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Browsing by study line "Pharmaceutical biology"

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  • Silén, Heidi (2021)
    Antimicrobial resistance is a growing problem worldwide. It has been shown that more than 70% of the bacteria that cause nosocomial infections are resistant to at least one antibiotic commonly used to treat them. Two concomitant phenomena that aggravate the diarrheal disease situation, especially in developing countries, are general contamination (spread of pathogens due to unclean water, poor sanitation, and malnutrition) and resistant bacterial strains (the adverse consequences of infections increase as infections prolong). According to the WHO, foodborne diseases (FBDs) were estimated to have caused approximately 91 million people to become ill and 137,000 deaths in Africa in 2010. The number is about a third of the deaths caused by FBD worldwide. Diarrhea caused about 70% of the FBD burden. Bacteria that cause food poisoning include Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus and Yersinia enterocolitica, some of which are discussed in more detail in this master’s thesis; antibiotics against which resistance has developed, how bacteria resist antibiotics, and the emergence of resistance in Africa. The antibiotic resistance of bacteria and the mechanisms of resistance against antimicrobial drugs are also discussed shortly. In addition to food poisoning, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus can cause difficult-to-treat infections such as wounds. In addition, this work has first dealt with antimicrobial plant derived compounds in general and their modes of action, and then focused on compounds, fractions and extracts of species of the genera Combretum, Terminalia, Pteleopsis and Anogeissus, as well as their antibacterial effects and uses in traditional medicine. In addition, the antibacterial mechanisms of action of different groups of compounds have been discussed in more detail. This work also deals with the combination studies of some plant extracts, fractions and compounds with antibiotics. Combination studies with antibiotics have generally been studied less than the antibacterial effects alone or the effects of combinations of many plant extracts, as used in African traditional medicine. The experimental part covers, among other things, the preparation and yield determination of crude extracts (water and methanol) as well as the agar diffusion method, the microdilution method, the Time kill tests and the checkerboard method in interaction tests to determine MIC, MBC and FIC values. Due to the Covid 19 pandemic, study results were obtained only by the agar diffusion method against Bacillus cereus. The most antimicrobial extracts were extracts of species of the genus Terminalia.
  • Silén, Jenna (2021)
    The life cycle of Chlamydia pneumoniae is a biphasic developmental cycle, as a obligate intracellular bacterium, it forms various morphological forms, including elementary bodies, reticulate bodies and aberrant bodies belonging to a persistent form. Due to the bacterial life cycle and the fact that chronication of C. pneumoniae infection and formation of persistent infection as well as pathogenesis is a complex problem involving multiple signaling pathways and affecting several different cells, it is useful to seek medication to influence infection from different stages of the bacterial life cycle. There are several different factors that induce persistence and thus models of persistence. Although the detection of aberrant RBs and thus aberrant bodies in C. pneumoniae infected tissues does not provide complete certainty about chronic infection, the bacterium has been linked to chronic health problems such as atherosclerotic cardiovascular disease and asthma. The aim of the study was to develop a persistence model induced by beta-lactam antibiotics, amoxicillin and penicillin G, in A549 cells by monitoring the size, shape, and number of inclusions using the IPA method and the immunofluorescence staining method for infection. In addition, the antibiotic sensitizing effect of three compounds on pulmonary chlamydial infection was studied. This effect was monitored by examining the recovery of persistent infection and by monitoring the protective effect of the compounds on beta-lactam-induced persistence. The work succeeded in finding an infection model that is well suited for studying beta-lactam persistence. Due to treatment recommendations, pulmonary chlamydial infections are practically treated with beta-lactam antibiotics. Based on the methods used, it was found that amoxicillin concentrations of 10 and 25 µg/ml and penicillin G concentrations of 100 U/ml and 250 U/ml were sufficiently effective to transfer bacteria to a state of persistence. It was found that the amoxicillin persistence model is reversible based on the increase in the size of the inclusions, especially at 25 µg/ml and quantitatively at 10 µg/ml. It was concluded that amoxicillin at a concentration of 10 µg/ml is sufficient to induce persistence in a beta-lactam antibiotic-induced persistence model. Further quantitative studies on the persistence model are needed, such as quantitative PCR based on the OmpA gene to determine more accurate dose-response relationships. Glutathione levels should also be monitored in the persistence model.
  • Rosqvist, Linn (2021)
    Marine invertebrates are a good and relatively unexplored source of bioactive compounds. These bioactive secondary metabolites can have unique structures and mechanisms of actions, since they are produced by organisms, which means their structures are not limited by the fantasy of chemists. Therefore, bioactive secondary metabolites isolated from marine invertebrates are attractive for drug development. Still, there are challenges regarding bioprospecting marine invertebrates. For example, the amount of material is limited and the environment as well as the biodiversity has to be taken into account when gathering the organisms. The aim of this thesis was to perform the first steps of bioprospecting marine invertebrates; extraction, fractionisation, analysis of bioactivity and identification of bioactive metabolites. The samples used in the experiment, gathered from three different locations, were of the sponge Caulophacus arcticus. The goal was not only to identify one or more bioactive metabolites for eventual further analysis, but also to compare the bioactivity of the samples gathered from different locations. The fractionisation was performed using flash fractionisation, which resulted in eight fractions of each extract. These fractions were tested for anticancer, antibacterial and biofilm inhibiting properties. The bioactivity of the fractions was analysed by performing cell viability assays (MTS assays) on four cell lines, antibacterial growth inhibition assays on five strains of bacteria and biofilm inhibition assays on biofilm of S. epidermidis. The active fractions, the fraction right before and after them and the corresponding fractions of the two other samples were further analysed using UHPLC-HR-MS, in order to identify eventually bioactive compounds and determine the elementary composition of these compounds. The most interesting fractions, from which one or more bioactive compounds were to be identified first, were prioritised based on the bioactivity assays. One compound, which was identified as potentially bioactive with a potentially novel elementary composition, was chosen as a target compound for further analysis. Based on the results, it was also possible to draw the conclusion that there were variations as well as similarities in the bioactivity of samples gathered from different locations. Still, further research is needed to determine if the bioactivity of the same fractions from different samples was caused by the same compounds or not. Even if there are challenges regarding bioprospecting of marine invertebrates, it is still useful to keep studying them in order to find new, bioactive compounds. There is a huge need of new drugs, especially for treating cancer and bacterial infections. Therefore, experiments such as this are relevant also in a bigger perspective. The target compound identified in the experimental part of this thesis might be further analysed in order to determine whether it is bioactive and whether it is profitable to develop it further.
  • Kylkilahti, Sanni (2022)
    Chilit ovat Capsicum-sukuun kuuluvia yleensä korkean kapsaisiinipitoisuuden omaavia paprikalajeja. Niitä käytetään mausteena. Lisäksi chilien sisältämillä kapsaisinoideilla on todettu olevan useita farmakologisia ominaisuuksia, kuten analgeettisia ja antioksidanttisia vaikutuksia. Niiden antimikrobisia ominaisuuksia on myös hieman tutkittu, mutta tutkimuksia on vielä verrattain vähän. Tämän työn tarkoituksena oli selvittää muutamien eri chililajikkeista valmistettujen uutteiden antimikrobisia vaikutuksia Escherichia colia ja Staphylococcus aureusta vastaan. Uutteet testattiin dimetyylisulfoksidiin (DMSO) ja veteen liuotettuina. Lisäksi testattiin myös kahden puhdasaineen, kapsiaatin ja solaniinin, vaikutuksia kyseisiä bakteereita vastaan. Antimikrobiakokeet suoritettiin 96-kuoppalevyllä noudattaen aseptisia työtapoja. Testattuja chiliuutteita oli 19. Uutteita valmistettiin eri chililajikkeiden versoista (1 kpl) siemenistä (3 kpl), lehdistä (10 kpl) ja hedelmistä (5 kpl). Dimetyylisulfoksidiin liuotetut uutteet testattiin pitoisuuksilla 2,0 mg/ml ja 4,0 mg/ml. Veteen liuotetut uutteet testattiin pitoisuudella 4,0 mg/ml. Solaniini- ja kapsiaattiuutteet testattiin kahdeksalla eri pitoisuudella (0,001172–0,15 μg/ml). Tutkimuksen tuloksena on, että testatut chiliuutteet eikä solaniini- ja kapsiaattiuutteet estäneet E. colin tai S. aureuksen kasvua. DMSO:iin liuotetuista uutteista korkeimmat estoprosentit kumpaakin bakteeria vastaan saatiin nuorilla Pimento-lehdillä. Veteen liuotetuista uutteista korkein estoprosentti E. colia vastaan saatiin Dulcen versoilla (30 % esto) ja S. aureusta vastaan Dulcen hedelmillä (50 % esto). Aiemmat tutkimustulokset chilien antimikrobisista vaikutuksista ovat ristiriitaisia, joten yhteneviä johtopäätöksiä chilien vaikutuksista bakteereihin ei voida tehdä. Johtopäätöksenä voidaan todeta, että chileillä on lukuisia terveysvaikutuksia. Antimikrobisen tehon varmistamiseksi tarvittaisi kuitenkin lisää tutkimuksia. Antibioottiresistenssi on maailmanlaajuinen ongelma, koska yhä useammat bakteerit ovat resistenttejä käytetyille antibiooteille. Tulevaisuudessa onkin erittäin tärkeää löytää uusia yhdisteitä bakteerien tappamiseksi, joten tutkimuksia uusien antimikrobisten aineiden löytämiseksi tarvitaan jatkuvasti lisää
  • Virtanen, Sonja (2020)
    Parenteral products are sterile products that are administered as injection, infusion or implantation. Administration of the contaminated parenteral product can cause severe consequences such as sepsis meningitis and even death. Most of the parenteral products used at the hospitals needs to be compounded (e.g. dissolved, diluted) before administration. Whenever possible, compounding should be done in biological safety cabinet using aseptic techniques. According to previous studies errors in aseptic techniques are quite common. Aim of this study was to compare three different environments as compounding area and their effect to the sterility of the compounded parenteral product. Based on the results of this study, changes to the protocols of the hospital could be made. Altogether 220 samples were compounded at two pediatric wards at HUS Helsinki University Hospital. Six volunteers (one pharmacist and five nurses) participated from both wards and each compounded 18 samples in three different environments (patient room, medicine room, biological safety cabinet). The samples were tested for the sterility by membrane filtration within 4 hours or after 24 hours of storage in the refrigerator. The investigator used an observation form to observe the compounding procedures. Environmental monitoring (settle plates) and monitoring of personnel (glove samples) were conducted. Almost all compounded samples (99%, n=213/215) were sterile. There were no significant differences in the contamination rate of the compounded samples between different environments. Five of the collected samples were excluded, because they were contaminated during the sterility test. According to observations, aseptic techniques were well followed. However, disinfection of the septum of the medicine bottle, hand hygiene and cleaning of the compounding area were observed to be deficiently completed. Even though there were lot of variation in the environmental and personnel monitoring the results were quite good. Results from the environmental monitoring were compared to the recommended limits of EU GMP for clean areas. One compounded sample was contaminated with Diezia maris and Corynebacterium mycetoides but the contaminants from the other contaminated sample could not be identified. Aseptic techniques were mainly well followed, however compounding should be done in the biological safety cabinet, since the environmental monitoring results show that the biological safety cabinet was only environment which was within the recommendation limits of the EU GMP for the compounding area of parenteral products. Protocols of the hospital could be changed, since there was no correlation between higher contamination rate of settle plates or compounded samples and not wearing mask and hair cover while compounding in the biological safety cabinet.
  • Lähdeniemi, Veera (2021)
    Drug metabolism is a series of enzyme catalysed processes that modify foreign compounds into a form that is more easily excreted from the body. Compounds can affect the activity of metabolizing enzymes and this may lead to toxic concentrations of a drug that is metabolized via the enzyme. With prodrugs, on the other hand, the drug might not achieve its biologically active form and therefore the treatment will not be effective. Recognizing and preventing metabolic interactions is important already in the early stages of drug discovery and development. Cytochrome P450 (CYP) enzyme inhibition is one of the major reasons for adverse drug-drug interactions (DDIs). The inhibition can be time-dependent (TDI), which means that the potency of inhibition increases over time. TDI may be reversible or irreversible, latter being more severe as new enzymes need to be produced in the body to restore the enzymatic activity. IC50 shift assay is a method that gives information of new compounds potential to cause TDI. IC50 shift assay does not show whether the TDI is reversible or irreversible, however further studies, e.g. dialysis assay, can be conducted to find it out. If the study compound is irreversibly bound to the enzyme, the enzyme activity should not recover in the dialysis. The aim of this master’s thesis was to develop a dialysis method that could determine the reversibility of the TDI observed in the IC50 shift assay. A dialysis method conducted with microsomes is described in earlier literature. Known inhibitors (both time-dependent and direct) for four CYP isoforms were studied in this work: CYP1A2 (furafylline and fluvoxamine), CYP2C9 (tienilic acid and sulphaphenazole), CYP2D6 (paroxetine and quinidine) and CYP3A4 (verapamil, azamulin and ketoconazole). IC50 shift assays were conducted to each inhibitor before the dialysis experiment. The studied compounds behaved in the dialysis assay mostly as assumed based on the literature. The workflow from IC50 shift assay to dialysis assay worked successfully and the IC50 shift data could be utilized when choosing the test concentrations for dialysis assay. Both the IC50 shift assay and dialysis assay were reproducible and the deviations between replicates and separate studies were relatively low. The method still requires some optimizing, but so far, the results are promising. In the future the dialysis method may be part of in vitro CYP inhibition studies at Orion Pharma.
  • Repo, Amanda (2022)
    The use of different methods of extended reality (xR) as a support in teaching has been under research for a long time. Although the use of various xR-technologies in other fields of healthcare, such as medical and nursing education, is already common, their use in pharmacy education is not yet well established. There is evidence that xR-technology has a positive impact for example on students’ motivation and learning outcomes. On the other hand, there are limiting factors that inhibit the technology becoming widespread, such as costs as well as a lack of knowledge about the technology usability. The aim of the study was to investigate usability and advantages of the augmented reality (AR) in a laboratory course as an educational supportive tool by using AR-glasses. The aim was also to investigate the learning outcomes of the students who participated in the study in three different phases: before carrying out the laboratory work (pre), immediately after the laboratory work (post) and in the course exam (delayed). Furthermore, the motivation of the students to use new technology in their studies was studied. The research was done in a collaboration with the Centre for University Teaching and Learning (HYPE) and with Sciar Company Oy. The researchers of HYPE were responsible for the pedagogical point of view, whilst the experts from the Faculty of Pharmacy were responsible for the study measurements of laboratory work related content knowledge. The research was implemented in two laboratory courses in Bachelor of Science level pharmacy studies: Medicinal product II and Pharmaceutical biology and asepsis in the fall of 2020. The students (n=18) prepared eye drops by using AR-glasses in the Medicinal product II -course and reference group (n=14) without AR-glasses. In the course of Pharmaceutical biology and asepsis, students (n=7) used AR-glasses to study the microbiological purity of the eye drops by utilizing membrane filtration method in cleanroom and reference group (n=9) without AR-glasses. ln addition, a serial dilution method was performed on a 96-well plate using an AR mobile application. The effect on learning outcomes was evaluated by using six open-ended questions measuring the understanding of content knowledge underlying the laboratory work, that were answered by the students at three different stages of the study (pre, post, delayed). To measure the usability of the AR equipment, a five-point Likert scale questionnaire studied the experimental groups students’ opinions on whether the AR mobile application could provide sufficient guidance and feedback while performing the laboratory work. In an open question, the students had the possibility to comment on the overall user experience of the AR mobile application. There were no statistically significant differences in learning outcomes between the AR-group and the reference group in both laboratory courses. The results showed indicative differences in short-term and long-term learning, with the AR-group achieving better learning outcomes in the short-term and the reference group in the long-term. In the course of Pharmaceutical biology and asepsis, the learning outcomes were the opposite. Students’ were found to be receptivity to the new technology that together with motivation supports positively the learning process. The use of AR-hardware increased certainty and reduced nervousness about the use of AR technology. As a conclusion, the study could not demonstrate the benefit of AR-technology in student learning outcomes. The study was limited by the small sample size. However, further studies are encouraged due to students’ positive attitudes and motivation towards AR technology. Regarding further studies, it is important to take into consideration the different backgrounds and learning methods of students. Thereby, the effects of xR-technologies on learning outcomes can be assessed as objectively as possible.
  • Kilpiö, Tommi (2021)
    Plant cell culture can be used for the production of valuable secondary metabolites. Inspired by the previous studies focusing on capsaicinoid production, this study aimed for establishing plant cell cultures of Capsicum chinense to produce capsinoids. Capsinoids are non-pungent capsaicinoid analogues with potential health benefits. Another aim of this study was to determine the α-solanine content in Capsicum plants and cell cultures to ensure that no toxic amounts are formed during the cell culture. Cell cultures of non-pungent Capsicum chinense cultivars, Trinidad Pimento and Aji Dulce strain 2, were established, and the cultures were fed with intermediates, vanillin and vanillyl alcohol, to enhance the production. In addition, cell cultures of extremely pungent Trinidad Scorpion cultivar were established and they were fed with vanillyl alcohol to study if this would result in formation of capsinoids instead of capsaicinoids. A high-performance liquid chromatography (HPLC) method with UV detection was validated for determining the capsiate contents of the cell culture samples and fruit samples for comparison. To analyze the α-solanine content of the cell culture samples and leaves and flowers of three cultivars belonging to three different Capsicum species, an HPLC-UV method was validated for this purpose as well. Despite validating a sensitive and specific method for capsiate analysis, no detectable amounts of capsiate were detected in any of the cell culture samples. Cell cultures of pungent cultivars did not produce detectable amounts of capsaicinoids either. Results from analyzing the real fruit samples were in accordance with previous literature reports, and Aji Dulce fruits were found to contain higher amounts of capsiate compared to Trinidad Pimento, although having only one indoor grown Aji Dulce fruit analyzed limits the reliability. The analytical method for determining α-solanine content had problems with internal standard and specificity. This method could be used for making rough estimates about the possible α-solanine content. No hazardous amounts were detected in any of the cell culture samples. Only one sample consisting of Aji Dulce young leaves could contain α-solanine slightly above the limits set for commercial potatoes. Results with flowers of Rocoto San Pedro Orange (C. pubescens) and Aji Omnicolor (C. baccatum) were inconclusive and it couldn’t be ruled out that they might contain large amounts of α-solanine. The reason why capsinoids, or even capsaicinoids, were not detected in the cell culture samples remains unsolved, but it could be speculated that capsinoids might degrade in the cell culture environment or that selection of cultivar or cell line is critical. This study gave further proof to the previous assumptions that chili leaves are safe and should not contain notable amounts of α-solanine.
  • Hämäläinen, Sanni (2019)
    Kasvavan antibioottiresistenssin vuoksi terveydenhuollossa tarvitaan uusia antibiootteja ja antibioottien apuaineita. Tästä syystä tässä työssä tutkittiin Combretaceae- ja Annonaceae-heimoihin kuuluvien tansanialaisten lääkekasvien antibakteerisia vaikutuksia. Kansanlääketieteessä näitä kasveja on perinteisesti käytetty mm. bakteerien aiheuttamien sairauksien ja oireiden hoitoon, mikä antaa viitteitä siitä, että ne sisältävät antibakteerisia yhdisteitä. Tutkimuksen tarkoituksena oli pyrkiä löytämään raakauutteita ja neste-nesteuutolla saatuja fraktioita, joilla on mahdollisimman hyvät estovaikutukset bakteerien kasvuun. Lisäksi oli tarkoitus selvittää Annonaceae-heimon lajien sisältämiä yhdisteitä, mikä voi osaltaan auttaa löytämään uusia antibiootteja. Antibakteerisia tutkimuksia tehtiin Combretaceae- ja Annonaceae-heimoihin kuuluvien Combretum-, Terminalia-, Friesodielsia- ja Hexalobus-sukujen lajien uutteille ja fraktioille käyttäen agardiffuusio- ja mikrodiluutiomenetelmiä. Yhteensä 45 eri uutteen ja fraktion estovaikutuksia tutkittiin ruokamyrkytyksiä aiheuttavien Bacillus cereus - ja Salmonella enterica -bakteerien kasvuun. Lisäksi tutkittiin Annonaceae-heimoon kuuluvien Friesodielsia obovata - ja Hexalobus monopetalus -lajien uutteiden ja fraktioiden sisältämiä yhdisteitä käyttäen HPLC-DAD - ja UHPLC/Q-TOF-MS -menetelmiä. Tutkimustulosten perusteella useilla Combretaceae- ja Annonaceae -heimoihin kuuluvilla lajeilla on antibakteerisia vaikutuksia grampositiivista B. cereus -bakteeria vastaan, mutta ei niinkään gramnegatiivista S. enterica -bakteeria vastaan. Aiemmissakin tutkimuksissa on usein, ei kuitenkaan aina, saatu parempia estovaikutuksia grampositiivisten kuin gramnegatiivisten bakteerien kasvuun. Tässä tutkimuksessa parhaimmat tulokset (MIC = 156 µg/ml) saatiin mikrodiluutiomenetelmällä B. cereus -bakteerin kasvun estoon Combretum fragrans -lajin lehtien kuumalla Soxhlet-metanoliuutteella ja Friesodielsia obovata -lajin lehtien metanoliuutteen veteen liukenemattomalla fraktiolla. Tarvitaan kuitenkin vielä paljon lisätutkimuksia, varsinkin H. monopetalus - ja F. obovata -lajien kohdalla, jotta voidaan selvittää, voidaanko tutkituista kasviuutteista ja niiden fraktioista eristää mahdollisia uusia antibiootteja tai antibioottien apuaineita ihmisten ja eläinten infektioiden lääkintään. Annonaceae-heimon lajeista löydettiin samantapaisia yhdisteitä kuin on löydetty aiemmissa tutkimuksissa, mutta 6,8-dimetyyli-monohydroksi-pinosembriiniä karakterisoitiin ensimmäistä kertaa F. obovata -lajista. F. obovata -lajin lehdistä tunnistettiin ensimmäistä kertaa (-)-krotepoksidi ja krotepoksidin johdannaisia. Lisäksi H. monopetalus -lajin juuresta karakterisoitiin ensimmäistä kertaa 3-(2',3'-dihydroksi-3'-metyylibutyyli)-5-(3''-metyylikrotonyyli)indolia, 3-(1,3-dihydroksi-3-metyylibut-2-yyli)-6-(2-hydroksi-3-metyyli-3-butenyyli)indolia sekä heksalobiini C:tä ja D:tä.