Farmasian tiedekunta

 

Recent Submissions

  • Mgbeahuruike, Eunice Ego (Hansaprint, 2019)
    Piper guineense is a medicinal plant that has wide application in African traditional medicine where it is often used in the treatment of bacterial and fungal infections. It is an economic plant with numerous health benefits which is also consumed regularly as a functional food. The fruits, leaves and seeds are used as spices and flavouring agents in commercial food preparations in West Africa. The extracts are also used for the treatment of various diseases ranging from diarrhea, intestinal diseases, rheumatoid arthritis, bronchitis, cough, stomach ache, asthma to febrile convulsions, fever and mental disorders. There is also recent interest on the biological and pharmacological properties of its bioactive compounds such as piperine, the main alkaloid constituents of P. guineense which is responsible for its pungent aroma. Based on these numerous ethnobotanical, traditional and economic uses of this plant, it became interesting to evaluate the bioactive compounds present in the extracts and to further screen the extracts against some selected human pathogenic bacterial and fungal strains so as to ascertain the efficacy of the extracts and its compounds as potent antibacterial and antifungal lead compounds. Microbial resistance to the currently available antibiotics is a global problem that has resulted to a constant search for a new antimicrobial drug with strong efficacy and low cost. There is need to screen the extracts and bioactive compounds from P. guineense for possible lead compounds for antibacterial and antifungal drug discovery. In this study, first, a method was developed for the chemical profiling, qualitative and quantitative analysis of P. guineense extracts and a good mobile phase composition was developed for the high performance liquid chromatography (HPLC) and thin layer chromatographic (TLC) analysis of the extracts. The effect of the chamber type on the separation was also evaluated using unsaturated horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber. Furthermore, the in vitro antibacterial activity of the extracts were evaluated using 8 pathogenic Gram-positive and Gram-negative bacterial strains. An ethnobotanical survey was also conducted on the use of P. guineense extracts in the treatment of fungal infections in West African traditional medicine. The study area was chosen to be Imo state, South Eastern Nigeria were P. guineense is mostly used by traditional healers for the treatment of fungal infections which is often common among those suffering from HIV and AIDS. The aim of the survey was to document the various methods of preparations and administrations of these extracts for the treatment of fungal diseases. From this ethnobotanical approach, the leaves and fruits extracts of the plant was further tested against 5 fungal strains including Cryptococcus neoformans which causes meningitis in immunocompromised individuals. HPLC and TLC methods were developed for the analysis of P. guineense extracts with emphasis on the shortest analysis time and minimal solvent consumption, and the best mobile phase giving favourable resolution of bands was found to be toluene: ethyl acetate (PS 6-4 corresponding to 60:40 % v/v). The result of the TLC analysis showed that the developing chamber conditions does not affect the TLC separation efficacy in the analysis of P. guineense extracts. The extracts were active against the tested bacterial and fungal strains with minimum inhibitory concentration (MIC) values ranging from 19 to 2500 µg/mL.
  • Fontana, Flavia (Helsingin yliopisto, 2019)
    Fontana F., 2019. Biohybrid Cloaked Nanovaccines for Immunotherapy Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis, 47/2019, pp.78 ISBN 987-951-51-5286-2 (Paperback), ISBN 978-951-51-5287-9 (PDF, http://ethesis.helsinki.fi), ISSN 2342-3161 Immunotherapy is revolutionizing cancer treatment achieving durable and long-term responses in patients. However, only subsets of patients treated experience a positive outcome, due to immunotherapeutic resistance. Combinations of immunotherapeutics can overcome the drug resistance; the administration of a cancer vaccine or an oncolytic virus followed by immune checkpoint inhibitors is under investigation. Thereby, there is an unmet need for powerful, yet safe vaccines. Nanoparticles, in particular porous silicon nanoparticles, present ideal characteristics to formulate nanovaccines, thanks to their size-specific targeting to the lymphoid organs, to their intrinsic adjuvant effect, and to the possibility to simultaneously load adjuvants and antigens. Moreover, biohybrid cell membrane technology has been proposed as an innovative antigenic source. Thus, the aims of the current thesis were to develop a biohybrid multistage nanovaccine formulation and to evaluate its anticancer efficacy in murine tumor models. Firstly, the parameters affecting the formulation of the biohybrid nanosystems were assessed, along with the elucidation of the influence of the cell membrane coating on the colloidal stability in physiological conditions and on the biocompatibility in different cell types. Secondly, the effect of the cell membrane-wrapping on the cellular uptake was evaluated in the presence of inhibitors of selective uptake pathways, to assess the differences between naked and coated nanoparticles. Then, a multistage nanovaccine was engineered by glass capillary microfluidics, followed by the cloaking with the cell membrane. The immunological profile of the nanovaccine was investigated in vitro, assessing the expression of co-stimulatory signals and the secretion of proinflammatory cytokines. The efficacy of the biohybrid nanovaccine as a monotherapy and in combination with an immune checkpoint inhibitor was then evaluated in melanoma murine models. Finally, the adjuvant core was changed from synthetic nanoparticles to oncolytic adenoviruses to investigate the translatability of the technique, the influence of the cell membrane-coating on the viral infectivity, and the preventive and therapeutic efficacy of the vaccine in different tumor models. Overall, porous silicon and adenovirus-based biohybrid nanovaccines were developed, providing new insights on the structure and efficacy of these systems as therapeutic cancer nanovaccines.
  • Julku, Ulrika (Helsingin yliopisto, 2019)
    Dopamine is one of the main neurotransmitters in the brain. Dopaminergic signalling regulates reward, memory, attention and motor functions. In the synapses of dopaminergic neurons, dopamine transporter (DAT) re-uptakes dopamine into the presynaptic nerve terminals after dopamine release terminating the dopaminergic signal and acting as one of the main regulators for kinetics of dopaminergic neurotransmission. Loss of dopaminergic neurons in the nigrostriatal pathway and protein aggregates called Lewy bodies are the main pathological findings in Parkinson´s disease. Lewy bodies are mainly composed of a protein called α-synuclein. The physiological role of α-synuclein has remained unclear but it has been suggested that the main function is regulation of dopaminergic neurotransmission since α-synuclein has been shown to participate in the regulation of dopamine synthesis, storage, release, and metabolism. α-synuclein-regulated functions in dopaminergic signaling are described in the literature review of this thesis. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and induces its aggregation. PREP inhibitors have beneficial effects in cellular and in vivo models of Parkinson´s disease by reducing α-synuclein aggregates and oligomers, and improving motor functions. Additionally, PREP inhibitors alter striatal dopamine level in mice and rats, and decrease immunoreactive DAT in the mouse striatum suggesting that PREP could have an effect on dopaminergic function. The aim of this study was to characterize the role of PREP in dopaminergic signaling and the effect of α-synuclein in PREP-mediated changes of the dopaminergic system. In the first study, the effect of PREP and α-synuclein on DAT phosphorylation and function was studied in DAT transfected HEK-293 cells. PREP altered DAT function and dopamine uptake, but the changes were not dependent on ERK phosphorylation or PKC activity. α-synclein had an effect on DAT phosphorylation in the absence of PREP but this was also independent of phosphorylation of ERK indicating that both α-synuclein and PREP are able to modulate DAT function via an ERK¬-independent mechanism. In the second study, the role of PREP in dopaminergic signaling was characterized in the nigrostriatal pathway of mouse. The influence of PREP was investigated by comparing the dopaminergic function of PREP knock-out mice and wild-type littermates. Lack of PREP elevated extracellular dopamine concentration, delayed re-uptake of dopamine, and increased phosphorylation of DAT in the mouse striatum indicating that PREP is able to regulate DAT function by modulating phosphorylation and localization of DAT. The effect of PREP inhibition on dopaminergic function, behavior, and α-synuclein in a Parkinson´s disease mouse model was investigated in the third study. Overexpression of α-synuclein was induced by supranigral microinjection of AAV-α-synuclein and mice were treated with the PREP inhibitor KYP-2047 after the onset of the behavioral symptoms. KYP-2047 treatment did not restore α-synuclein-induced reduction in striatal dopamine but behavioral improvement and reduction in α-synuclein oligomers indicated restoration of dopamine release and recycling. The aim of the fourth study was to investigate if α-synuclein-induced toxicity in the nigrostriatal pathway is dependent on PREP expression. The main finding was that α-synuclein toxicity was reduced in the absence of PREP and restoration of PREP expression increased toxicity in the behavioral tests. However, nigrostriatal dopamine level was not affected suggesting that lack of PREP protects dopamine release and recycling from α-synuclein-induced toxicity. In conclusion, PREP regulates DAT function in cells and in the mouse nigrostriatal pathway, but the mechanism is not dependent on ERK and PKC activation. Deletion of PREP or PREP inhibition do not have effects on α-synuclein-induced dopaminergic cell loss, but they are able to restore behavior and dopaminergic function in the mouse brain suggesting that PREP inhibitors could provide a novel treatment for Parkinson´s disease.
  • Albert, Katrina (Helsingin yliopisto, 2019)
    The neurodegenerative disorder Parkinson’s disease is diagnosed when motor symptoms appear, which is caused by death of the substantia nigra dopamine neurons. Most disease cases are idiopathic, and there are currently no disease-modifying therapies. Since the mechanism underlying Parkinson’s disease is still unknown, bringing treatments to the clinic has been difficult. Alpha-synuclein (α-syn) is a protein found abundantly in the central nervous system of vertebrates. Its importance for Parkinson’s disease was confirmed when it was discovered that mutations in the gene led to an autosomal dominant disease form and that it is the majority protein in what is considered a pathological marker of the disease, Lewy bodies. Cerebral dopamine neurotrophic factor (CDNF) is a conserved protein with neurotrophic-like properties. It has been shown to protect dopamine neurons in toxin models of Parkinson’s disease and is currently in Phase I/II clinical trials. It has not been tested in α-syn animal models and therefore the aim was to model α-syn-based Parkinson’s disease and to test whether CDNF can intervene with α-syn aggregation and has a therapeutic effect. We generated two models that used α-syn to model sporadic Parkinson’s disease and test CDNF on: adeno-associated virus (AAV) and preformed fibrils. We used an AAV to overexpress human wild-type α-syn and were able to model nigrostriatal dopamine loss accompanied by behavioural deficits. However, the variation in the success of the model was too high to consider it feasible to test CDNF on. This, combined with concerns about controls, led us to conclude that it may not be an ideal model of sporadic Parkinson’s disease. Using a preformed α-syn fibrils model to seed endogenous α-syn, we observed modest behavioural deficits that were ameliorated by CDNF, however the model did not result in dopamine neuron loss with the measures used. Although, we were able to model the spreading of Lewy body- and neurite-like inclusions that were positive for phosphorylated α-syn. From parallel in vitro studies we can conclude that CDNF is affecting the preformed α-syn fibrils model, but further studies are needed to clarify this. Since CDNF has been successful in the 6-hydroxydopamine (6-OHDA) model after striatal injection, we tested injection to the substantia nigra and characterized the injection in naïve rats to further study CDNF. We expected similar effects of CDNF on dopamine neurons and behaviour using nigral injection, however issues with the injection paradigm and that CDNF was given as a single injection meant only minor behavioural effects and no restoration of dopamine neurons. Though when CDNF was injected to the substantia nigra of naïve rats, it was not transported to the striatum, but rather diffused around the midbrain. Lastly, we used a proteasomal inhibitor, the lactacystin toxin. When lactacystin was injected we observed a buildup of α-syn, nigrostriatal dopamine loss, neuroinflammation, and mild behavioural deficits. In general, this was repeated successfully and could be used for therapeutic studies. In conclusion, we used four different methods to model Parkinson’s disease to varying degrees of success in order to test CDNF. Our results indicate the importance of having proper controls and outcome measures. Additionally, we had success in modeling the progressive spreading of Lewy-like pathology, a phenomenon that is occurring in Parkinson’s disease. Notably, CDNF had some success and future studies will explore this further.
  • Kohtala, Samuel (Helsingin yliopisto, 2019)
    Major depressive disorder is a common and devastating psychiatric disorder. While pharmacotherapy and psychotherapy can be effective, a significant proportion of patients remain treatment resistant. Traditional antidepressants need to be taken for several weeks or months before the therapeutic effects become evident. For treatment-resistant patients, electroconvulsive therapy (ECT) is still the most effective treatment. Postictal slowing of electroencephalogram (EEG) activity has been associated with the therapeutic effects of ECT, but the mechanistic basis of this remains poorly studied. For decades this has encouraged researchers to investigate the antidepressant effects of isoflurane anesthesia with promising, but inconsistent, results. More recently, evidence of the rapid-acting antidepressant effects of subanesthetic doses of ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist and a dissociative anesthetic, has sparked a renewed interest in the development of novel antidepressant therapies. Another treatment to show positive results is nitrous oxide (N2O), a gaseous anesthetic with NMDAR antagonist properties. One of the proposed mechanisms of ketamine’s action is related to its ability to increase glutamatergic signaling, leading to further changes in synaptic potentiation and in the function of neuronal networks. These changes have been suggested to involve the actions of brain-derived neurotrophic factor (BDNF) signaling via its receptor TrkB. Downstream of TrkB, the inhibition of glycogen synthase kinase 3β (GSK3β), the induction of mammalian target of rapamycin (mTOR) mediated protein synthesis, and the consolidation of synaptic changes have been implicated in ketamine´s actions. The first aim of this study is to investigate the molecular changes induced by isoflurane anesthesia in the adult mouse hippocampus using phosphoproteomics in the absence of a priori information. We find that brief isoflurane anesthesia induces 318 phosphorylation changes in a total of 237 proteins. While confirming the phosphorylation alterations on selected proteins, we also discover that various anesthetics, including urethane and ketamine, regulate these targets in a similar manner. In the second part, we investigate the effects of N2O on molecular signatures implicated in ketamine’s action. Findings reveal that N2O produces cortical excitation, followed by the rebound emergence of slow EEG activity following gas cessation, which coincide with the phosphorylation of TrkB, GSK3β and p70S6k (a kinase downstream of mTor). Moreover, we demonstrate that these pathways become regulated during the postictal period after flurothyl-induced seizures or during slow EEG activity induced by hypnotic agent medetomidine. Notably, medetomidine is not effective in the learned helplessness test. Finally, we investigate the dose-dependent changes induced by ketamine in TrkB signaling. An acute administration of sedative-anesthetic doses of ketamine, accompanied by increases in slow EEG activity, is found to increase the phosphorylation of the investigated pathways. These changes appear independent of ketamine’s metabolite hydroxynorketamine, an agent shown to have antidepressant-like behavioral effects in rodents.
  • Leino, Sakari (Helsingin yliopisto, 2019)
    No cure exists for Parkinson’s disease (PD), a disease marked by the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNC), a loss of dopamine in the dorsal striatum, and resulting motor symptoms. Furthermore, treatment of PD with levodopa is often complicated by abnormal involuntary movements (levodopa-induced dyskinesia, LID). Novel treatment options for PD and LID are thus greatly needed. Nicotinic acetylcholine receptors represent one possible novel treatment target, given the complex control they exert over dopaminergic neurotransmission, protective effects of smoking against PD, and extensive preclinical evidence of neuroprotective and antidyskinetic effects by nicotinic receptor ligands. Nicotinic receptor subtypes essential for nigrostriatal dopaminergic neurotransmission include those containing the α5 subunit, which have not been previously studied in the context of PD. In this thesis, further preclinical investigations of the role of nicotinic receptors in PD and LID were carried out. An extensive in vivo and ex vivo characterization of the role of α5-containing receptors in mouse models of PD was performed. The effects on LID by chronic nicotine treatment in drinking water and other drug treatments were studied in vivo utilizing mouse models of both moderate and severe PD and LID. The mechanisms of action underlying LID and the antidyskinetic effects of nicotine were studied by ex vivo measurements of striatal dopamine release and corticostriatal brain-derived neurotrophic factor (BDNF). In parallel, methods for stereotactic surgery and postoperative care were significantly improved. Mice lacking α5-containing nicotinic receptors were found to be less susceptible to unilateral nigrostriatal neurodegeneration, the resulting interhemispheric motor imbalance, and LID. Striatal dopamine uptake measurements suggested reduced dopamine transporter function as a possible mechanism of neuroprotection. Nicotine was found to inhibit LID, with findings suggesting a role for α6β2* nicotinic receptors. However, neither nicotinic receptor agonists nor the clinically used drug amantadine alleviated severe LID associated with near-total dopaminergic denervation. The findings also confirmed a correlation between striatal BDNF and LID. The present findings suggest the potential usability of α5-containing nicotinic receptors as a drug target against PD and LID. The findings also confirm the preclinical potential of nicotine as an antidyskinetic drug while suggesting limited efficacy in advanced PD. In addition, the findings expand previous knowledge on the possible mechanisms of LID and the antidyskinetic effects of nicotine.
  • Deng, Feng (Helsingin yliopisto, 2019)
    Breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2), and permeability glycoprotein (P-gp, also known as multidrug resistance protein 1 or MDR1) are all well-studied efflux drug transporters that are expressed in the cells of many physiological barriers, where they restrict the entry and facilitate the elimination of various harmful compounds. Their substrates are chemically diverse, including drugs, dietary compounds, and endogenous metabolites. Due to their protective nature, a disruption of their transport function, by drug-drug interaction for instance, may lead to an altered exposure and adverse effects of their substrates. Therefore, it is necessary to evaluate transporter inhibition early on in drug development. Vesicular transport (VT) assay is a widely used in vitro technique in substrate and inhibition studies. In this thesis, the optimization of the VT assay with the addition of albumin was explored. Up to a two-fold improvement in transport activity was observed, but in relation to many sources of variability that affect the VT assay, such as interlaboratory variability and addition of cholesterol in membrane vesicles, the improvement was rather modest and likely insignificant for IVIVE. While the inhibition of drug transporters by food is well reported, less is known about food additives that are abundant in all processed food. A set of 26 food additives, including colorants, preservatives, and sweeteners, was examined using the VT assay for drug transporter inhibition. The MRP2 and BCRP, in particular, were strongly inhibited by the azo dyes used as colorants. The results indicated that food additives may have an impact on the bioavailability of oral medications but further whole cells studies are required to confirm the impact of these findings. Although numerous drug transporter inhibitors are known, the structural and physicochemical basis of inhibition remains generally undiscovered. The structure-activity relationship (SAR) of 114 compounds, divided into 5 subgroups, were studied using the vesicular transport assay in order to identify important structural features of MRP2 inhibition. In addition, pharmacophore models for each subgroup were also developed. The SAR of BCRP, MRP2, and P-gp inhibition was further studied with a smaller set of compounds and computational docking. The results suggested that anionic charge and halogen substitution were beneficial but not required for MRP2 inhibition. On the other hand, while lipophilicity increased P-gp inhibition, the negative charge and halogen substitution were detrimental for it. In addition, docking scores were found to have a good correlation with in vitro activity in certain subgroups and single protein residue interactions important for inhibition were identified.
  • Troberg, Johanna (Helsingin yliopisto, 2019)
    UDP-glucuronosyltransferases (UGTs) are a group of important conjugation enzymes that transfer a sugar moiety from UDP-glucuronic acid onto a nucleophilic group in substrate compounds. This glucuronidation reaction converts compounds into more water-soluble forms, and thereby enhances their excretion into bile, feces or urine. Frequently prescribed drugs are often eliminated through glucuronidation. Normally, several of the 19 human UGTs may participate in the glucuronidation of a certain drug. Occasionally, however, the formed glucuronide originates from the activity of just one particular UGT. Therefore, the published papers in this thesis aimed not only to determine the overall formation of glucuronides, but to characterize the activity of individual UGTs. The experimental design was to express UGTs as recombinant proteins in insect cells and to test their individual activities with several drugs or probe substrates. In some publications, results with recombinant UGTs were compared to results from glucuronidation activity in tissue microsomes. The results revealed that UGT1A4-P24T polymorphism affects the signal sequence cleavage and the length of the mature protein. The observed kinetic behavior suggests that the variant enzyme consists of active and inactive forms of UGT1A4. Similarly, an inactive form of an enzyme could explain the difference in activities between the commercial UGT1A10 and our highly active UGT1A10. The results indicated that the role of UGT1A10 in the small intestine is significantly more substantial than previously thought. Activity of UGT1A10 was also investigated in another article, which identified UGTs that glucuronidate two structurally similar environmental toxins, bisphenols S and F. Bisphenol S is a good substrate for UGT1A9, whereas bisphenol F is a better substrate for the highly homologous UGT1A10. Catalytic differences between many UGTs were studied in the presence or absence of bovine serum albumin and differences were found in a compound-dependent manner. Difference in activities of dog and human UGT1As was the subject of another paper. Results revealed that, perhaps with the exception of UGT1A6, human and dog UGT1A counterparts do not exist. Consistently, in liver and intestinal microsomes, there were large differences between dog and human in glucuronidation rates for a set of test substrates.
  • Svarcbahs, Reinis (Helsingin yliopisto, 2019)
    Neurodegenerative disorders are characterized by accumulation of toxic protein species that are followed by a gradual loss of neurons in certain brain regions and person’s loss of movement and dementia. The cause of the protein accumulation is not fully understood but is partially influenced by the disturbances in the protein degradation pathways, post-translational protein modifications that facilitate either gain-of-toxicity or loss-of-function of these proteins. In Parkinson’s disease, the best-known aggregation prone protein is alpha-synuclein (aSyn) that is the main component of Lewy bodies, the histopathological hallmarks of Parkinson’s disease and other synucleinopathies. Several studies suggest that aSyn aggregates can damage neurons by various mechanisms, and propagate toxicity by cell-to-cell transfer thus making it a tempting target for drug therapy. Current drug therapies can only relieve symptoms of neurodegenerative diseases but do not address, for example, protein aggregate clearance or pharmacological deceleration of the inclusion formation. In previous studies, prolyl oligopeptidase (PREP) has been shown to enhance the aggregation of aSyn. PREP inhibitors have been shown to reduce the aggregation and increase the clearance of aggregates via enhanced autophagy. However, the mechanisms of how PREP affects aSyn aggregation and regulates autophagy, and if this has a long-term impact on aSyn toxicity, have not been studied. The aim of this study was to investigate the role of PREP deletion, restoration, overexpression, and catalytical inhibition on the cellular signaling pathways and aSyn aggregation. The first part of the work was done in PREP knockout cells and knockout mice where the aSyn protein was overexpressed alone or together with PREP. We showed that absence of PREP decreases aSyn-overexpression mediated behavioral and cellular toxicity in mouse brain. Additionally, we found that PREP knockout cells exhibit reduced stress response and toxicity in the presence of protein overload, have increased autophagic activity, and remove excess aSyn into the cell media. The second part studied effects of chronic PREP inhibition by KYP-2047 on aSyn aggregation and on motor disturbances in the aSyn viral vector overexpression PD mouse model. The main finding showed that after chronic PREP inhibition, animals lost pathological unilateral motor behavior due to reduction in aSyn oligomer species in the nigrostriatal pathway. The third part concentrated on mapping the role of PREP in the pathways responsible for the autophagy initiation. The main finding was discovery of PREP’s role in negatively regulating one of the most important protein phosphatase complexes, protein phosphatase 2A (PP2A), via direct protein-protein interaction. Besides, this interaction could be altered with PREP inhibitor treatment that resulted in upregulation of PP2A activity and explained the functional results of autophagy induction after PREP inhibition. In summary, the findings of this study underline mechanisms through which PREP might be mediating aSyn related pathology and underlines the potential of PREP inhibition as an attractive drug target in reducing aSyn aggregate formation and boosting clearance from the affected brains. PREP involvement in the PP2A network regulation and additional functional data warrants further PREP investigation in the context of other neurodegenerative disorders and PP2A-related ailments.
  • Tähkä, Sari (Helsingin yliopisto, 2019)
    Microfluidic devices have been noticed potential platforms for bioanalytical research. The small size enables rapid analyses, reduced sample and reagent consumption and microtechnology allow integration of multiple analytical units on-chip. Despite the progress made in the past years, the current materials for these devices have challenges with the cost and/or mass production possibilities, unit compatibility and reproducible performance. Thiol-ene polymers have been proposed as alternative materials for microsystems to address the above-mentioned challenges. The aim of this thesis was to study the feasibility of thiol-enes for out-of-cleanroom replica molding of analytical devices. Thiol-ene photopolymerization was studied in the absence of a photoinitiator. Without the initiator the bulk properties were noticed relatively similar compared with the initiated-thiol-enes, but the curing time was shown to have a notable impact on the surface properties in the absence of the initiator. This allowed control over the surface chemistry and bonding of even two alike thiol-ene layers. These obtained properties of thiol-enes were utilized in enzyme microreactor, in microchip electrophoresis (MCE), and in mass spectrometric (MS) applications. Proteolytic peptide digestion was obtained with immobilized enzymes on the microreactor. Straightforward enzyme immobilization was developed by the use of thiol-rich surfaces and gold nanoparticles via thiol-gold chemistry. In MCE, the controllability of the surface properties allowed production of devices with stable and high electroosmotic flow. Owing to the good solvent stability of thiol-enes, MCE separations could be conducted even in non-aqueous media with enhanced resolving power. For online MS applications, the microdevices were integrated with on-chip electrospray tip. With optimized polymer composition, stable electrospray and good chip-to-chip reproducibility was achieved. The tunable surface chemistry allowed development of inert surfaces by polyethylene glycol coating to fully eliminate nonspecific surface interactions. This thesis work increases our understanding of the material and surface properties of thiol-enes with a view to their use in mass spectrometry based bioanalysis as well as in other fields of microfluidics. The impact of the work increases the customization possibilities in low-cost microfabrication.
  • Kolsi, Laura (Helsingin yliopisto, 2019)
    Natural products and their derivatives are excellent starting points in drug design and widely studied as promiscuous anticancer agents. Moreover, they represent a big part of the current anticancer drugs in clinical use. Pancreatic cancer is one of the most fatal cancers with an extremely low five-year survival rate and increasing incidence. This cancer is particularly hard to target because of the huge number of genetic mutations per cancer demonstrating an urgent need for new multi-target treatment strategies. The tumor microenvironment and inflammation greatly promote tumor development and metastization. Chronic inflammation is, indeed, generally recognized as one of the hallmarks of cancer and offers a great target for drug development. This thesis focuses on the semisynthesis of abietane-type diterpenoids and studies about their anticancer and anti-inflammatory activities. The development of novel methods focusing on catalysis and sustainability within diterpenoid chemistry was also an objective of this thesis. Dehydroabietic acid, the starting material for these studies, exists in the rosin of coniferous trees and exhibits a wide range of biological activities including anticancer and anti-inflammatory activity. Moreover, due to its commercial availability, it provides a good and inexpensive starting material for the semisynthesis of complex naturally occurring abietanes and novel diterpenoids aiming towards improved bioactivities. Benzylic oxidation of aromatic abietanes is a key chemical transformation for the functionalization of the abietane core. To replace the environmentally hazardous and noxius chromium(VI) reagents, widely used for this oxidation, we studied sodium chlorite in combination with aqueous tert-butyl hydroperoxide. This method enables the preparation of 7-oxo, 7-oxo-15-hydroperoxy and 7-oxo-15-hydroxy derivatives of various aromatic abietanes. For 12-substituted aromatic abietanes the method is regioselective providing the 7-oxo products in good yields. A short semisynthesis is possible with this method to obtain the naturally occurring picealactones A, B and C which exist in the heartwood of Picea morrisonicola Hayata and represent potential compounds for the treatment of endocrine cancers. A 13-propenyl side chain exists in naturally occurring aromatic abietanes such as angustanoic acid E and aquilarabietic acid H, the former possessing antiviral and anti-inflammatory activities and the latter expressing antidepressant activity in vitro. Previous studies report the use of halogenated reagents and hazardous solvents such as benzene for the incorporation of this moeity onto aromatic abietanes. In our studies, bismuth(III) triflate was identified as the most efficient catalyst to perform the dehydration of tertiary alcohols to access this chemical entity, with high yields and good selectivity. Furthermore, by modifying the reaction conditions, namely solvent and temperature, dimerization and cyclization occurred. Expansion of the method to cover other non-terpenoid compounds showed that the method is applicable to compounds from different chemical classes. In addition, it enables a short semisynthesis of natural products from Pinus massoniana Lamb, presented for the first time in this work. Overall, this thesis resulted in the synthesis of 41 new dehydroabietic acid derivatives providing new information for both diterpenoid chemistry and biology. Novel dehydroabietic acid derivatives were tested against pancreatic cancer and inflammation. In vitro studies revealed that dehydroabietic oximes inhibited the growth of pancreatic cancer cells and nitric oxide production with IC50 values in the low micromolar range. In addition, they were able to induce cancer cell differentiation as well as downregulate cyclin D1 expression with upregulation of p27 levels, consistent with cell cycle arrest at the G1 phase. Furthermore, according to a kinase profiling study, one of the oximes showed potential in selectively inhibiting p90 ribosomal S6 kinase 2 (RSK2), an AGC kinase involved in cellular invasion and metastasis.
  • Välimäki, Mika (Helsingin yliopisto, 2018)
    Acute myocardial infarction is a life-threatening condition that occurs as a result of reduced blood flow in the cardiac muscle, eventually leading to tissue damage. In infarcted areas, cardiomyocytes have insufficient ability to proliferate and replace the injured cells, which is associated with a deficient pumping capacity. A strictly regulated combinatorial interplay of transcription factors, e.g., GATA4, NKX2-5, TBX5, and MEF2C, orchestrates cardiac type gene expression during the cardiomyocyte differentiation and maturation processes. The aim of the present study was to (i) characterize the protein-protein interaction of the cardiac transcription factors GATA4-NKX2-5, (ii) evaluate the chemical agents that modify the synergy of GATA4-NKX2-5 in vitro, (iii) examine the capacity of the lead compound to promote myocardial repair in vivo after myocardial infarction and other cardiac injuries and (iv) study the structural features of the compound important for metabolism and cytotoxicity. Integration of the experimental mutagenic data with computational modeling suggests that the structural architecture of the GATA4-NKX2-5 interaction resembles the protein structure of the conserved DNA binding domain of nuclear receptors. Fragment-based screening, reporter gene-based optimization and pharmacophore searching were utilized to identify the most potent lead compound targeting the GATA4-NKX2-5 interaction: N-[4- (diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide. This compound presented anti-hypertrophic effects in vitro and cardioprotective effects in vivo. In addition, structural analysis of the lead compound revealed the signature molecular features for metabolism and cytotoxicity. Current drug treatments are able to delay, but not prevent the progress of the heart failure; therefore, modulators of protein-protein interactions of key transcription factors may represent a novel class of pharmaceuticals for cardiac remodeling and repair.
  • Hintikka, Laura (Helsingin yliopisto, 2018)
    Anabolic-androgenic steroids (AAS) are synthetic compounds that are structurally related to testosterone. AAS are an important class of drugs that are commonly abused in sport and are classified as prohibited substances according to the World Anti-Doping Agency (WADA). AAS are metabolized extensively in the human body and excreted to urine mostly as glucuronides or sulphates. These conjugated compounds are present in human specimens in low concentrations and for a limited period. Sensitive and specific analytical methods are thus highly important to be able to detect these compounds for an extended time period in athletes. These compounds are usually analyzed from biological matrixes after cleavage of the glucuronide or sulfonate moiety. Direct analysis of intact conjugated steroid metabolites is an attractive option that involves more straightforward sample preparation. Nonetheless, the commercial availability of the conjugated reference material is limited. The first aim of this study was to produce glucuronide-conjugated metabolites of AAS to be used in the development of methods for doping control. Glucuronide-conjugated metabolites of anabolic androgenic steroids were produced by in vitro enzyme-assisted synthesis. The method offers a simple and stereospecific procedure of producing small amounts of reference material, which are needed in the development and application of analytical methods for AAS metabolites. Secondly, these AAS glucuronides were utilized in the development of a liquid chromatography – mass spectrometry (LC-MS) method for detection of glucuronide-conjugated AAS metabolites in human urine. Novel method was validated, and the robustness and transferability of the developed method was studied in an interlaboratory comparison among seven laboratories. The developed method offers simpler and a more straightforward as well as sensitive approach to the analysis of exogenous steroids, without a hydrolysis process and time-consuming sample preparation. Another approach for a more sensitive and specific method for analysis of anabolic steroids from urine, was to combine gas chromatography (GC) featuring good resolving power to softer ionization in atmospheric pressure (API). A microchip-based miniaturized heated nebulizer provides easy interfacing for GC-API-MS with high ionization efficiency. Two sensitive and selective gas chromatography - microchip atmospheric pressure photoionization - tandem mass spectrometry (GC-µAPPI-MS/MS) methods were developed, validated and successfully applied to the analysis of anabolic steroids in authentic excretion urine samples. These methods offer valuable tools for anti-doping laboratories where new analytical strategies are needed to be able to detect an increasing number of prohibited compounds with various physico-chemical properties.
  • Karhu, Lasse (Helsingin yliopisto, 2018)
    The orexinergic system is a key regulator of the sleep-wake cycle, and as such, presents a prominent target for drug development against ailments such as insomnia and narcolepsy. The system comprises two G protein-coupled receptors (GPCR), OX₁ and OX₂, and two neuropeptides, orexin-A and orexin-B. In the beginning of the study presented here, several antagonists (blockers) of the receptors were available but drug-like agonists (activators) were not. The search for the latter was hampered by the poor understanding how the endogenous ligands, the orexin peptides, activate their receptors. The main objective for the thesis research was to elucidate the binding mode of orexin peptides at their cognate receptors, along with the activation determinants, using both computational and traditional experimental methods. We produced homology models for the OX₁ receptor based on related GPCRs, and subsequently adopted the orexin receptor structures reported during the study. Peptide binding mode was probed through rigid-body docking, which resulted in two alternative binding modes. These were followed up by extensive molecular dynamics simulations within membrane environment, accompanied with simulations of small-molecule binding. Deriving from the simulations, we proposed a single, well-defined binding mode for the orexin peptide C-terminus within the canonical GPCR binding site. In addition, we observed that the small-molecular antagonist was remarkably stable within the binding site, whereas the recently reported agonist Nag26 was more mobile. The pool of simulations allowed us to observe differences between the agonists and the antagonist, leading to suggestions on determinants of agonist and antagonist binding. To assess the bioactive conformation of orexin peptides, we produced conformationally constrained orexin peptide variants. These showed that the stabilization of the straight α-helical conformation of the orexin-A is detrimental to potency, but not necessary to efficacy, at least with the utilized stapled peptides and α-aminoisobutyric acid insertions at the tested sites. We assume that the modifications were directly incompatible with the binding interactions, or the stabilized conformation was sub-optimal. The literature review focuses on the functions and characteristics of GPCRs and the orexinergic system, and provides insight into computational tools used in the study.
  • Sirenius, Ilari (Helsingin yliopisto, 2018)
    Wishes and illusions related to drugs and drug-like products – a psychodynamic perspective. This study examines the wishes and illusions related to drugs and drug-like products from a psychodynamic perspective. The study’s literature review examines the development of drug information and drug consultation as well as attitudes and levels of commitment to treatment; while also looking at psychodynamic thinking related to drug use including mindscapes and their development and regularities. In the context of drug use attitudes towards drug use in society are examined, also focusing on the conflict of interest between drugs and therapies. The empirical section of this thesis is based on data from a nationwide questionnaire survey conducted in 1984 among 4,100 respondents of at least 15 years of age. Sampling was arranged using a central population register maintained by Finland’s Population Register Centre. The survey’s response rate was 60 %. The study used a Likert response scale. Age and gender and levels of education were used as background variables. No great differences between the genders were noticeable. The oldest age group (60 – 95 years) were the largest users of medications. Their health status was the worst. Respondents with only basic education tended to be in a poorer state of health than those had completed upper secondary school or obtained university degree. Medications were most important to the oldest age group and to those with only basic education. Some users hoped that drugs could have “magical” effects such as maintaining their ability to work, making them live longer, or generally making them feel good. Respondents’ perceptions of the efficacy of medicines were most markedly shaped by the views of medical doctors and pharmacists. Details relating to how medicines actually take effect were often unclear to respondents, and this tended to intensify the various illusions they held in relation to the impacts of medicines. Knowledge of the harmful effects of drugs most significantly influenced the drug-using behavior of the younger and more educated respondents. Among older respondents drugs were more perceived as replacing deteriorated bodily functions, so they were more often considered as indispensable and users paid less attention to their harmful side-effects. Psychological factors related to drug use may ultimately lead to drugs being left unused. This deliberate or unintented non-use of drugs is a topic that merits further investigation. Due to the immense progress made in the field of medicine, people increasingly hope that all the problems in lives could be solved by using drugs. Non-pharmacological treatments accepted within mainstream medicine are less known in pharmacies.
  • Galli, Emilia (Helsingin yliopisto, 2018)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are expressed ubiquitously in the body and show protective effects on neurons and other cell types. Although the effects of MANF and CDNF administration have been studied in various animal disease models, such as neurodegenerative diseases, ischemia, and diabetes, it is largely unknown whether their endogenous levels and expression patterns are changed in relation to the pathology of these diseases. This thesis describes the development and validation of enzyme-linked immunosorbent assays (ELISAs) for the specific and sensitive quantification of MANF and CDNF in biological samples including serum, tissues, and in vitro cell samples. Since MANF removal leads into a clear diabetic phenotype in mice, we studied the circulating MANF levels in relation to human type 1 diabetes. The aim was to find out whether MANF expression and/or secretion is differently regulated before and at the onset of clinical symptoms of the disease, thus giving insights into its association to type 1 diabetes development in humans. Multiple studies have demonstrated neuroprotective and neurorestorative effects of MANF and CDNF. Since they are unable to penetrate the blood brain barrier, they need to be delivered within the central nervous system. In this thesis, the effects of intracranial gene therapy of human CDNF were studied in animal models of Parkinson’s and Alzheimer’s disease. Transgene expression depends on multiple variables, such as transduction efficiency, stability, and virus titer. Thus, the quantification of expressed protein is important in the interpretation of its effects. The species-selectivity of the developed ELISAs was utilised when analysing human CDNF levels in rodent brain. In addition to gene therapy, long-term protein delivery can be implemented by encapsulated cells that secrete the therapeutic protein to the target site. The developed ELISAs were used for the quantification of secreted CDNF from cells. Despite the successful production of several cell clones with high CDNF expression, its secretion was found to decrease markedly once the cells were confluent in cell culture plates or in polymeric microcapsules. This thesis describes modifications that were done to the CDNF coding sequence to improve its secretion, aiming at efficient delivery of CDNF by encapsulated cell therapy. The developed sensitive, specific, and thoroughly validated ELISAs for the quantification of MANF and CDNF can be utilised in future studies aimed at understanding the regulation and functions of these proteins in health and disease. In addition, the ELISAs hold the potential for analysis of these proteins as biomarkers.
  • Turku, Ainoleena (2018)
    The main objective of this dissertation was to gain an understanding of orexin receptor activation at the molecular level and apply it in discovery of novel orexin receptor ligands. As non-peptide orexin receptor activators were almost completely unknown at the start of this study, attention was focused on them. To accomplish these goals, I utilized a combination of molecular modelling and pharmacological in vitro studies. First, I studied the known orexin receptor ligands by structure- and ligand-based computational methods, and assembled the hypothesized activation features for a pharmacophore model. The model was utilized in a virtual screening, and a hit list of 395 compounds continued to a pharmacological screening phase, wherein I assessed their activities in a functional Ca2+-based screening assay developed particularly for that purpose. I validated the screening hits in the competition binding and Ca2+ elevation assays; six compounds showed weak agonist activity and Ki’s in the 1−30 µM range (Publication I). Antagonists with sub-micromolar binding affinities were also identified. Retrospective docking simulations of these agonistic hits and known non-peptide orexin receptor agonists (Nag26 and Yan7874, the latter of which was pharmacologically characterized in Publication II) were used to devise a working hypothesis of the binding pocket regions important for orexin receptor activation. Interactions in the antagonist binding region and two additional sub-pockets—one between TM5 and TM6, and the other approximately one helical turn above the antagonist binding site close to TM7—would be needed for orexin receptor activation (Publication I). Relying on this, I constructed a targeted azulene-based combinatory compound library accessible to in-house chemistry. The azulene library was virtually screened at the crystal structure of OX2 receptor, and compounds selected from the hit list were synthesized and screened in vitro. I validated the hits as above, and novel antagonists, weak agonists and compounds potentiating the actions of orexin-A were identified (Publications III and IV). The literature review focuses on the concept of GPCR activation and the orexin system: its structure, functions, and pharmaceutical applications thereof.
  • Leino, Teppo (Helsingin yliopisto, 2018)
    Orexin peptides, orexin-A and orexin-B, and orexin receptor 1 and orexin receptor 2 form the orexin signaling system. The most studied part of the orexin system is its key role in sleep-wake regulation, although it is linked also to other physiological functions, such as addiction and nociception. To date, a large number of orexin receptor antagonists have been developed with one, suvorexant, having reached the market in the treatment of insomnia. However, much less attention has been paid to the development of small-molecular agonists of the orexin receptors, and only a few are known in the literature. Agonists might be beneficial for patients with narcolepsy or certain types of cancers. The aim of this thesis was to develop small molecules based on the azulene scaffold for targeting orexin receptors. Azulene is an unexplored ring structure in medicinal chemistry, however, it resembles other bicyclic aromatics such as indole and naphthalene, which are frequently found in drug molecules. The small number of existing general synthetic routes for azulenes possessing three or more substituents has most likely hindered the use of azulene-based compounds in medicinal chemistry. Due to this, the study was initiated by developing two different synthetic routes to access 1,3,6-trisubstituted azulenes. In the developed methods, the azulene scaffold was first synthesized from simple, readily available and inexpensive starting materials. Then the scaffold was functionalized via versatile synthetic handles, such as a halogen atom or a formyl group, which allow a facile generation of compound series. The efficiency of the synthetic routes was demonstrated with test substances, which gave good overall yields. The developed methods were used in the synthesis of azulene-based compounds, whose biological activity was assessed on the orexin receptors. The first series of compounds was based on the results from virtual screening of the library of 70 000 synthetically accessible azulene-based compounds. The second series was designed based on the results from the biological evaluation of the first series. With this approach, novel azulene-based ligands for orexin receptors were identified. The two most promising binders had Ki values in the low micromolar range and five other compounds acted as weak orexin receptor agonists. In addition, compounds potentiating the response of orexin-A to OX1 receptors in a concentration-dependent manner were discovered. These novel azulene-based compounds offer an interesting starting point for further development of antagonists, agonists and potentiators for orexin receptors.
  • Paukkonen, Heli (Helsingin yliopisto, 2018)
    The main role of excipients is to ensure the safety and efficacy of the whole pharmaceutical formulation throughout its shelf-life and administration. Formulation design and development as well as material testing are the key components for successful drug delivery. This is becoming increasingly complicated as new active pharmaceutical ingredients typically have poor solubility and/or bioavailability. Due to this, there is an ever increasing need to explore new excipients and material combinations as innovative formulation solutions are required. Furthermore, modified release formulations are needed to control the release rates and to adjust the desired therapeutic effects, raising even more demand for effective formulations. The main aim of this thesis was to evaluate the performance of plant based materials nanofibrillar cellulose (NFC) and anionic carboxylated nanofibrillar cellulose (ANFC) as pharmaceutical excipients for modified release formulations and bioadhesive films. These materials are widely available from renewable sources; biocompatible with relatively low toxicity combined with high mechanical strength and large surface area available for encapsulation. NFC and ANFC, together with HFBII protein, were used as emulsion stabilizers for encapsulation and release of poorly water-soluble drugs. The synergistic stabilization mechanism achieved with these biopolymers improved emulsions stability with extremely low concentrations. In another study, ANFC hydrogels were evaluated as matrix reservoirs for diffusion controlled drug release. Their rheological and drug release properties were shown to be preserved after freeze-drying and reconstruction. The ANFC hydrogels controlled the release kinetics of small molecular weight drugs moderately, whereas significant control was obtained in the case of large proteins. In a comparative study, three new grades of microcrystalline cellulose (MCC) hydrogels were evaluated for diffusion controlled drug release. MCC matrices efficiently controlled the release of both large and small compounds, indicating great potential for drug release applications in a similar manner to the ANFC hydrogels. Bioadhesive NFC and ANFC based films were prepared by incorporating bioadhesive polymers mucin, pectin and chitosan into the film structure. The bioadhesive properties of the films combined with good mechanical and hydration properties, together with low toxicity makes them a feasible option for buccal drug delivery applications. In conclusion, NFC and ANFC were shown to be versatile excipients applicable for several types of dosage forms. In the future, it is seen that these materials may be used systematically as functional excipients for modified release dosage form.
  • Laurén, Patrick (Helsingin yliopisto, 2018)
    Hydrogels are emerging as an important source for current biomaterial design, as they often possess intrinsic physical and mechanical similarities with soft tissue, are non-toxic and biocompatible. However, many hydrogel-based biomimetic materials are either derived from limited sources, or require external activators to achieve functionality, such as chemical crosslinking or environmental cues. Furthermore, many cross-linkers used with hydrogels are toxic, and environmental cues invoke slow responses. Therefore, to function as a rational biomaterial design for a biomedical application, these properties are preferably avoided, or improved with a composite system containing two or more polymer components to overcome these limitations. Plant-derived nanofibrillar cellulose (NFC) possesses the same intrinsic properties as many other hydrogels derived from the components of extracellular matrices (ECM). Therefore, NFC shares the biocompatibility and non-toxicity aspects of biomimetic materials. However, additional features of NFC can be exploited, such as shear-thinning properties, spontaneous self-gelation and chemical modification capabilities. Additionally, the source of NFC is practically inexhaustible, and is environmentally biodegradable, bearing no ecological burden. Therefore, when designing hydrogel-based biomaterials, NFC offers versatility, which enables the fabrication of potential biomedical applications for various purposes in an environmentally safe way. In this thesis, a wide range of potential applications of NFC-based hydrogels were investigated. These include 3D cell culturing, in vivo implantation and coating systems for drug and cell delivery, controlled drug delivery and local delivery as a bioadhesive system. These methods offer insight into the versatility of NFC-based hydrogels, which could improve the future design of biomaterials, for a safer and more efficient use in biomedical applications.

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