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  • Eronen, Janne (2023)
    Adequate vaccine coverage and vaccine refusal have been prominently featured in the media during the COVID-19 pandemic. The decision-makers have considered adequate vaccine coverage important for maintaining the capacity of healthcare. The purpose of this study is to produce population-based research data about the factors affecting the willingness to take the COVID-19 vaccine. With the help of that information, it is possible to identify the factors that influence individual’s decision-making about whether to take the COVID-19 vaccine. The data of the study is the first Kansalaispulssi-data of the year 2022. Kansalaispulssi is a survey, made by the assignment of the State Council, which is used to research Finnish citizen’s views of the current topics. The research design is cross-sectional. The analysis of the study is done by crosstabulation using the Chi-square test. Age and financial situation of the person were related to the willingness to take the COVID-19 vaccine and the person's view of the role of the vaccine in preventing severe corona disease and getting rid of the COVID-19 pandemic. Age also influenced the person's view of the importance of the vaccine in preventing the COVID-19 disease. Gender, province, or educational level had no effect on a person's vaccine views in this study. Based on the research, Finnish citizens are very pro-vaccine. They also understand well the role of the vaccine in preventing a serious illness caused by the disease.
  • Neuvonen, Emilia (2024)
    Tutkimuksen tausta: Avohuollon apteekit tekivät monia toimenpiteitä varmistaakseen apteekkipalveluiden jatkuvuuden ja lääkkeiden saatavuuden COVID-19-pandemian aikana. Kriisijohtamisen prosessiteoria antaa rakenteellisen viitekehyksen kriisien ymmärtämiseen ja hallintaan. Tavoite: Tutkimuksen tavoitteena oli tutkia suomalaisten avohuollon apteekkien kriisijohtamisprosessia COVID-19-pandemian aikana käyttäen kriisijohtamisen teoriaa teoreettisena viitekehyksenä. Menetelmät: Poikkileikkauskyselytutkimus kehitettiin kriisijohtamisen prosessiteorian pohjalta ja lähetettiin suomalaisille avohuollon apteekkareille sekä yliopistoapteekkien johtaville proviisoreille toisen pandemia-aallon aikana loka–marraskuussa 2020. Logistisen regressioanalyysin avulla tutkittiin, oliko johdon riskinäkemyksellä yhteyttä kriisivalmiuteen, ja miten kriisisuunnitelmat, tiimit ja yhteistyö ulkoisten sidosryhmien kanssa olivat yhteydessä pandemian vaikutuksiin avohuollon apteekeissa. Avoimista vastauksista saatu laadullinen aineisto ryhmiteltiin samankaltaisuuksien perusteella. Tulokset: Kyselyyn vastasi yhteensä 221 apteekkaria ja johtavaa proviisoria (vastausprosentti 36,7 %). Apteekeista 79,6 %:lla oli olemassa pandemiasuunnitelma ennen kriisiä. Pandemiakriisitiimi oli nimetty 35,3 %:lla apteekeista ja 33,5 % lisäsi tai paransi yhteistyötä muiden apteekkien tai lääkehuollon toimijoiden kanssa. Kollektiivinen päätöksenteko ja ulkoisen yhteistyön lisääminen tai parantaminen olivat yhteydessä vähäisempiin negatiivisiin vaikutuksiin johdon jaksamisessa. Lisäksi kollektiivinen päätöksenteko oli yhteydessä vähäisempiin negatiivisiin vaikutuksiin organisaation taloudessa. Kirjallisuudesta poiketen olemassa olevalla pandemiasuunnitelmalla oli enemmän negatiivisia vaikutuksia organisaatioiden resursseihin. Yhteyttä apteekkarin riskinäkemyksen ja apteekkien kriisivalmiuden välillä ei havaittu. Johtopäätökset: Apteekkien tekemät toiminnalliset muutokset ja infektion ehkäisy- ja torjuntakäytännöt mahdollistivat keskeisten palvelujen tarjoamisen pandemian asettamista haasteista huolimatta. Oppimista ja sopeutumista tapahtui reaaliajassa kriisin aikana. Kollektiivisen päätöksenteon kehittäminen ja yhteistyö kollegoiden ja muiden lääkehuollon toimijoiden kanssa voivat parantaa apteekkarien jaksamista ja apteekkien taloutta tulevissa kriiseissä. Jatkotutkimuksissa voitaisiin hyödyntää laadullisia tutkimusmenetelmiä ja tutkia tarkemmin kollektiivista päätöksentekoa sekä vaikuttavan kriisisuunnitelman sisältöä avohuollon apteekkien kontekstissa.
  • Laurikkala, Nella (2022)
    Digitalization of health care and the corona pandemic have increased availability and use of online services provided by community pharmacies. In Finland, willingness to use online pharmacy services has been studied from population approach. Less is known about the user satisfaction with the core online pharmacy services such as dispensing and medication counseling services. This study aimed to investigate satisfaction with the University Pharmacy’s online services (ya.fi) from customers’ approach. Primarily, customer satisfaction with dispensing and medication counseling services was assessed. In addition, characteristics affecting customer satisfaction were analyzed. The conceptual framework of the study was Andersen's Model of Health Services Use. The data for this study was collected by a cross-sectional survey conducted in August 2020 among University Pharmacy’s online pharmacy customers who had made a purchase during the last three months. The survey instrument consisted mainly of structured Likert-scale questions, which were used to form two sum variables: satisfaction on online dispensing services (3 variables, Cronbach's alpha 0.803) and satisfaction on online counseling services (2 variables, Cronbach's alpha 0.883). Satisfaction on online dispensing services was studied through willingness to recommend and use the services in the future. Satisfaction on online counseling services was studied through a comparison of medication counseling on an online pharmacy and a conventional pharmacy. IBM SPSS (28) -software was used for statistical analysis consisting of bivariate (Kruskal-Wallis and Mann-Whitney U tests) and multivariate (generalized linear model) analyses to identify factors affecting satisfaction with dispensing and medication counseling services. Of 15 172 invitations sent to fill out the survey, 2555 eligible responses were received (16 %). Of the respondents, 92 % had concomitantly used the services of a conventional pharmacy. . The mean of satisfaction on online dispensing services on a scale from 1 to 5 (5 being the most positive option "completely agree") was 4.3 (SD 0.8). Similarly, the mean of satisfaction on online counseling services was 3.7 (SD 0.9). According to the multivariate analyses, significant characteristics affecting satisfaction on online dispensing services were age, form of living (alone/family with children/couple), purchase of prescription or OTC medicine, frequency of internet use and previous visits to a conventional pharmacy. Characteristics affecting satisfaction on online counseling services were education, purchase of prescription medicine, use of chat information service, frequency of internet use and previous visits to a conventional University Pharmacy outlet. The services on ya.fi online pharmacy rated most important by the respondents were services about medicine availabilities (in conventional University Pharmacy outlets and during a shortage) and information about medicines (prices, Kela reimbursements and information about customers' prescriptions). Customers were satisfied with online dispensing and counseling services. Online dispensing services received a higher satisfaction rate than online counseling services. Customers who had used the chat service and purchased a prescription medicine online were more likely to assess online counseling services to be equal or better than in a conventional pharmacy. Active use of internet and purchases of medicines online were factors connected to higher satisfaction with online dispensing services. Most online pharmacy customers had also visited conventional pharmacies. The results from this study can be utilized in the development of online and other pharmacy services.
  • Heiskanen, Suvi (2010)
    Cyclin dependent kinase 5 (Cdk5) is studied to take part in the migration neurons and development of brain. It is proven to participate also in the mediation of endothelial cell migration and angiogenesis. Angiogenesis is a crucial physiological mechanism in mediating wound healing and menstrual cycle among other functions. It is also important in some patophysiological processes like diabetic retinopathy and tumour outgrow. Tumour is shown to need its own vascularisation after reaching a size of 2-3 mm as a diameter in order to proceed growing. This makes Cdk5 a potential therapeutic target in regulating angiogenesis. In order to be activated, Cdk5 forms a complex together with its neuronal activator p35 or p39, or with their respective cleavage products p25 or p29. The mechanisms, how Cdk5 is activated in human ehdothelial cells has not been studied before. This master thesis is to evaluate the existence of Cdk5 activators p35 and p39 and their respective cleavage products in spreading human umbilical vein endothelial cells (HUVECs) to mimic the cell migration by freshly plating the cells. In our studies we performed western blot analysis and quantitative PCR analysis to investigate the expression of p35 and p25 in spreading HUVECs in different time points. We also performed an immunofluorescence assay to investigate the localisation of p35 and p25 in spreading HUVECs using confocal laser scanning microscopy (CLSM). The expression of p35 and p25 was also studied after growth factor stimulation (VEGF, FGF). The expression of the activator p39 in spreading HUVECs was studied using quantitative PCR method. Finally we investigated the interaction of Cdk5 with its activator p35 and its cleavage product p25 in spreading HUVECs using immunoprecipitation (IP). We were able to show in our studies that the activators p35 and p25 are expressed in HUVECs and that their expression is changing in spreading HUVECs in different time points. Additionally we were able to show, that p35 and p25 are partly localized in periphery in spreading cells. We were able to show that also the activator p39 is expressed in spreading HUVECs, but its relative amount was shown to be only a small portion of p35 in HUVECs. We were able to prove the interaction of Cdk5 with its activator p35 and p25 using immunoprecipitation, although the result could not be completely verified. Stimulation with growth factors showed no appreciable changes in the expression of p35 or p25. Based on the results, we can state that both the activator p35 and p39, and at least p25, the cleavage product p35, are expressed also in HUVECs. As they are neuronal activators of Cdk5 and Cdk5 has shown to participate in the mediation of angiogenesis and endothelial cell migration, the results amplify our hypothesis that also these activators might have a role in mediating endothelial cell migration and angiogenesis. Nevertheless to assure it, to specify the possible different roles of each activators and their interaction with Cdk5, further studies are needed.
  • Gruzdaitis, Päivi (2011)
    Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine's effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.
  • Koskela, Outi (2012)
    Pharmacogenetics is the study of variations in DNA sequence as related to drug response, i.e. pharmacokinetics, drug efficacy and adverse effects. The literature review of the thesis covers pharmacogenetics of analgesics. The most studied genetic variations affecting the analgesics response are the 118A>G variant of µ-opioid receptor gene (OPRM1) and several variations in the genes coding for cytochrome (CYP) P450 enzymes. Also variations in the COMT gene and the ABCB1 gene coding for P-glycoprotein have been shown to modify the response to analgesics. Genetic polymorphism of CYP2D6, CYP3A4 and CYP3A5 enzymes was studied in the experimental part of the thesis. The aim of the study was to determine if the allele and haplotype frequencies of the CYP2D6, CYP3A4 and CYP3A5 gene variations are different between Finnish breast cancer patients and healthy volunteers. The results will be further used to explore whether the genetic polymorphism of these metabolic enzymes affects the response to a certain drug substance. The study population consisted of 996 Finnish breast cancer patients. Common genetic variants affecting the enzymatic activity of CYP2D6, CYP3A4 and CYP3A5 were studied. In addition to gene copy number, ten single nucleotide polymorphisms (SNP) of the CYP2D6 gene were genotyped. For CYP3A4 gene, genotyping was done for intron 6 SNP rs35599367 shown to decrease CYP3A4 gene expression. CYP3A5 SNP 6986A>G leading to splicing defect and premature STOP codon was also genotyped. Genotyping and copy number determination was done using PCR-based TaqMan® 5'-nuclease method. CYP2D6 haplotype analysis and phenotype predictions were derived based on genotype data. According to CYP2D6 enzyme activity individuals are commonly classified as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) or ultra-rapid metabolizers (UM). The frequencies of CYP2D6 phenotypic classes in our study population were the following: PM, 2.8%; IM 2.0 %; EM 87.7% and UM 7.6%. The haplotype and phenotype frequencies determined for breast cancer patients coincide with the values observed earlier for Finnish healthy volunteers. In our study population, the minor allele frequency (MAF) of the CYP3A4 rs35599367 SNP was 2.7% and the MAF of the CYP3A5 6986G>A SNP 7.6%. The MAF of CYP3A5 6986G>A SNP found in our study is in line with the previous findings for Finnish healthy volunteers. There are no previous publications on the frequency of CYP3A4 rs35599367 SNP in Finnish population. In conclusion, no differences were detected in the frequency of the studied CYP2D6 and CYP3A5 genetic variations between Finnish breast cancer patients and healthy volunteers. Frequency of CYP3A4 rs35599367 SNP in Finnish healthy volunteers should be determined in order to compare it with our findings in the population comprising of breast cancer patients. The results of this study can be further used to explore the effects of CYP2D6, CYP3A4 and CYP3A5 genetic polymorphism on drug response.
  • Suominen, Laura (2020)
    Background: Alzheimer’s disease (AD) is a worldwide challenge for health care professionals and researchers. Every year, AD causes dementia for millions of patients. No preventive or curative medication is available despite continuous research. Amyloid-beta (Aβ) deposits in brain are one of the main pathological findings in AD. Accumulating Aβ peptides are thought to be the reason behind further disease progression. If the Aβ accumulation could be restricted or Aβ degradation increased their toxic effects would be prevented. Soluble oligomers and protofibrils are the most toxic species of Aβ. Most of the Aβ targeting drugs developed so far have not specifically targeted these toxic species. Neprilysin (NEP) is a major Aβ degrading enzyme that targets mostly the smallest species (monomers and dimers) of Aβ. Another common challenge for protein drugs has been passing the blood-brain barrier (BBB). Different strategies, such as utilising transferrin receptor (TfR) mediated transcytosis, have been studied for drug transport. For example, a rat anti-mouse TfR antibody, 8D3, or its fragments can be used for drug transportation. Objectives: To produce a recombinant protein, sNEP-scFv8D3, combining soluble NEP and single chain variable fragment of 8D3. Testing its ability to degrade different species and isoforms of Aβ in vitro and study in vivo brain uptake. Evaluate whether it is a promising model for future AD treatments. Methods: The recombinant protein was expressed in Expi293 cells and purified with affinity chromatography. The TfR binding was studied with TfR ELISA and enzymatic activity with MCA assay. Aβ ELISA was used for determining the Aβ degradation. Recombinant protein was compared to sNEP. In in vivo studies the brain uptake and blood half-life of radiolabeled sNEP-scFv8D3 of were studied on NLGF mice. Immunohistochemical analyses of brain cryo sections were done to evaluate the co-localisation of Aβ aggregates and sNEP-scFv8D3. Results and discussion: sNEP-scFv8D3 bound to TfR and showed similar enzymatic activity as sNEP. Both sNEP-scFv8D3 and sNEP were able to degrade monomeric Aβ-40 and Aβ-42 but no significant effect was seen on larger aggregates. In mice brain, sNEP-scFv8D3 was detected in same areas as Aβ aggregates. Compared to sNEP, our recombinant protein had better brain uptake. The blood half-life of sNEP-scFv8D3 was approximately 9.5 h and it was cleared fast from the brain. Already 6 h post injection, levels in the brain had dropped more than by half. Further studies are needed to determine whether sNEP-scFv8D3 is effectively transported across the BBB and if it can reduce brain Aβ levels in vivo. Conclusions: In the future, sNEP-scFv8D3 or its improved version could be used at the earliest stages of AD to prevent disease progression. Since sNEP-scFv8D3 degrades only small Aβ aggregates it could be combined with another drug targeting larger oligomers. Together they would decrease the total Aβ deposition in brain.
  • Aksela, Laura (2016)
    Even though cancer treatment modalities have improved during last decades, there is still lack of specific, efficient and curative treatments especially in case of advanced and metastatic cancers. One relatively new approach is to use oncolytic adenoviruses, which selectively infect and kill cancerous cells leaving healthy cells unharmed. These viruses have shown to be effective especially when administered intratumorally and in combination with chemotherapeutics. However this approach has multiple challenges like rapid clearance by antibody neutralization in systemic administration. Another challenge is the cell entry of oncolytic adenovirus, which is mainly mediated by the Coxsackie-Adenovirus receptor and this receptor is downregulated in various cancer cells. Rapid clearance and reduced cell entry thus lead to decreased amount of oncolytic adenovirus in target cells and decreased efficacy. In order to overcome these limitations, this study explored the possibility to use cancer cell derived extracellular vesicles (EVs) as drug delivery system for oncolytic adenovirus. Since oncolytic adenoviruses have shown to be effective especially in combination with chemotherapeutics, the ability of EVs to deliver both oncolytic adenoviruses and chemotherapeutic drug paclitaxel was studied. The aims of this study were to i) study whether oncolytic adenoviruses could be encapsulated inside EVs (EV-virus complex) and load this complex with paclitaxel (EV-virus-PTX complex), ii) discover whether the surface charge or size distribution of EV-virus and EV-virus-PTX complexes differs from the control EVs and iii) study the infectivity/efficacy of EV-virus and EV-virus-PTX complex in comparison to noncapsulated adenovirus in vitro. Since this is a novel approach, the literature review focused on the characteristics, advantages and challenges of oncolytic adenoviruses and EVs. In order to determine whether cancerous cell are able to encapsulate oncolytic adenoviruses inside EVs, A549 lung cancer and PC-3 prostate cancer cells were infected with oncolytic adenovirus and the formed EVs were isolated form conditioned media using differential centrifugation. Paclitaxel was loaded into these EV-virus complexes with incubation. EV-virus complexes were imaged using transmission electron microscopy (TEM) (i). The characteristics of these EV-virus and EV-virus-paclitaxel complexes were studied by determining the surface charge by electrophoretic light scattering and the size distribution by nanoparticle tracking analysis (ii). In order to determine the infectivity/efficacy of these complexes in autologous use, three in vitro level assays were performed (cell viability, immunocytochemistry and transduction assay) (iii). In addition confocal microscopy was used to observe the localization of EV-virus complexes inside the cell. These studies pointed out that both cell lines were able to encapsulate oncolytic adenovirus inside EVs, which was observed by TEM. The size distribution of these EV-virus and EV-virus-PTX complexes may support this observation and the size was in range 50-500 nm. In addition the determined surface charge was shown to be similar in EV-virus and EV-virus-PTX- complexes when compared to control EVs derived from noninfected cells - however more specific assays in order to characterize the surface properties of EV-virus complexes are needed. As a main finding, these EV-virus and EV-virus-PTX complexes were shown to significantly increase the efficacy of oncolytic adenovirus in comparison to free oncolytic adenovirus, paclitaxel and paclitaxel+virus combination in all three in vitro assays. In addition localization of the EV-virus complex was seen with confocal microscopy imaging. These results indicate that EVs may enhance the delivery of oncolytic adenovirus into cancerous cells. Using EVs as a drug delivery system for both oncolytic adenovirus and chemotherapeutic drug paclitaxel was shown to increase the efficacy of oncolytic adenovirus in comparison to free virus. This characteristic could potentially enhance the targeting ability to cancerous cells and thus lead to decreased amount of side-effects of healthy tissues especially in case of chemotherapeutics. These promising results of this novel approach are however preliminary due to relatively low number of repetitions (n~3) and more research is needed especially in order to characterize, purify and concentrate the EV-virus complexes.
  • Flink, Anu (2011)
    Here, we demonstrate the application of desorption atmospheric pressure photoionization (DAPPI) as a screening method at the Criminal Laboratory of the Finnish National Bureau of Investigation for samples confiscated by the Finnish criminal police. DAPPI is a fast mass spectrometric technique to analysis compounds directly from the sample surface in ambient atmosphere. In DAPPI, the sample is thermally desorbed from the sample surface using hot solvent vapor, after which the analytes are ionised in the gas-phase by photon-initiated gas-phase reactions. DAPPI was applied to the direct analysis of confiscated drugs, anabolic steroids and explosives of various matrices without any sample preparation. Confiscated drug samples included e.g. tablets, powders, herbal mixtures, herbal products [Catha edulis (khat) leaves, opium, Cannabis sativa, Psilocybe mushrooms] and ampules and tablets containing anabolic steroids. Powders were sprinkled on a 2-sided tape on a microscope slide, after which the excess powder was shaken away from the tape surface. Liquid samples were analysed from a kitchen paper, after application of 1 Äl of oil from ampules. Other samples were analysed by simply placing them on the DAPPI sampling stage and by directing the solvent plume on the sample surface. DAPPI proved to be a fast and specific analysis technique to this type of forensic analysis. DAPPI does not require any sample preparation, which therefore is well suited for fast forensic analysis, especially for plant samples and oily anabolic steroids, which are considered very challenging with conventional methods. Contamination of the mass spectrometer could be avoided by adjustment of the distance of the sample from the mass spectrometer inlet. Memory effects or contamination of the MS instrument were not observed even after several weeks of DAPPI measurements. DAPPI was also used for trace detection of the explosives trinitrotoluene (TNT), nitroglycol (NK), nitroglycerine (NG), penitrit (PETN), cyclonite (RDX), octogen (HMX) and picric acid. These organic explosives are nitrated compounds, which are divided based on their chemical structure into nitroaromatics (TNT and picric acid), nitroamines (RDX and HMX) and nitrate esters (PETN, NG and NK). Explosive dilutions were analysed with DAPPI from a polymer surface [poly(methyl methacrylate), PMMA] after application and drying of 1 Äl of sample. Also forensic analysis of post-blast residues from different matrices were done. DAPPI was effective in the ionisation of nitroamines and nitrate esters as their adducts with anions such as nitrate, acetate, formate and acetate. TNT used to form negative molecular ions through electron capture and picric acid formed deprotonated molecules through proton transfer. A DAPPI-MS method was developed for all explosives but the identification of the very low concentration explosive traces from wild variety of matrices proved to be difficult.
  • Aalto, Henni (2011)
    Lipids are fat soluble compounds that are derived from living tissues. Lipids have many important physiological functions. Developing methods for efficient lipid analysis is important since lipids can function as biomarkers in diseases. Additionally these methods can be used for the discovery of the biological processes of disease development. Lipids comprise of molecules with different polarity and structure. Several mass spectrometric ionization methods have been used in the analysis of lipids but they usually require sample preparation prior to the analysis. Desorption electrospray ionization-mass spectrometry (DESI-MS) and desorption photoionization-mass spectrometry (DAPPI-MS) are novel ionization methods that allow sample analysis straight from the matrix, such as tissue, usually without any sample preparation. DESI-MS has already been used in the analysis of different lipids, but DAPPI-MS has only been used in the analysis of steroids. The ionization of a range of lipid compounds (phospholipids, triglycerides, fat soluble vitamins, fatty acids, and steroids) by DAPPI-MS and DESI-MS was studied. Analysis conditions were optimized for all the different lipid classes with both DAPPI and DESI using standard samples. Some lipids were also analysed straight from pharmaceutical preparations. There were differences in the suitabilities of DAPPI-MS and DESI-MS for the ionization of different lipid classes. DAPPI-MS worked well for the ionization of nonpolar lipids like triglycerides, vitamins and fatty acids, but the phospholipids fragmented in the DAPPI-MS process and showed no molecular ion. Previous studies have shown that DESI-MS works well in the ionization of phospholipids, and this study showed that it works reasonably well for other lipid groups as well, with the exception of some of the nonpolar lipids. New knowledge was acquired especially about the suitability of DAPPI-MS for the analysis of different lipids. Based on the results it can be said that DAPPI-MS works equally well or better than DESI-MS in the ionization of most lipid classes. The DAPPI method should still be further developed so that phospholipids, which are very important lipids in human physiology, could be analysed by DAPPI-MS. As lipids were not analysed straight from a tissue sample, there are no conclusions about the suitability of DAPPI-MS for the analysis of lipids straight from tissue samples.
  • Lindfors, Pia (2010)
    The most important part in bioanalysis is the sample cleanup process which is usually the most laborious and time consuming part of the analysis and very susceptible to errors. A functional bioanalysis has to be quick, easily automated, sensitive, selective and stable. It also needs to be suitable for high throughput analysis. Desorption atmospheric pressure photoionization (DAPPI) is a novel direct desorption/ionization technique for mass spectrometry that enables direct analysis of solids from surfaces or liquid samples from a suitable sample plate often without any sample preparation. The suitability of DAPPI-MS for biological samples was investigated by measuring the limits of detection for selected opioids and benzodiazepines and screening them from authentic urine samples. Limits of detection were measured for standard solutions and spiked urine. Opioids and benzodiazepines were analyzed from post mortem urine samples with an optimized DAPPI-MS method. Post mortem urine samples were analyzed with and without sample preparation. Sample preparation improved the sensitivity of the method remarkably. About 50 % of the analytes were detected without sample preparation and almost 100 % after sample cleanup. It is however difficult to estimate the suitability of DAPPI-MS as a screening method because not all analyte concentrations of the urine samples were known. Therefore we cannot be certain weither the results obtained without sample preparation are caused by the suppression of the urine matrix or if the concentrations of the analytes are below the limits of detection. The reliability of the method can further be improved by investigating the metabolites of the analytes and improving the system towards automation. On grounds of this research DAPPI-MS should be used cautiously as a screening method for urine samples without sample preparation and with only high enough analyte concentrations. DAPPI-MS shows promise as a screening method for opioids and benzodiazepines from urine when the sample cleanup is used before the analysis.
  • Granqvist, Riikka (2021)
    Parkinson´s disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer´s disease. There is still no drug that alters the state of the disease. It has been found that Endoplasmic reticulum (ER) stress is one mechanism in PD. ER stress occurs due to accumulation of unfolded proteins. ER stress triggers Unfolded protein response (UPR) that protects against ER stress by decreasing unfolding of proteins. In the beginning, UPR has protective effect, but in prolonged ER stress UPR triggers apoptotic cell death. There are several key mediators in the UPR pathway. Characterisation of ER stress in PD models may be important for the current and future drug development of PD. If ER stress is a significant factor that affects the disease development, it would be important to find a drug that alters these mechanisms and UPR. This may be a way to halt the disease development. Different animal models of PD, like 6-OHDA (6-hydroxydopamine) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model, have similarities in their mechanisms. It has been found that ER stress occurs both in the brain of PD patients and animal models of PD. That is why studying and further characterisation in animal models is relevant. The aim of this study was to characterize ER stress in 6-OHDA rat model. The expression of some key mediators of the UPR were determined in this study. There were male and female Spraque Dawley rats in this experiment. 6-OHDA or saline was injected intrastriatally in 3 spots by stereotaxic surgery. Two weeks after 6-OHDA lesions, amphetamine-induced rotation test was conducted to the rats. The rats were divided into groups based on lesion size according to the results. For this study, the rats were euthanised at week 2 or week 4 post lesion. The rats were euthanised by carbondioxide, and the death was confirmed by decapitation. The brains were collected and stored in -80°C. Striatum and substantia nigra were collected later. Total RNA was isolated from these samples. Part of the RNA sample was used to conduct cDNA synthesis. Finally, the gene expression of Atf4, Ire1α, Xbp1s, Xbp1t, Grp78 and Chop was measured from these cDNA samples by qPCR (quantitative polymerase chain reaction). The qPCR data describes the expression of exact gene. The data was processed prior to statistical analysis. By statistical analysis, it was possible to compare the expression of these genes between 6-OHDA group and vehicle group. In addition, comparison was made between 6-OHDA treated groups at week 2 and 4. According to the results, only Chop expression had increased in 6-OHDA lesioned rats at week 2 compared to the vehicle group. In other genes there were no statistical differences, unlike in several other studies where the expression was found to be increased. Thus, the characterisation of this model requires further studying, possibly by increasing the sample size and studying later time points as well.
  • Parkkinen, Ilmari (2018)
    MicroRNAs are ~22 nucleotide long RNA strands which regulate gene expression by binding to the 3’UTRs of messenger RNAs. MicroRNAs are predicted to regulate about a half of all protein-coding genes in the human genome thus affecting many cellular processes. One crucial part of microRNA biogenesis is the cleaving of pre-miRNA strands into mature microRNAs by the type III RNase enzyme, Dicer. Dicer has been shown to be downregulated due to aging and in many disease states. Particularly central nervous system disorders are linked to dysregulated microRNA processing. According to the latest studies, Dicer is crucial to the survival of dopaminergic neurons and conditional Dicer knockout mice show severe nigrostriatal dopaminergic cell loss, which is a hallmark of Parkinson’s disease. By activating Dicer with a small-molecule drug, enoxacin, the survival of dopaminergic cells exposed to stress is significantly improved. However, enoxacin, which is a fluoroquinolone antibiotic, activates Dicer only at high concentrations (10-100 μM) and is polypharmacological, which may cause detrimental side effects. Therefore, enoxacin is not a suitable drug candidate for Dicer deficiencies and better Dicer-activating drug candidates are needed. The aim of this work was to develop a cell-based fluorescent assay to screen for Dicer-activating compounds. Assays which measure Dicer activity have already been developed, but they have some pitfalls which don’t make them optimal to use for high-throughput screening of Dicer-activating compounds. Some are cell-free enzyme-based assays and thus neglect Dicer in its native context. The RNA to be processed by Dicer does not represent a common mammalian RNA type. Most assays do not have internal normalizing factors, such as a second reporter protein to account for e.g. cell death, or the analysis method is not feasible for high-throughput screening data. Considering these disadvantages, the study started by designing a reporter plasmid in silico. The plasmid expresses two fluorescent proteins, mCherry (red) and EGFP (green), and a mCherry transcripttargeting siRNA implemented into a pre-miR155 backbone which is processed by Dicer. Thus, measuring the ratios of red and green fluorescence intensities will give an indication on Dicer activity. The plasmid also has additional regulatory elements for stabilizing expression levels. The plasmid was then produced by molecular cloning methods and its functionality was tested with Dicer-modulating compounds. The assay was optimised by testing it in different cell lines and varying assay parameters, and stable cell lines were created to make large-scale screening more convenient. Finally, a small-scale screen was done with ten pharmacologically active compounds. Transiently transfected, in Chinese hamster ovarian cells, mCherry silencing was too efficient for reliable detection of improvement in silencing efficiency due to floor effect. With an inducible, Tet-On, system in FLP-IN 293 T-Rex cells, the expression could be controlled by administering doxycycline and the improvement in silencing was quantifiable. The assay seemed to be functional after 72 hours and 120 hours of incubation using enoxacin (100 μM) as a positive control. However, the screening found no compounds to significantly reduce mCherry/EGFP fluorescence ratio and, additionally, the effect of enoxacin was abolished. Therefore, a more thorough analysis on the effects of enoxacin was done and, although statistically significant, enoxacin was only marginally effective in reducing mCherry/EGFP fluorescence ratio after 72 hours of treatment. It should be noted from the small-scale screening that metformin and BDNF, compounds previously shown to elevate Dicer levels, showed similar effects to enoxacin. The quality of the assay in terms of high-throughput screening was determined by calculating Zfactors and coefficients of variations for the experiments, which showed that the variability of the assay was acceptable, but the differences between controls was not large enough for reliable screening. In conclusion, the effects of metformin and BDNF should be further studied and regarding the assay, more optimisation is needed for large-scale, high-throughput, screening to be done with minimal resources.
  • Grazhdankin, Evgeni (2018)
    We have developed a software for homology modelling by satisfaction of distance restraints using MODELLER back-end. The protocols used extend exploration of distance restraints and conformational space. We drive the models in optimization cycle towards better structures as assessed by the used metrics on DOPE score, retrospective distance restraint realization and others. Hydrogen bond networks are optimized for their size and connectivity density. The performance of the method is evaluated for its ability to reconstruct GPCR structures and an extracellular loop 2. The software is written in object-oriented Python (v.2.7) and supports easy extension with additional modules. We built a relational PostgreSQL database for the restraints to allow for data-driven machine and deep learning applications. An important part of the work was the visualization of the distance restraints with custom PyMOL scripts for three-dimensional viewing. Additionally, we automatically generate a plethora of diagnostic plots for assessing the performance of the modelling protocols. The software utilizes parallelism and is computationally practical with compute requirements on an order of magnitude lower than those typically seen in molecular dynamics simulations. The main challenges left to be solved is the evaluation of restraint goodness, assigning secondary structures, restraint interconditioning, and water and ligand placement.
  • Reunanen, Saku (2020)
    Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic neurons that form the nigrostriatal pathway gradually die. This causes the main motor symptoms of Parkinson’s disease: tremor, rigidity and bradykinesia. While PD affects 1-2% of total population, all currently used medicines are symptomatic, and there is no disease modifying therapy available at present. Although several different animal models for Parkinson’s disease exist, the lack of adequate animal models is often cited as a major obstacle for predicting the clinical success of potential drug candidates. Lewy bodies (LBs) are abnormal aggregates that develop and spread inside nerve cells of human PD patients, their main structural component being α-synuclein. Because α-synuclein is thought to play a major role in the pathology of PD, much research has been focused on it. Different α-synuclein-based animal models of PD exist today, of which the most recent are based on using direct injections of preformed α-synuclein fibrils (PFFs). These new α-synuclein based disease models have helped to understand the disease process in PD better, but cell death in these models takes longer to achieve and is often less pronounced compared to traditional neurotoxin based animal models of PD. The aim of this study was to participate in the development and characterization of a novel mouse model of PD. This new model combines PFF-injections with the commonly used neurotoxin 6-OHDA, which should result in more robust dopamine pathway degeneration than what is seen with the current PFF-based models. The main hypothesis of this study was that the combination of intrastriatal injections of PFFs and a low dose of 6-OHDA would cause gradual spreading of the α-synuclein aggregation pathology in the nigrostriatal dopamine pathway and progressive dopamine neuron loss leading to motor deficits. C57BL/6 mice were stereotactically injected unilaterally with both PFF and 6-OHDA, and their performance was assessed every other week with different behavioral tests until week 12. At the end, brains were collected and optical density of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was measured from striatal sections, and TH and DAT positive cells in the substantia nigra were counted. The amount of Lewy bodies present in the brain slices was also counted from the cortex and substantia nigra areas of the brain. In the histological assays, statistically significant reductions of both TH and DAT were found in the brain sections of the PFF + 6-OHDA combination group and the amount of TH and DAT positive cells were lower in this group compared to the group receiving vehicle treatment only. However, the results of behavioral tests were non-significant, although a non-statistical positive trend in the amphethamine-induced rotations test was observed where mice receiving PFF + 6-OHDA rotated the most. Taken together, combination model that utilizes both PFF and 6-OHDA injections seems like a promising candidate in modelling PD in mice, but much more research and further development of the model is required before this combination model is ready and robust for use in drug development.
  • Anttila, Emmi (2021)
    Mild traumatic brain injury (TBI) is defined as an injury that disrupts the normal functioning of the brain and is the result of external force to the head. It is the most common type of traumatic head injury, and it is common especially in contact sports and within military personnel. Mild TBI typically causes no clear structural changes to the head, but it can induce persistent clinical symptoms, as well as microscopic pathological changes to the brain that may eventually lead to neurodegeneration and increase the risk for several diseases. Mild TBI is a risk factor for several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy. The primary objective of this study was to develop a repetitive mild TBI mouse model for future research purposes in the field of head trauma and neurodegeneration. The injury was induced as a closed head injury with an electromagnetic impactor. Literature and pilot experiments were used to define the parameters of the impactor required to induce a brain injury of desired severity. The characterization criteria of the mild TBI model considered the criteria used to define human mild TBI, as well as long term effects often reported after repetitive mild TBI: neurodegeneration as tau protein related pathology, neuroinflammation, and memory deficits. The secondary objective of this study was to tentatively test a prolyl oligopeptidase (PREP) inhibitor on the behavioral and histological effects of mild TBI. The functioning of the mild TBI model was studied by histopathological and behavioral assessments. After baseline behavioral assessment and repetitive (1 injury every 24 hours altogether 5 times) mild TBI inductions, the mice were monitored for approximately 3 months, during which several rounds of behavioral tests were performed. Barnes maze and novel object recognition tests were used to assess memory functions, and locomotor activity test was used to assess general locomotor activity. After euthanasia, brain histopathology was performed to study the amount of tau protein and the level of neuroinflammation. Due to the low number of animals in the study, the results are directional and need to be confirmed in subsequent studies. The histopathology showed greater amount of neuroinflammation and tau protein in the brains of injured mice, but statistical evaluations could not be made. Memory functions were slightly worse in the injured mice compared to controls, but significance of the results is unclear. Locomotor activity was not influenced by the mild TBIs. PREP inhibition treatment increased the locomotor activity of the mice, but the significance is unclear. The mild TBI model seems promising and the characterization criteria were partially met. The results of the study need to be verified in subsequent studies with a greater amount of animals. The model developed here can be used to study the involvement of head trauma in neurodegeneration, as well as treatment alternatives to changes caused by mild TBIs. As there currently are no curative treatments to neurodegenerative diseases, research regarding neurodegeneration and its risk factors is highly important.
  • Katajamäki, Jani (2021)
    Cytochrome P450 (CYP) enzyme inhibition is one of the most common reasons for adverse drug-drug interactions. An especially harmful form of inhibition is time-dependent inhibition (TDI) in which the inhibition potency increases over time and persists even after discontinuation of the drug. Both direct and time-dependent inhibition can be efficiently screened with the so-called cocktail method containing several CYP-selective probe substrates in a single reaction mixture. This method is practical especially in ADME studies of drug development, as it offers lower costs, consumption of fewer reagents and faster implementation in comparison to conventional methods. In addition, the cocktail method can be used to establish new diagnostic CYP inhibitors in vitro. The aim of this Master’s thesis was to participate in the development and optimization of a new cocktail assay method. The method was developed for screening of major drug-metabolizing CYP enzymes in vitro both in a direct and time-dependent manner using pooled human liver microsomes. Based on preliminary testing, included probe substrates were divided into two cocktails to avoid significant inter-substrate interactions: cocktail I containing tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4, and cocktail II containing coumarin/CYP2A6, (S)-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2. First, cocktail incubation conditions were optimized, followed by the determination of probe reaction kinetics, kinetic parameters (Km, Vmax) and inter-substrate interactions with single- or dual-substrate incubations. Finally, suitable probe substrate concentrations and the composition of cocktails was evaluated based on the obtained results. As a result of assay optimization, optimal incubation conditions for yet unoptimized cocktail II were established. In optimized incubation conditions, all probe reactions exhibited saturable Michaelis-Menten kinetics except for tacrine 1-hydroxylation (CYP1A2), which exhibited biphasic kinetics instead (Km1: 7.36, Km2: 517). The selected probe substrate concentrations were all below or near their respective Km values except for (S)-mephenytoin 4’-hydroxylation (40 µM vs. Km of 12.5 µM); however, its concentration could not be reduced in order to maintain sufficient metabolite formation for UHPLC-MS/MS-analysis. Dual-substrate incubation assays demonstrated a need for the reduction of bupropion concentration below 100 µM due to its inhibitory effects on CYP2C8 and CYP3A4. In addition, chlorzoxazone/CYP2E1 and testosterone/CYP3A4 were tested as complementary probe substrates for the cocktails; however, they proved to be unsuitable for both cocktails due to significant interactions (>40% inhibition). Prior to the deployment of the method, some adjustments of probe substrate concentrations are still required in addition to consideration of the suitability of less commonly used CYP3A4 and CYP2E1 probe reactions to improve cocktail coverage. Lastly, validation of the method with known time-dependent model inhibitors should also be conducted. Besides to improvement of the cocktails, new information was generated on inter-cocktail probe-probe interactions and enzyme kinetics of probe reactions, especially for the less-studied astemizole O-demethylation (CYP2J2) and tacrine 1-hydroxylation (CYP1A2). Generated information can be used, for example, in the development of new cocktails.
  • Juntunen, Maiju (2020)
    Cancer immunotherapy refers to therapy strategies that utilise the mechanisms of the immune system to treat cancer patients. The benefits of the approach include the possibility for specific targeting and utilisation of the host immune system. The treatment methods include cancer vaccines, oncolytic viruses (OVs), cell-based immunotherapies and antibodies. The interplay between the cancer and the immune system has been observed crucial for the progress of the cancer and the success of immunotherapies. An immune inflamed tumour microenvironment has been observed beneficial for the success of several therapy methods. Many immunotherapy methods rely on detecting tumour specific antigens that are used to guide the therapy agent to the target site. This strategy poses challenges when considering tumour immune evasion mechanisms, which can cause downregulation of target antigens, and heterogeneity of tumour cells and patients. OVs have the advantage of not requiring predetermined target structures to exert their effect to the tumour cells. They cause direct tumour cell lysis and induce immune responses, and may be modified to express additional genes, including immunostimulatory agents. However, virus-related immunosuppressive mechanisms and a rapid viral clearance may limit their effects. A Western Reserve (WR) Vaccinia virus (VACV) is a highly oncolytic virus strain but the virus has been observed to suppress the function of the cyclic guanosine monophosphate adenosine monophosphate synthase – stimulator of interferon genes (cGAS STING) innate immune pathway which has been shown to have a significant role in anti-tumour immune responses. The aim of this study was to create a WR VACV encoding a dominantly active (D A) STING and to determine whether the virus is capable of activating the cGAS STING pathway. The effects were compared to a corresponding virus vvdd tdTomato that does not have the STING encoding gene. The pathogenicity of viruses was controlled by a double deletion of the thymidine kinase and vaccinia growth factor genes which restricts the virus replication to tumour cells. Transgene fragments were cloned from template plasmids by polymerase chain reactions (PCRs) and joined together in a Gibson Assembly (GA) reaction to form a STING-P2A-eGFP gene insert. The insert was attached to a shuttle vector pSC65-tdTomato by restriction enzyme digestion, ligation and transformation in Escherichia coli. The correct transgene plasmid construct was verified by Sanger sequencing and PCRs. The transgene was inserted to a modified WR VACV vvdd-tdTomato-hDAI by a homologous recombination. The newly created VVdd STING-P2A-eGFP virus was purified by plaque purification. The STING protein expression was studied by an immunocytochemistry (ICC) assay. The immune signalling pathway activation was examined by testing nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation in RAW-Blue cells and dendritic cell activation and maturation in JAWS II cells. The cell viability after iinfection was studied with four cell lines; A549, B16-F10, HEK293 and MB49. The D-A STING expressing virus was produced successfully. The ICC experiment verified the capability of the VVdd STING-P2A eGFP to produce the STING protein in the infected cells. The preliminary findings indicate that the VVdd STING-P2A-eGFP virus activates the NF-κB signalling in the RAW-Blue cells and that the activation is dependent on the STING expression. The activation level is relative to the infection concentration at MOI range 0,001 to 0,1. The findings suggest that the VVdd-STING-eGFP virus can induce innate immune signalling via the STING pathway. The reference virus did not activate the signalling. The in vitro experiments also indicated that the STING virus may induce DC activation and maturation. We observed a trend of CD86 and CD40 expression upregulation on the JAWS II DCs. The effects to the cell viability were inconclusive. More studies should be conducted to verify the results. The effects of the virus should be studied in more advanced cancer models that take into account the complexity of the immune system. These preliminary results indicate the that the VVdd-STING-P2A-eGFP virus could stimulate the immune signalling through the STING pathway.
  • Uoti, Arttu (2021)
    Background and objectives: Cancer is one of the leading causes of death worldwide, and resistance to current treatments demands the continuous development of novel cancer therapies. Cancer immunotherapy aims to induce anticancer immune responses that selectively target cancer cells. Viruses can also be harnessed to elicit tumor-specific immune responses and to improve the response rates of other concomitant cancer therapies. The purpose of this study was to develop a novel viral vector-based cancer vaccine for intratumoral immunotherapy. By using the previously developed PeptiENV cancer vaccine platform, the vector viruses were coated with cell-penetrating peptide (CPP) sequence-containing tumor peptides in an attempt to further drive the immune responses elicited by the vector against cancer cells. The efficacy of the PeptiENV complex as a cancer vaccine was assessed by following its effects on tumor growth and the development of local and systemic antitumor immune responses. Methods: The PeptiENV complex formation was assessed by a surface plasmon resonance (SPR) analysis. Dendritic cell (DC) activation and antigen cross-presentation were studied using the murine JAWS II dendritic cell line. The development of cellular immune responses against tumor antigens was first studied by immunizing mice with the PeptiENV complex. The antitumor efficacy and immunity of intratumoral PeptiENV administration were then studied using the murine melanoma models B16.OVA and B16.F10.9/K1. In addition to intratumoral PeptiENV treatment, some of the B16.F10.9/K1-implanted mice were also treated with an anti-PD-1 immune checkpoint inhibitor (ICI) to study the PeptiENV complex as a biological adjuvant for ICIs. Results: The SPR analysis confirmed that CPP-containing peptides can be stably anchored onto the viral envelope of the viral vector. The in vitro results showed that the PeptiENV complex does not hamper the presentation of antigens at the surface of DCs. Additionally, the viral vector was found to activate DCs seen as a change in the cells’ morphology and surface protein expression. Immunizing mice with the PeptiENV complex induced a robust antigen-specific cytotoxic T cell response. Upon intratumoral administration in vivo, the PeptiENV cancer vaccine was not capable of inducing tumor growth control against B16.OVA melanoma, although it did still elicit robust systemic and local antitumor T cell responses. In the treatment of B16.F10.9/K1 melanoma, however, the PeptiENV complex induced efficient tumor growth control, which resulted in a significant survival benefit. Additionally, co-administration of anti-PD-1 resulted in an additive therapeutic effect. Discussion and conclusions: The present study describes a novel, highly immunogenic viral vector-based cancer vaccine that has the potential to be used as an adjuvant treatment for ICI therapy. Subsequent studies could be conducted to gain a deeper understanding of the immunological mechanisms underlying the antitumor efficacy of the cancer vaccine complex. Moreover, this novel PeptiENV complex could also be further developed as an infectious disease vaccine platform against emerging pandemics. However, the effects of pre-existing antiviral immunity on the efficacy of the cancer vaccine should be explored in future studies.
  • Pusa, Reetta (2024)
    The potential of extracellular vesicles (EVs) as diagnostic markers and drug delivery vehicles has been studied increasingly in recent years. One of the challenges in this field has been the isolation of EVs from complex biological fluids such as blood. The methods widely used for the isolation process include for example size exclusion chromatography (SEC) and ultracentrifugation (UC). As these methods use size and density of the particle, the have not been efficient enough in isolating EVs from certain particles such as lipoproteins. Due to the challenges related to these methods, other isolation methods have been sought to improve the efficiency of EV isolation. One of these methods is ion-exchange chromatography (IEC). From the two forms of IEC, anion-exchange chromatography has been studied more in EV isolation due to the negative net charge on EV particles. However, in this study the functionality and efficiency of cation-exchange chromatography (CEC) in EV isolation was studied as very little research has been done on this method. In this study, two CEC-resins were studied to define their applicability in EV isolation. A standard strong cation-exchange chromatographic resin SP Sepharose Fast Flow was compared to a strong tentacle-type resin. In addition to this, we studied the possibility to use a magnesium gradient to separate different forms of lipoproteins from EVs through dextran-sulfite precipitation. Tentacle-type CEC-resin was found to be more efficient in capturing EVs compared to the standard-type resin without magnesium. These EVs could then be eluted from the column with sodium chloride. The use of magnesium gradient allowed the separation of apolipoproteins in the samples. Higher concentrations of magnesium also reduced the number of lipoproteins in the samples altogether but resulted in the loss of EVs as well. These results were promising and showed that cation-exchange chromatography can be used in EV isolation. Tentacle-type resin seemed to be most efficient in removing impurities and capturing EVs. While more research is needed before these findings can be applied to clinical use, these results prove that cation-exchange chromatography can be used in EV isolation as a new, efficient and up scalable method.