likely, TRPV1 does not mediate the effect of CBD on HED intake. Furthermore, no significant effects on water intake were observed. In this study, the core aims were to evaluate whether CBD affects homeostatic feeding or binge eating behavior in mice. The results provided a novel insight into the effects of CBD. The findings indicate that the acute systemic administration of CBD reduces HED intake, and possibly, simultaneously increases chow intake, suggesting a balancing effect on feeding in bingeing mice. However, the role of TRPV1 in this effect remains unclear, and further studies are needed.
  • Paukkonen, Heli (2013)
    Casein based formulations are promising materials for controlled drug release. Caseins are the major milk proteins, and their biocompatibility, low toxicity and natural metabolism in physiological systems make caseins extremely suitable materials for pharmaceutical formulations. Polyelectrolyte complex nanoparticles can be prepared under very mild conditions, and they are stable in the gastrointestinal tract, which makes them suitable carrier materials for oral delivery and controlled release of peptide and protein drugs. Aim of this work was to synthesize casein-poly(acrylic acid) polyelectrolyte complex nanoparticles in different mass ratios, and to study the release profile of a model compound rhodamine 6G from these nanoparticles. The casein shell of the nanoparticles was crosslinked with two different crosslinkers, because the objective was to study the effect of surface modification on size of nanoparticles as well as on the release profile of the model compound. The goal was to achieve controlled release of the model compound by modifying the thickness and the density of the casein shell structure. Size and size distribution of nanoparticles was studied by dynamic light scattering. Surface charge was studied by electrophoretic mobility measurements. Morphology was characterized with electron microscopy, and the effect of the casein shell thickness on the release of rhodamine 6G was studied with dialysis method. The synthesized nanoparticles had spherical morphology, but the size distribution was wide. The release of rhodamine 6G was slower from the nanoparticles when compared to the release of reference free rhodamine 6G, but the effect of casein shell thickness on the release of loaded rhodamine 6G remained partially unclear. However, it seems possible to achieve controlled release of encapsulated compounds from casein-poly(acrylic acid) nanoparticles with optimal surface modification in the future.
  • Silmu, Veera (2021)
    Parkinsonin tauti on hitaasti etenevä hermorappeumasairaus, jossa mustatumakkeen dopamiinihermosolut tuhoutuvat. Taudille on tyypillistä dopamiinihermosoluissa esiintyvät Lewyn kappaleet, jotka koostuvat pääasiassa väärin laskostuneesta ja kasautuneesta alfasynukleiiniproteiinista. Myös neuroinflammaation uskotaan olevan osa Parkinsonin taudin patofysiologiaa. Nykyiset lääkkeet vaikuttavat ainoastaan taudin oireisiin, joten tarve uusille lääkkeille on suuri. Pilottikokeen tarkoituksena oli selvittää aiheuttaako adenoassosioidun virus- (AAV) vektorin alfasynukleiinin ja alfasynukleiinifibrillien yhdistelmämalli rotilla liikehäiriöitä ja tyrosiinihydroksylaasi- (TH) positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa sekä saadaanko mallilla aikaan neuroinflammatorinen vaste. Varsinaisen pitkän kokeen tarkoituksena oli selvittää aivojen dopamiinihermokasvutekijän (CDNF) mahdollinen neurorestoratiivinen vaikutus tässä mallissa. Alfasynukleiinin kasautumispatologian tasoa ja CDNF:n neurorestoratiivista vaikutusta selvitettiin käyttäytymiskokeilla sekä mustatumakkeen ja aivojuovion TH-vasta-ainevärjäyksillä. Yhdistelmämallista aiheutuvaa neuroinflammatorista vastetta selvitettiin ionisoidun kalsiumia sitovan adapterimolekyylin 1 (Iba1) ja gliaalisen fibrillaarisen happaman proteiinin (GFAP) vasta-ainevärjäyksillä. Pilottikokeen sylinterikokeessa yhdistelmämalli ei indusoinut liikehäiriötä, mutta pitkän kokeen askel- ja sylinterikokeessa mallin osoitettiin aiheuttavan unilateraalille leesiolle tyypillinen liikehäiriö. Pilottikokeen ja pitkän kokeen TH-vasta-ainevärjäyksissä mallin osoitettiin aiheuttavan TH-positiivisten dopamiinihermosolujen tuhoutumista mustatumakkeessa ja hermopäätteiden tuhoutumista aivojuoviossa. Nämä tulokset osoittavat, että yhdistelmämallilla saadaan aikaan alfasynukleiinin kasautumispatologiaa. Pilottikokeessa osoitettiin myös, että yhdistelmämallilla saadaan aikaan neuroinflammatorinen vaste, mikä osoittaa, että malli soveltuu hyvin uusien lääkkeiden vaikutuksen tutkimiseen Parkinsonin tautiin liittyvässä neuroinflammaatiossa. Pitkän kokeen sylinterikokeessa AAV-CDNF:llä ei ollut vaikutusta mallista aiheutuvaan liikehäiriöön. Sen sijaan askeltestissä kämmenen suunnan mittauksessa AAV-CDNF korjasi liikehäiriötä. AAV-CDNF ei kuitenkaan suojannut TH-positiivisia hermosoluja mustatumakkeessa tai hermopäätteitä aivojuoviossa, minkä perusteella johtopäätöstä CDNF:n neurorestoratiivisesta vaikutuksesta ei voida tehdä.
  • Tallberg, Thomas (2017)
    Transactive DNA Response Element Binding Protein 43 (TDP-43) is a RNA binding protein participating in gene expression on a transcriptional level. It is localized in the cell nucleus. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons. In most ALS patients TDP-43 becomes localized into the cytoplasm of neurons and glia cells. The TDP-43 rat ALS model provide insight in ALS disease progression and molecular mechanisms. This animal model has been characterized previously in the literature. Cerebral Dopamine Growth Factor (CDNF) is a neuroprotective and restorative protein in rat animal model of Parkinson's disease. CDNF may have an impact on disease progression in ALS. One of the goals in this work was to recharacterize the TDP-43 rat ALS model and to try repeat published data. The other aim of this work was to treat TDP-43 rats with intraventricular chronic infusion of CDNF, and to compare symptom progression with TDP-43 rats treated with phosphate buffered saline. Behavioral assays were done trice a week and when rats reached endpoint, spinal cords were removed. Motor neuron counting and detection of stress granule formation were investigated in spinal cords with immunohistochemistry. Also, the volume of CDNF diffusion in rat brain after chronic intraventricular CDNF infusion was investigated with immunohistochemistry. In the characterization part, symptom progression was repeated in a similar manner as it has been reported previously. CDNF treatment could not stop the symptom progression nor slow down the progression of symptoms in TDP-43 rats. Motor neuron counting revealed a heavy loss of motor neurons in the lumbal part of the spinal cord in both treatment groups. Diffusion of CDNF was very poor in the rat brain. Higher doses of CDNF and proper administration depth in the brain or route of administration should be reconsidered in the future.
  • Montonen, Ella (2015)
    Endoplasmic reticulum stress (ER-stress) is the result of accumulation of unfolded and misfolded proteins in the ER. The unfolded proteins activate the unfolded protein response (UPR), which seeks to reduce the protein load in the ER and reduces ER-stress. When ER-stress is prolonged, the UPR will activate apoptosis. Amyotrophic lateral sclerosis (ALS) is a rare, progressive neurodegenerative disease that affects lower and higher motorneurons. The cause of ALS is unknown but ER-stress is known to play a role in the disease progression. CDNF is a new neurotrophic factor, which is known to play a role in protein folding in the ER. CDNF is neuroprotective and neurorestorative in animal models of Parkinson's disease. Thus, CDNF is a potential new drug candidate for treating ALS. The aim of this work was to examine the effect of CDNF on disease state and life span in transgenic SOD1(G93A)-mice. CDNF or PBS was injected into the mouse's ventricle in stereotaxic surgery when the mice were about 90 days old. Clinical status and motor coordination was monitored twice a week throughout the study. The mice were dissected when they reached the end point that was set for the study. Deepfrozen gastrocnemius muscles were stained with antibodies, to examine the integrity of the neuromuscular junctions (NMJ). Quantitative PCR (qPCR) was executed on deepfrozen spinal cord and motor cortex samples to measure the expression of ER-stress genes. The results showed that CDNF improves motor coordination and delays disease progression in SOD1 female mice. The NMJs were notably more damaged in SOD1 mice than in wild type mice, but CDNF did not have any significant effect on NMJ integrity. ER-stress could be observed in the spinal cord and motor cortex of SOD1 mice and CDNF decreased ER-stress in the motor cortex. CDNF did not decrease ER-stress in the spinal cord where the expression of apoptosis related genes was increased. Thus, CDNF is a potential new drug candidate for treating ALS and it should be studied further.
  • Järvinen, Erkka (2016)
    UDP-glucuronosyltransferases (UGTs) catalyse glucuronidation reactions between glucuronic acid and drug molecules, which contain nucleophilic groups, mostly hydroxyls, amines or carboxylic acids. Glucuronidation is the most important reaction in the conjugative drug metabolism. Because these conjugates are not usually able to cross cell membranes passively, they need active efflux transport. Efflux transporters mostly belong to superfamily of ATP-binding cassette transporters (ABC). Subfamily C of ABC transporters (ABCC) are known to be involved in efflux transport of glucuronides. Especially MRP2 (ABCC2) and MRP3 (ABCC3) play key roles in the elimination of glucuronide conjugates of drugs. MRP2 is localized in the apical membranes of hepatocytes and enterocytes, whereas MRP3 is localized in the basolateral membranes of the respective cells. On the other hand, UGT1A1 and UGT2B7 are highly expressed in liver and small intestine and are the most important UGTs in drug metabolism. It is known, that UGTs and efflux transporters work together forming interplay to eliminate drugs. Therefore, studying both of them in the same in vitro system is in important focus of drug metabolism studies. The Madin Darby canine kidney cell line (MDCK) is one of the standard in vitro tools in drug metabolism studies. In this study, MDCK was chosen for a cell line to co-express UGTs (UGT1A1 or UGT2B7) and efflux transporters (MRP2 and MRP3 simultaneously. Therefore, cloning of the UGT2B7 cDNA and the ABCC3 cDNA encoding MRP3 was aimed in this study. On the other hand, the UGT1A1 cDNA was already cloned in-house and MRP2 expressing MDCK cells were established earlier. Cloning of the UGT2B7 cDNA was not successful in this study despite of several different strategies such as PCR-amplification of the cDNA fragment using kidney or liver sscDNA as template. Cloning of the ABCC3 cDNA encoding MRP3 was achieved and a mammalian expression vector containing this cDNA was constructed. In addition, the mammalian expression vector containing the UGT1A1 cDNA was used to establish MDCK-UGT1A1 cells and this cell line was characterized regarding the expression of UGT1A1 mRNA and UGT1A1 protein amount. Furthermore, establishment of MDCK-UGT1A1-MRP2 cell line was attempted in this study without success. The mammalian expression vector containing the ABCC3 cDNA encoding MRP3 could be used for future experiments to achieve novel cell lines such as MDCK-UGT1A1-MRP3 and MDCK-UGT1A1-MRP2-MRP3 for drug metabolism studies. In addition, the novel cell line MDCK-UGT1A1 could be used for drug metabolism studies in further experiments, but also as a cell line for further establishment of above cell lines. On the other hand, the cloning of the UGT2B7 cDNA needs optimization and several different strategies should be used to achieve the mammalian expression vector containing this cDNA.
  • Holvikari, Kira (2015)
    MRP2 is an efflux-transporter from the group of ABC-transporters located in the apical side of cell membranes mainly in the liver, intestine, kidneys and lungs. This transporter is associated with multidrug resistance, a phenomenon where the absorption of a drug to the cell is prevented by the transporter as it transports the compound out of the cell. To overcome this phenomenon, inhibitors and substrates for MRP2 are constantly studied. Several flavonoids have been presented of being inhibitors and the research of these compounds continues. Pharmaceutical excipients are also another major group of compounds that possess inhibitory effects towards MRP2. Excipients, as well as flavonoids, are an increasingly studied section of drug interactions and today it may be evaluated that excipients are not thought as inert compounds as has been presented for several years. For now the research of MRP2 interactions focuses mainly on in vitro studies. In the experimental part of this thesis the effects of natural compounds and pharmaceutical excipients are studied towards MRP2 with the vesicular transport assay (VT-assay) with MRP2- Spodoptera frugiperda 9 (Sf9)-membrane vesicles using 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF) as probe. A total of 157 compounds are screened using this in vitro method and hits are further experimented studying IC50 and Ki values. Potential compounds are also tested with two types of particle size measurements (Dynamic light scattering and nephelometer) to evaluate inhibition caused by microaggregates. Some compounds are also studied with liquid chromatograph-mass spectrometry (LC-MS) to determine possible substrates for MRP2. 19 (12%) hits were found from the library of 157 compounds. These hits included 6 stimulators (CDCF transport increased ≥ 150%) and 13 inhibitors (CDCF transport decreased ≤ 50%). IC50 determination was conducted for 12 inhibitors with best-fit values of: Ellagic acid 10.4 µM, gossypin 17.4 µM, morin dihydrate 19.4 µM, myricetin 27.1 µM, nordihydroguaiaretic acid (NDGA) 36.2 µM, octyl gallate 20.3 µM, silybin 52.3 µM, pluronic ®F98 6.9 µM, lutrol F127 ~ 8.2 µM and tannic acid 1.99 µM. Ki determination was conducted for 3 compounds where best-fit values were myricetin 42.9 ± 47.4 µM, gossypin 19.4 ± 12.5 µM and tannic acid 0.0538 ± 0.0398 µM. Ki determination allowed determination of inhibition type: competitive inhibition for tannic acid and gossypin, noncompetitive inhibition for myricetin. Particle sizes studied with dynamic light scattering (DLS) and a nephelometer did not show any significant aggregate formation and inhibition by that mechanism can be ruled out granted that the measurement method should be optimised. Stimulators baicalein, baicalin, digitoxigenin and inhibitors myricetin, gossypin and tannic acid were studied finally with the VT-assay with LC-MS as detector in search of substrates for MRP2. With significant changes in ‚àíATP and +ATP at 50 µM was gossypin. To conclude, gossypin possesses competitive inhibition towards MRP2 and exhibits sings of being a substrate for the transporter as well. Further studies need to be performed to confirm these findings.
  • Sainio, Janne (2011)
    Lactose is probably the most used tablet excipient in the field of pharmacy. Although lactose is thoroughly characterized and available in many different forms there is a need to find a replacer for lactose as a filler/binder in tablet formulations because it has some downsides. Melibiose is a relatively unknown disaccharide that has not been thoroughly characterized and not previously used as an excipient in tablets. Structurally melibiose is close to lactose as it is also formed from the same two monosaccharides, glucose and galactose. Aim of this research is to characterize and to study physicochemical properties of melibiose. Also the potential of melibiose to be used as pharmaceutical tablet excipient, even as a substitute for lactose is evaluated. Current knowledge about fundamentals of tableting and methods for determinating of deformation behavior and tabletability are reviewed. In this research Raman spectroscopy, X-ray powder diffraction (XRPD), near-infrared spectroscopy (NIR) and Fourier-transform infrared spectroscopy (FT-IR) were used to study differences between two melibiose batches purchased from two suppliers. In NIR and FT-IR measurements no difference between materials could be observed. XPRD and Raman however found differences between the two melibiose batches. Also the effects of moisture content and heating to material properties were studied and moisture content of materials seems to cause some differences. Thermal analytical methods, differential scanning calorimetry (DSC) and thermogravimetry (TG) were used to study thermal behaviour of melibiose and difference between materials was found. Other melibiose batch contains residual water which evaporates at higher temperatures causing the differences in thermal behaviour. Scanning electron microscopy images were used to evaluate particle size, particle shape and morphology. Bulk, tapped and true densities and flow properties of melibiose was measured. Particle size of the melibiose batches are quite different resulting causing differences in the flowability. Instrumented tableting machine and compression simulator were used to evaluate tableting properties of melbiose compared to α-lactose monohydrate. Heckel analysis and strain-rate sensitivity index were used to determine deformation mechanism of melibiose monohydrate in relation to α-lactose monohydrate during compaction. Melibiose seems to have similar deformation behaviour than α-lactose monohydrate. Melibiose is most likely fragmenting material. Melibiose has better compactibility than α - lactose monohydrate as it produces tablets with higher tensile strength with similar compression pressures. More compression studies are however needed to confirm these results because limitations of this study.
  • Mäki, Toni (2020)
    The human immune system can provide a powerful tool in developing therapies against various cancers. Even though the idea of an immune system actively searching for and disposing of potential mutated tumor cells is over a century old, only recent developments in various fields such as mass spectrometry, immuno-checkpoint blockade strategies and in silico modelling have enabled the realization of the full potential of recruiting immune system to fight cancer and the possibilities of personalized therapies. These therapeutic methods, including but not limited to oncolytic virus therapies, T-cell therapies and cancer vaccines, are based on the body’s ability to recognize mutated antigen peptides presented on the cell surface by MCH-receptors (also known as HLA-receptors in humans) and the disposal of the malignant cells by cytotoxic T-cells. Thus, the capability to map the individual HLA-presented peptidome and differentiate the immunogenic peptides is a foundation for this plethora of therapies and is in focus of ongoing research. This master thesis is a part of a project aiming to set up immunoaffinity-purification/MS based method in order to analyse the ligandome and determine T-cell recognized cancer associated antigens from tumor cells. Objectives of the work: 1. Characterizing tumor cell lines. 2. Immunological assay set up. 3. Collecting cell culture material for the ligandome affinity purification. 4. In silico prediction if the immunogenicity of selected peptides and assessing their source proteins. Methods used: 1. Cell culture. 2. FACS-analysis. 3. MTS-viability assay. 4. Immunological assays (ELISA, ELISPOT). 5. Immunological bioinformatics analysis tools (IEDB) and database search (UniPROT). Results: 1. Flow cytometric analysis provided essential information of the cell line HLA-1 expression. Additional information of PD-L1 expression can be used to evaluate cell line’s immune-evasion abilities. Preliminary MTS assay is used to determine linear range and optimal time frame for the PBMC/cancer cell co-culture killing assay. 2. Interferon γ cytokine secretion was determined by ELISPOT to assess PBMC response against known antigens in a preliminary experiment to approximate usable range for the following antigen specific PBMC assays. ELISA is used to confirm the presence of HLA-I receptors in the ligandome affinity purification eluates and to estimate the efficacy of purification. 3. Feasibility of in silico methods in the prediction of immunogenic peptides was explored. The experiments provided information that can be applied to the further development of the immune ligandome discovery project. In silico methods were successfully used to characterize previously identified HLA-restricted peptides and one previously identified immunogenic T-cell epitope. Even if the data acquired in silico can be considered only nominally verified at this stage, the results are encouraging.
  • Korpelainen, Anna (2019)
    Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease in which both upper and lower motor neurons degenerate gradually. The disease leads to a total paralysis of almost all skeletal muscles and to death within 3-5 years after onset. At the moment there are two disease modifying medicines available, riluzole and edaravone. Neither is able to cure the disease or even to stop or remarkably slow down its progression. Endoplasmic reticulum (ER) stress has been proposed as one of the pathophysiological mechanisms underlying ALS. During ER stress misfolded of unfolded proteins accumulate in ER lumen. As a defense mechanism, the cell launches unfolded protein response (UPR). UPR response aims to reduce the protein load in ER and restore cell’s normal functions. If the damage is already beyond repair, UPR signal cascades lead to programmed cell death. Neurotrophic factors (NTFs) regulate the growth of nervous tissue and participate in repairing processed. Many of the known NTFs have first seemed promising in the preclinical models of ALS but however failed in clinical trials. Cerebral dopamine neurotrophic factor (CDNF) differs drastically both in structure and function from conventional NTFs. CDNF has seen to relieve ER stress and improve motor behavior in the animal models of Parkinsons’s disease. Recently CDNF entered clinical trials in Parkinson’s patients. Since ER stress is believed to be present not only in ALS but also in Parkinson’s disease and other neurodegenerative diseases, it might have an effect in treating ALS patients. SOD1-G93A is a well-established animal model of ALS in which the animals show typical motor impairments comparable to human disease. In this study we used a novel mouse line obtained from crossing traditional SOD1-G93A model and CDNF knock out models. The study aimed to evaluate the effect of endogenic CDNF loss in survival, onset of symptoms, motor behavioral and spinal motor neuron degeneration in the new line. ER-stress and autophagy marker levels were studied with quantitative polymerase chain reaction (CNDF) and western blotting techniques. Spinal motor neuron loss was examined by anti-choline acetyltransferase antibody (ChAT) stainings. SOD1-G93A CDNF knock out animals were observed to have more severe motor impairments in the early stages of the disease compared to the traditional SOD1-G93A mice. In addition, the degeneration of spinal motor neurons appeared to be more severe in the new line. There were no statistically significant differences in ER stress between the genotypes although a trend of increased ER stress was observed. Endogenous CDNF loss had no effect on the healthy animals. The results suggest that CNDF is a potential treatment for ALS and it might have only little side effect since it does not seen to affect healthy tissue. In medical usage, CDNF might be most effective when administered immediately after disease onset. However, this might be difficult because of the challenges in ALS diagnosis.
  • Kylkilahti, Sanni (2022)
    Chilit ovat Capsicum-sukuun kuuluvia yleensä korkean kapsaisiinipitoisuuden omaavia paprikalajeja. Niitä käytetään mausteena. Lisäksi chilien sisältämillä kapsaisinoideilla on todettu olevan useita farmakologisia ominaisuuksia, kuten analgeettisia ja antioksidanttisia vaikutuksia. Niiden antimikrobisia ominaisuuksia on myös hieman tutkittu, mutta tutkimuksia on vielä verrattain vähän. Tämän työn tarkoituksena oli selvittää muutamien eri chililajikkeista valmistettujen uutteiden antimikrobisia vaikutuksia Escherichia colia ja Staphylococcus aureusta vastaan. Uutteet testattiin dimetyylisulfoksidiin (DMSO) ja veteen liuotettuina. Lisäksi testattiin myös kahden puhdasaineen, kapsiaatin ja solaniinin, vaikutuksia kyseisiä bakteereita vastaan. Antimikrobiakokeet suoritettiin 96-kuoppalevyllä noudattaen aseptisia työtapoja. Testattuja chiliuutteita oli 19. Uutteita valmistettiin eri chililajikkeiden versoista (1 kpl) siemenistä (3 kpl), lehdistä (10 kpl) ja hedelmistä (5 kpl). Dimetyylisulfoksidiin liuotetut uutteet testattiin pitoisuuksilla 2,0 mg/ml ja 4,0 mg/ml. Veteen liuotetut uutteet testattiin pitoisuudella 4,0 mg/ml. Solaniini- ja kapsiaattiuutteet testattiin kahdeksalla eri pitoisuudella (0,001172–0,15 μg/ml). Tutkimuksen tuloksena on, että testatut chiliuutteet eikä solaniini- ja kapsiaattiuutteet estäneet E. colin tai S. aureuksen kasvua. DMSO:iin liuotetuista uutteista korkeimmat estoprosentit kumpaakin bakteeria vastaan saatiin nuorilla Pimento-lehdillä. Veteen liuotetuista uutteista korkein estoprosentti E. colia vastaan saatiin Dulcen versoilla (30 % esto) ja S. aureusta vastaan Dulcen hedelmillä (50 % esto). Aiemmat tutkimustulokset chilien antimikrobisista vaikutuksista ovat ristiriitaisia, joten yhteneviä johtopäätöksiä chilien vaikutuksista bakteereihin ei voida tehdä. Johtopäätöksenä voidaan todeta, että chileillä on lukuisia terveysvaikutuksia. Antimikrobisen tehon varmistamiseksi tarvittaisi kuitenkin lisää tutkimuksia. Antibioottiresistenssi on maailmanlaajuinen ongelma, koska yhä useammat bakteerit ovat resistenttejä käytetyille antibiooteille. Tulevaisuudessa onkin erittäin tärkeää löytää uusia yhdisteitä bakteerien tappamiseksi, joten tutkimuksia uusien antimikrobisten aineiden löytämiseksi tarvitaan jatkuvasti lisää
  • Reijonen, Visa-Aleksi (2020)
    Making the treatment of these infections even harder is the fact, that Chlamydia pneumoniae can produce persistent forms of itself, which are immune to antibiotic treatment. When the bacteria sense a stress factor, for example the presence of a β-lactam antibiotic or interferon γ, they start producing these persistent forms called aberrant bodies. When the stress factor is removed, the bacteria can switch back to their replicating form and start infecting the tissues again. It is also known, that C. pneumoniae bacteria will trigger persistence when the bacteria migrate from lung epithelia into monocytes. Interestingly the onset of this mode of persistence does not require any other triggers besides the invasion of the monocyte. These persistence mechanisms enable latent, quiet, and recurring infections. This master’s thesis aimed to study the coculture of lung epithelial (HL cells) and monocytes (THP-1 cells), and by utilising the magnetic separation method presented by Kortesoja et al, to find a positive control compound in the prevention of Chlamydia pneumoniae internalisation into the THP-1 cells for said protocol. In these cocultures the inhibitory effect of different compound groups such as lignans present in Schisandra chinensis plant, MAPK-inhibitors, and β2,2-amino acid derivatives in C. pneumoniae migration from HL cells to THP-1 cells was assessed. Statistic relevance was observed in JNK inhibitor SP600125, MAPKAP-kinase-2 inhibitor SB203580, and ERK1/2 inhibitor FR180204 compounds. These compounds inhibited the internalisation of Chlamydia pneumoniae into THP-1 cells in the cell coculture by 61,05 ± 16,63 % (p = 0,0001), 54,06 ± 16,02 % (p = 0,0002), and 36,76 ± 10,33 % (p = 0,009) respectively. SP600125 and SB 203580 compounds also had an inhibitory effect on the internalisation of C. pneumoniae into the THP-1 cells in a cell monoculture (39,98 ± 18,92 %, p = 0,026 and 37,89 ± 19,47 %, p = 0,035 respectively), whereas FR180204 had no statistical significance, even though it inhibited the internalisation of C. pneumoniae into the THP-1 cells in cell monoculture by 27,53 ± 21,17 %. From the compounds used in the experiments, only MAPK inhibitors had an effect in inhibiting the C. pneumoniae internalisation into the THP-1 cells. The most potent compound in said inhibition was the JNK inhibitor SP600125. JNK pathway has been thought to take part in chlamydial infections but only little research has been done. The results of this master’s thesis’ experiments support the thought of JNK enzyme taking part in chlamydial infections but determining how exactly it affects the infection cycle of C. pneumoniae bacteria still needs further investigation.
  • Bäckström, Mia (2017)
    Background: Dexmedetomdine is a α2-adrenergic receptor agonist, which by binding to the α2-adrenergic receptor in the sympathetic nervous system exhibits sedative effect. Additionally, it has an analgesic and anxiolytic effect. Dexmedetomidine is registered as a sedative for use in the intensive care unit and in USA, additionally, in surgical settings. The study was conducted to characterize the pharmacokinetics in healthy volunteers through pharmacokinetic analysis methods. Methods: The clinical study was conducted on healthy 10 voluntary subjects each receiving dose of 1 µg/kg both intravenously (IV) and subcutaneously (SC). The study session lasted for 10 hours, with a wash-out period of at least 7 days between consecutive administrations. Arterial blood samples were taken to determine the plasma concentrations of dexmedetomidine. The pharmacokinetics of the IV and SC dose were determined by noncompartmental analysis (NCA) and, additionally, population modeling using nonlinear mixed effects model (NONMEM) was used to determine the pharmacokinetics of the IV dose. Results: The population's mean clearance after the IV dose was 40.0 L/h and for SC 45.6 L/h. The elimination half-life was 2 hours for IV, whereas terminal half-life was 9 hours for the SC dose. The SC bioavailability was 120 %. From the population modeling the typical elimination clearance, volume of distribution in central compartment, inter-compartmental clearance, and volume of distribution in the second compartment were 39.6 L/h, 13.7 L, 116 L/h, and 77 L, respectively. Conclussion: The obtained pharmacokinetic parameter values from NCA for IV were in line with the results from previous studies. For the SC dose the pharmacokinetic parameter values had high SD indicating high inter-individual variations. However, when the 8th subject was excluded from data analysis less SD was obtained and the result resembled more the results from other extravascular studies. The pharmacokinetic population results for IV dexmedetomidine were similar to previous studies on healthy subjects. Weight was used as a covariate, and was modeled by allometrically scaling the parameters. From the results it is shown that the covariate improved the model's goodness of fit.
  • Sipola, Kirsi (2021)
    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons in brain and spinal cord. The degeneration of motor neurons leads to muscle atrophy and paralysis. Currently there is no cure for ALS. Available drugs for ALS can lengthen the survival time by a couple of months. Several factors involve the pathophysiology of ALS, such as endoplasmic reticulum stress and neuroinflammation. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a protein which has shown neuroprotective effects on animal models of Parkinson disease and brain ischemia. C-terminal fragment of MANF can cross the blood-brain barrier, allowing it to be administered subcutaneously instead of injected directly into the brain. The experimental part consists of two parts. The aim of the first part was to study the pharmacokinetic properties of next generation MANF (C-MANF). The aim of the second part was to elucidate the effect of twice a week administered subcutaneous injection of C-MANF in genetic SOD1-G93A mouse model and its neuroprotective effects by assessing protection of lumbar motor neurons. Pharmacokinetic properties of C-MANF were determined in wild type mice after a single subcutaneous injection of C-MANF at different time points by using indirect ELISA assay. The effects of C-MANF in SOD1-G93A mouse model were assessed by subcutaneous injection of either C-MANF or PBS twice a week and by monitoring clinical score and motor behavior of mice from 10 weeks of age to clinical endpoint. Hematoxylin eosin staining was used to study neuroprotective effects of C-MANF. C-MANF administered subcutaneously is absorbed into the blood circulation and the highest serum concentration of C-MANF is after 60 minutes of dosing. Subcutaneously injected C-MANF also crosses the blood-brain barrier and reach the brain in 120 minutes. C-MANF did not preserve motor function or ameliorated ALS symptoms in SOD1-G93A mouse model. In this study C-MANF did not increase the survival of SOD1-G93A mice. C-MANF did not significantly protect motor neurons from degeneration even though there was a slight trend between the groups. No beneficial effects were observed with C-MANF in SOD1-G93A mouse model and therefore the dose and frequency of administration of C-MANF were not optimal. Subcutaneously injected C-MANF provides a safer dosing option for neurodegenerative disorders.
  • Cavonius, Karin (2021)
    Huntington’s disease (HD) is a rare but devastating neurodegenerative disease, progressively culminating in severe brain atrophy and death. The disease is caused by an inherited mutation resulting in a CAG trinucleotide repeat expansion in the huntingtin gene, leading to the production of a neurotoxic protein, known as mutant huntingtin, with an abnormally long polyglutamine stretch. Even though the genetic background of HD is known, the cellular pathways affected in the disease are complex and not completely understood. Increasing evidence indicates that endoplasmic reticulum (ER) stress – a condition of disturbances in normal ER activity, leading to accumulation and aggregation of misfolded proteins in the ER lumen – is a central factor in the pathogenesis of HD and other neurodegenerative diseases. In the literature review of this thesis, known pathogenic cellular mechanisms of HD and how these cellular mechanisms are connected to ER stress, are discussed. Unpublished data from previous studies in our laboratory have indicated that the ER luminal protein canopy homolog 2 (CNPY2) could play a role in the regulation of neuronal survival, including the viability of mutant huntingtin expressing neurons. The aim of the experimental part of this study was to gain insight into a possible function of CNPY2 in HD, by examining the levels of the protein in neuronal models of HD under various conditions, such as ER stress, and by searching for potential interacting partners of CNPY2 amongst known ER stress regulators. The obtained results show that the levels of CNPY2 are increased in striatal neurons expressing mutant huntingtin, and that the secretion of CNPY2 is increased by these neurons, compared to control neurons expressing normal huntingtin. Further, we show that CNPY2 interacts with the major ER stress regulator binding immunoglobulin protein (BiP) in human neuroblastoma cells treated with the ER stress inducer tunicamycin, and that the intracellular levels of CNPY2 are altered by tunicamycin treatment. Together, these findings indicate that CNPY2 could be involved in the pathogenesis of HD. However, further research on the functions of CNPY2 and its role in ER stress regulation is required to understand the nature of this involvement.
  • Nurmi, Kurt (2022)
    Viral promoters are an essential part of a normally functioning virus. Their main task is to drive the transcription of genes which govern hijacking of cell function and replication of viral particles. In addition to supporting normal function of a virus, they can be used to drive the transcription of transgenes which can be used in different therapies. In oncolytic therapies, transgenes can be used to prime the host system against neoplasms which has been shown to generate long term anti-tumour immunity. Human adenoviruses (Ad) are commonly used as a platform for oncolytic virotherapies. Human Ad’s replicate poorly in mouse tumour cell lines, yet some promoters, which are included in the viral constructs to drive the transcription of beneficial transgenes, are able to function. Currently it is unknown whether E3, the native promoter of adenovirus 5 of the E3 region, is capable of functioning in murine cell lines. In this thesis we study whether human cytomegalovirus promoter (CMV) and E3 differ in their efficacy to drive the transcription of the mOX40L and mCD40L transgenes. In the experimental part of this thesis, we compared the efficacies of two viral promoters, AdE3 and AdCVM, in transcribing mOX40Land mCD40L in vitro. Efficacy of transcription was assessed through immunofluorescence and flow cytometry in human and murine cell lines. Furthermore, the effects of promoters on viral infection, killing and replication were evaluated in burst assay and the colorimetric MTS proliferation assay. MTS and burst assay were conducted to confirm if viral infection, killing and replication occurs in human and murine cell lines. Both AdE3 and AdCMV were able to infect and kill human cell lines and cell viability decreased in correlation to the number of viral particles used. In murine cell lines, no decrease in cell viability was detected in the 4T1 cell line. In burst assay, viral replication was observed for both AdE3 and AdCMV in the human MDA-MB-436 cell line. In murine CT26 cell line, no replication was observed for AdE3 or AdCMV constructs. Immunofluorescence assay was performed to visualize transgene expression and localization. Results indicated that mOX40L was localized on cell surface while mCD40L was detected both outside and inside of the cytosolic compartment. Flow cytometry results revealed that both AdE3 and AdCMV constructs are capable of efficiently transcribing mOX40L in human cell lines. In the flow cytometry results for AdE3, two large cell populations with different fluorescence intensities were detected. AdCMV lacked this feature which is postulated to be due to higher lytic activity of the viral construct. In murine cell lines, HCMV could produce mOX40L, but production in murine cell lines was severely attenuated compared to human cell lines. mOX40L produced by the AdE3 construct did not differ from the baseline and was deemed incapable of producing mOX40L in murine cell lines. For the purpose of studying novel virotherapeutics the results of this thesis would indicate that human CMV can be used to drive expression of transgenes in murine cell lines. Despite this, it is preferable to use host specific viruses and promoter sequences for a better translation between mice and humans. Viruksen promoottorit ovat keskeisessä osassa toimintakykyisessä viruksessa. Virus promoottorin päätarkoituksena on geenien transkriptio, mitkä vastaavat solun keskeisten toimintojen kaappaamisesta ja virus partikkeleiden replikaatiosta. Näiden toimintojen lisäksi promoottoreita voidaan käyttää transgeenien transkriptiossa, mitä voidaan hyödyntää sairauksien hoidossa. Onkolyyttisissä terapioissa transgeenejä voidaan käyttää virittämään kehon immuunipuolustus taistelemaan kasvainkudosta vastaan. Ihmisen adenovirusta käytetään usein onkolyyttisten viroterapioiden alustana. Ihmisen adenovirus (Ad) replikoituu hyvin heikosti hiiren syöpäsoluissa, mutta osa adenovirukseen sisälletyistä eksogeenisistä promoottoreista, joita käytetään terapeuttisten transgeenien transkription ajamiseen, kykenee toimimaan ja tuottamaan haluttua proteiinia. Tällä hetkellä ei tiedetä, kykeneekö E3, joka on adenoviruksen E3 lokuksen promoottori, toimimaan hiiren solulinjoissa. Tässä tutkielmassa selvitämme ihmisen sytomegalovirus promoottorin (CMV) ja E3 eroa niiden tehossa ajaa mOX40L ja mCD40L transgeenien transkriptiota. Kokeellisessa osuudessa vertailimme kahden virus promoottorin, E3 ja CMV, eroa niiden tehossa ajaa mCD40L ja mOX40L transkriptiota in vitro. Transkription tehoa tutkittiin immunofluoresenssin ja virtaussytometrian avulla ihmisen ja hiiren syöpäsolulinjoissa. Tämän lisäksi promoottorien vaikutusta virus infektioon, replikaatioon ja kykyyn tappaa soluja arvioitiin burst kokeella ja kolorimetrisellä MTS menetelmällä. MTS ja burst kokeiden avulla varmistettiin AdE3 ja AdCMV virusten kyky infektoida, tappaa ja replikoitua ihmisen ja hiiren syöpäsolulinjoissa. Sekä Ad3 ja AdCMV todettiin kykenevän infektoimaan ja tappamaan ihmissyöpäsoluja ja solujen viabiliteetin lasku korreloi virus partikkeleiden määrän kanssa. Hiiren 4T1 syöpäsoluissa ei todettu solujen viabiliteetin laskevan. Burst kokeessa havaitsimme sekä AdE3 että AdCMV kykenevän replikoitumaan ihmisen MDA-MB-436 solulinjassa. Hiiren CT26 solulinjassa kummankaan viruksen ei havaittu kykenevän replikoitumaan. Immunofluoresenssi kokeessa visualisoimme transgeenien ilmentymisen ja paikantumisen. Tulokset osoittivat, että mOX40L paikantui solun pinnalle. mCD40L havaittiin puolestaan sekä solun ulkopuolella että sytosolissa. Virtaussytometria kokeen tulokset osoittivat, että sekä AdE3 ja AdCMV pystyivät tehokkaasti ilmentämään mOX40L ihmisen solulinjoissa. AdE3 virtausytometria tuloksissa löydettiin kaksi solupopulaatiota, joilla oli toisistaan poikkeavat fluoresenssi intensiteetit. Tätä ilmiötä ei havaittu AdCMV:lla infektoiduilla soluilla, mikä saattoi johtua korkeammasta lyyttisestä aktiivisuudesta. Hiirisolulinjoissa CMV kykeni ilmentämään mOX40L, mutta transkription teho oli selvästi alhaisempi verrattuna ihmissolulinjoihin. E3 promoottorin ilmentämä mOX40L ei eronnut kontrollista ja sen todettiin olevan kykenemätön tuottamaan mOX40L hiirisolulinjoissa. Tuloksemme osoittavat, että ihmisen CMV promoottori kykenee ilmentämään transgeenejä hiiren 4T1 ja CT26 solulinjoissa. On kuitenkin huomattava, että isäntälajille natiivien virusten ja promoottorien käyttö olisi tarkoituksenmukaisempaa tulosten käännettävyyden kannalta hiiristä ihmisiin.
  • Turku, Ainoleena (2010)
    The aims of this work were (1) to compare the three dimensional structures of different S- adenosylmethionine (SAM)-dependent methyltrasferases and (2) to screen in silico a commercial library for potential methyltransferase inhibitors. In this work we decided to focus on DNA methyltransferase-like enzyme (DNMT2) and catechol-O-methyltransferase(COMT). There were two different parts in my work. The first part was to analyze the 3Dstructures of DNMT2 and COMT in relation with their amino acid sequences. The structures of DNMT2 and COMT were compared together by means of superimposition with Sybyl 8. The ligand binding properties were studied by manual and automatic docking of known inhibitors in order to understand the binding specificity of these methyltransferases. The softwares I used for docking were Autodock 4.2 and Gold 4.0. The sequence alignments and superimposition of the known crystal structures showed that the structures of DNMT2 and COMT share a similar fold. Furthermore the main similarities between the structures of these enzymes are in the co-enzyme binding sites. The only significant difference in the binding sites is the place of one tyrosine residue, which causes a slight change in the conformation of the bound co-enzyme. Unlike co- enzyme binding sites, the substrate binding sites of DNMT2 and COMT are different. There is indeed a bound magnesium ion in the substrate binding site of COMT but not in the substrate binding site of DNMT2. Because the substrate binding sites are more different than the co-enzyme binding sites, we decided to screen the potential active ligands only at the substrate binding sites. The second part of the work was virtual screening. I used a subset of 20.000 molecules of ChemBridge DIVER Set that can be purchased commercially. The softwares I used for library preparation were CONCORD and Balloon, from which Balloon created more reasonable 3D structures for the docking. I did two parallel screenings to the crystal structure of COMT (PDB code 3BWM) with docking program GOLD 4.0, which is the only program that can take account metal coordination. To DNMT2 I did two sets of screenings, one with GOLD 4.0 and another with Autodock 4.2. I used known COMT inhibitors as control in the COMT run and known DNA methyltransferase inhibitors as control in DNMT2 run. Before docking to the three dimensional structure of DNMT2, one loop near the substrate binding site had to be modeled. I used Swiss-Modeler and Modeller softwares for that. Docking to COMT was successful according to the rank of the known COMT inhibitors compared to the subset of the FIMM library that was screened. I created the hitlist of 60 compounds based on the scores of these compounds, pharmacophore search and visual examination. 30 of these compounds were purchased and are currently being tested. The results of the DNMT2 run were not as reliable as the results of COMT run mentioned before, since the DNMT2 run was unable to retrieve known inhibitors better than random. The reason for that can be the quality of the model of the missing loop or the chosen controls. Furthermore only one of the ten small molecules that we used as controls is proved to be DNMT2 inhibitor, the others are DNMT1 and DNMT3 inhibitors and while the binding sites of DNMT1, DNMT2 and DNMT3 are very similar, they are, however, not completely identical.
  • Backman, Heidi (2020)
    Theoretical framework: The consolidated pharmaceutical market is becoming increasingly global and the same international pharmaceutical companies operate around the world in different countries, responsible for drug development and production. The high costs of developing novel medicines and the motive for higher profits has led to elevating price level of pharmaceuticals and health care services. Finland and the U.S. offer two extremes at the pharmaceutical market. The pharmaceutical market field in Finland is very structural and rigid, and medicine prices are regulated by law. In the U.S. the prices are based on the laws of supply and demand and the prices differ by different states, retailers and insurance policies. A small-scale longitudal price comparison is also reviewed to showcase the effect of continuously rising medicine prices. Study objective: The idea of this study is to describe and compare pricing mechanisms of pharmaceuticals and price differences between two very different market structures and review how these might affect the cost-effectiveness of national health care spending. These divergences are also mirrored to survey recent global pharmaceutical market problems such as drug shortages, possibly due to less appealing markets of higher price regulation policies. Materials and methods: Price data were collected from national, official, open-source databases. National health care expenditure and comparison to GDP was collected from publications by the OECD. All monetary values have been presented in both currencies (EUR and USD) to present more comparable values. Results: When compared to other OECD-countries the U.S. spent distinctly the largest amount of funds on health care per capita. Finland’s national health care costs were thousand times minor in total spending and less than a half per capita when compared to those of the U.S. With lower expenditure Finland manages to offer access to public, government-funded health insurance program. Meanwhile the prices of prescription medicines in Finland have decreased significantly, the prices for have continuously elevated in the U.S. Conclusions: The outcome of this study is that free markets and a complex supply chain, compared to more regulated markets with more transparency, have higher overall price level in pharmaceuticals and health care services. Free markets and sufficient intellectual property rights are more enticing to pharmaceutical companies. They promote new innovations and developing of much-needed novel therapies to modern health problems, such as AIDS and the global threat of worsening situation of antibiotic resistance. More regulated markets may create problems such as drug shortages and are often considered complex and less appealing market systems due to high level of administrative work but conserve the cost-effectiveness of the use of public funds.
  • Virtanen, Sonja (2020)
    Parenteral products are sterile products that are administered as injection, infusion or implantation. Administration of the contaminated parenteral product can cause severe consequences such as sepsis meningitis and even death. Most of the parenteral products used at the hospitals needs to be compounded (e.g. dissolved, diluted) before administration. Whenever possible, compounding should be done in biological safety cabinet using aseptic techniques. According to previous studies errors in aseptic techniques are quite common. Aim of this study was to compare three different environments as compounding area and their effect to the sterility of the compounded parenteral product. Based on the results of this study, changes to the protocols of the hospital could be made. Altogether 220 samples were compounded at two pediatric wards at HUS Helsinki University Hospital. Six volunteers (one pharmacist and five nurses) participated from both wards and each compounded 18 samples in three different environments (patient room, medicine room, biological safety cabinet). The samples were tested for the sterility by membrane filtration within 4 hours or after 24 hours of storage in the refrigerator. The investigator used an observation form to observe the compounding procedures. Environmental monitoring (settle plates) and monitoring of personnel (glove samples) were conducted. Almost all compounded samples (99%, n=213/215) were sterile. There were no significant differences in the contamination rate of the compounded samples between different environments. Five of the collected samples were excluded, because they were contaminated during the sterility test. According to observations, aseptic techniques were well followed. However, disinfection of the septum of the medicine bottle, hand hygiene and cleaning of the compounding area were observed to be deficiently completed. Even though there were lot of variation in the environmental and personnel monitoring the results were quite good. Results from the environmental monitoring were compared to the recommended limits of EU GMP for clean areas. One compounded sample was contaminated with Diezia maris and Corynebacterium mycetoides but the contaminants from the other contaminated sample could not be identified. Aseptic techniques were mainly well followed, however compounding should be done in the biological safety cabinet, since the environmental monitoring results show that the biological safety cabinet was only environment which was within the recommendation limits of the EU GMP for the compounding area of parenteral products. Protocols of the hospital could be changed, since there was no correlation between higher contamination rate of settle plates or compounded samples and not wearing mask and hair cover while compounding in the biological safety cabinet.
  • Karhu, Lasse (2012)
    The orexinergic system is a central regulator for sleep-wake rhythm and energy homeostasis. Dysfunction of the system is at least one of the reasons behind narcolepsy, in addition to which insomnia, obesity and certain cancers could be treated by targeting orexin receptors. The orexin system in human comprises two receptor subtypes, orexin receptor 1 and 2 (OX₁R and OX₂R respectively) as well as two cognate ligands, peptides orexin-A and -B. In this study the focus is on OX₁R and orexin-A. The aims of the study are (1) to propose a binding mode for orexin-A to OX₁R and (2) to understand the molecular interactions of OX₁R leading to receptor activation. I order to create 3D molecular models of OX₁R, a sequence alignment of the eight G proteincoupled receptors (GPCRs) that have been crystallized up to date was first generated by ClustalX and adjusted based on the superimposition by SYBYL-X. Structurally conserved regions were deduced from the alignment and used to add the orexin receptors. Five different models built with MODELLER were selected for their large binding cavity among a large pool of models. These models were constructed based on the chemokine receptor 4 (PDB Id:3ODU), as such and a modified version where TM3 was moved by 1 Å further from the center of the binding cavity, from the β₂-adrenoceptor (PDB Id: 2RH1) and from the adenosine receptor A2A (PDB Id: 2YD0), as such and with rotamer changes to few binding site residues. Orexin-A with straight conformation found by NMR (PDB Id:1WSO) was docked to these models using ZDOCK and RDOCK. In addition, an in-house docking protocol was implemented, but could not be validated. Docking poses were scored by purpose built knowledge based scoring function and clustered. High scoring clusters were then used to converge to three different binding modes. As a result, we suggest that the binding site of OX₁R consists of two hydrophobic walls, one from TM3 and TM5, the other from TM6 and TM7. Binding modes include a hydrogen bond network between the ligand and especially binding site residues Gln1263.32, Thr2235.46, Asn3186.55, Lys3216.58 and Tyr3117.43. Based on the binding modes, it is suggested that the OX₁R is activated by similar binding site contraction as β-adrenoceptors and adenosine A2A. The contraction in could result from the hydrogen bonds between ligand, Gln1263.32, Thr2235.46 and Asn3186.55. The hydrogen bonding of Thr2235.46 can also disrupt interactions between TM5 and TM3, an interaction which is identified as an important factor in keeping the receptor in the inactive state. The role of other ligand residues would be to direct ligand binding and keep the ligand in the helical conformation.