Browsing by master's degree program "Master 's Programme in Microbiology and Microbial Biotechnology"
Now showing items 21-40 of 57
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(2023)Dietamoeba fragilis is a very common human intestinal parasite in the world and also in Finland. D. fragilis causes dientamoebiasis in humans, and symptoms of infection may vary from mild bowel symptoms to prolonged diarrhoea and weight loss. Some of the infected patients are completely asymptomatic. Despite its prevalence, insufficient data exist on the life cycle and host organisms of D. fragilis. Interestingly, D. fragilis has been found in dogs in a few studies. Thus, more research is needed on the role of household pets as part of the parasite’s life cycle and choice of host organism. Comprehensive study on the role of dogs in the life cycle of D. fragilis is warranted. The purpose of this thesis was to design a study on the role of dogs in the D. fragilis parasite life cycle “The presence of Dientamoeba fragilis parasites and multiresistant ESBL-bacteria in dog owners and their pets”, to present research methods and to apply research permit from the ethics committee. The aim of the study is to analyze the prevalence of D. fragilis in dogs in Finland and the role of the dog in the spread of D. fragilis parasite within humans. In addition to D. fragilis, the study combines collection of samples for ESBL bacteria analyses, but this thesis focuses only on the part of study D. fragilis. Two different real-time PCR methods are tested in this project: an in-house method and an Amplidiag® Stool Parasites method, out of which Amplidiag® Stool Parasites method is selected to be used in the study. In the thesis documents for the ethics committee's referral are written and ethical permit to conduct the study from the HUS ethics committee is applied. A favourable opinion will allow a comprehensive study of dogs and humans to be carried out. Ethics permit is need to recruit participants for research on the basis of a laboratory-confirmed D. fragilis -discovery. The committee confirms that the study is ethically acceptable. During the thesis project, the committee gave the study a conditional favourable decision. The implementation of the study will start in the spring of 2023, when the final assent is received.
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(2022)Dietary data is essential in creating dietary guidelines and interventions, but the traditional data collection methods can be biased and costly. Wastewater metagenomes present a potential new way to collect dietary data with the utilization of microbial markers. In this study, potential microbial markers for fiber and meat intake were identified from literature. The abundances of these markers and their associations with the corresponding dietary data were analyzed using a previously published global wastewater metagenome dataset covering 58 countries. Majority of these potential markers were detected in the analysed wastewater metagenomes. Of the identified markers, Prevotella and Prevotella copri showed significant associations with whole grain intake, Alistipes and Alistipes putredinis showed significant associations with processed meat intake, and Faecalibacterium showed significant association with red meat intake. In addition, associations between dietary data and both taxonomic and functional annotations of the metagenomes were determined to identify any additional potential markers. Multiple additional species, genera and gene families showed significant associations with red meat and processed meat intakes. Future research should include finer resolution data to validify these results and further investigate the potential of these taxa and genes as markers. In conclusion, microbial markers present a promising way to collect dietary data from wastewater metagenomes.
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(2023)Carbapenemase-producing Enterobacteriaceae (CPE) resist carbapenem class of antibiotics in addition to the other type of antibiotic resistances they usually carry. The blaKPC-3 gene is one of the genes causing the carbapenemase production in bacteria. The aim of this study was to establish the Oxford Nanopore Technologies MinION sequencing method and integrate it with Illumina sequencing into a hybrid assembly for investigating the location of the blaKPC-3 gene in the bacterial genome. We used 14 isolates suspected of plasmid-mediated gene transfer and prepared three libraries using different DNA extraction methods. The contigs from the hybrid assembly sequences were used to annotate the target genes, which were then included in a phylogenetic tree and distance matrix analysis. DNA extraction method had an impact on the amount of data and the length of reads produced in MinION and the Smart DNA prep extraction kit with InnuPure C16 touch extraction device produced the best results. The blaKPC-3 gene was located on plasmids in each isolate, and the similarity of the plasmid sequences indicated that horizontal plasmid-mediated gene transfer has likely occurred between different species. However, the clonal spread cannot be excluded, and further research is needed to confirm these findings.
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Exploring the potential of lactic acid bacteria for sensory improvement of plant protein ingredients (2022)One of the greatest challenges of our time is securing the global protein supply for the growing population in a sustainable manner. Fermentation with lactic acid bacteria has a long history of successful employment for the production of fermented foods and beverages. During this study, the ability of diverse lactic acid bacteria for fermentation and sensory improvement of leguminous and cereal protein concentrates was investigated.The main aim of this study was to overcome the sensory limitations of these plant protein ingredients by finding suitable candidates for the design of new starter cultures for their fermentation. A collection of 82 lactic acid bacteria was screened for fermentation of leguminous and cereal protein concentrates with different nutrient supplementations. Most strains required additional nutrients to adequately acidify the leguminous protein concentrate during a 24-h fermentation, while the cereal-based substrate appeared to be a more complete growth substrate. Descriptive sensory analysis also revealed differences in the aroma perceived by a panel depending on the matrix, supplementation and fermenting strain employed. Three of the strains that produced the most desirable aromas and acidified sufficiently the test matrixes were further studied. All three strains preferentially fermented glucose to lactic acid rather than any other sugar. The concentration of hexanal, one of the volatile compounds involved in grassy and beany off-flavor formation, reduced during fermentations in favor of 1-hexanol, a compound with a significantly higher odor threshold. However, only two of the cultures were able to prevent the growth of contaminating bacteria during fermentation. The results of this study can provide guidance for selecting potential starter cultures and fermentation substrate composition to improve the aroma of plant protein ingredients. Two of the selected strains especially have shown potential to be used as starter cultures for the leguminous protein concentrate. Further studies are required to optimize the performance of the selected strains in the test matrixes and to quantitatively characterize their effect on the substrates’ volatile profile, taste and antinutritional factor content
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(2023)Membrane-bound pyrophosphatases (M-PPases) catalyse the reversible hydrolysis of pyrophosphate into two inorganic phosphate molecules. This hydrolysis is coupled to the transport of protons and/or sodium ions across the biological membrane, generating an electrochemical gradient, which can be utilized by the host organism as an energy source under different stress conditions. The essential physiological roles of M-PPases in agriculturally relevant plants and various human pathogens, such as Bacteroides fragilis, Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei, make them an important research area. Despite the biochemical studies conducted with several M-PPases and structural characterization of Thermotoga maritima and Vigna radiata M-PPases, there remain several questions regarding the biochemistry and catalytic mechanism of M-PPases, one being the basis of K+ dependence. Mutational studies have suggested that a single residue at position 495 is the sole determinant of K+ dependence in M-PPases. In K+-dependent M-PPases this residue is an alanine and a K+ ion binding in close proximity of it, has been shown of having stimulatory effect on K+- dependent M-PPases by increasing the maximal rate of pyrophosphate hydrolysis. In K+-independent M-PPases, a lysine residue resides at 495 position, where the ε-NH3+ group of the lysine residue has been suggested, based on structural modelling and AlaàLys (A495K) mutational studies, to structurally and functionally mimic the activating function of K+ in K+-dependent M-PPases. In order to provide structural basis to this, in this study, a M-PPase belonging to a hyperthermophilic deep- sea bacterium T. maritima containing a single A495K point mutation was expressed in Saccharomyces cerevisiae, solubilized using the high-temperature (“Hot-solve”) method, purified with metal affinity chromatography and structurally characterized using X-ray crystallography. As suggested, the ε-NH3+ group of the introduced lysine was shown to occupy the same space and form the same interactions at the active site as the K+ ion in K+-dependent M-PPases. However, these structural findings were in contradiction with the conducted hydrolytic activity assay which showed that the A495K mutation, besides abolishing K+ dependence, significantly reduced the overall activity compared to the wild type by ~2-10-fold, depending on the K+ concentration. These findings suggest that additional factor(s) besides the 495 residue determine the K+ dependence in M-PPases.
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(2022)The popularity of fermented beverages is on the rise due to signature flavours, associated health and nutritional benefits and a 100% natural label. Research in this sector is currently focused on industrial-scale production of traditional homemade fermented beverages such as Kombucha, Kefir and Kvass. To expand consumer choice beyond these traditional beverages and to provide more nutritional and flavor diversity, it is essential to develop novel products by using new microbial communities and new substrates. The industrial scale-up of fermented beverages produced using microbial communities is challenging as the flavour complexity and functionality of the beverage depends on the complex fermentation processes and interactions between the microbiota species. Fermentation systems that can separate the metabolic stages into separate fermentation steps would be needed to simplify and make the complex fermentation more efficient, scalable, and reliable. The aim of the thesis was to develop and compare different fermentation strategies to control the complex fermentation of previously isolated microbes to produce a bio-flavoured, low-alcohol, malt beverage with a signature fruity flavour and aroma. During the study, green-malt microbial species: Lactiplantibacillus plantarum, Saccharomyces cerevisiae and Saprochaete suaveolens were identified as significant contributors to the development of aroma and flavour compounds in the malt fermentates. Using the optimal cell concentration of the selected species, three different fermentation strategies: simultaneous inoculation, sequential inoculation and sequential fermentation were adopted to design five different fermentation systems. Cocktail blends of individual fermentates were also created and tested for flavour and aroma. All potential production methods were compared in contrast for parameters such as ease of operation, time-efficiency, flavour and aroma, and future scalability. The results showed that complex fermentation of the novel and low alcohol malt beverage could be controlled by selecting organoleptically significant microorganisms from the complex community, controlling the time and order of inoculation and using a stagewise or modular fermentation system. Sequential fermentation produced the desired low alcohol level and flavorful, fruity malt beverages. However, this system required centrifugation at each step and thus resulted in limited ease of operation. Sequential inoculation was an optimal and efficient method of controlling the fermentation since it required a single vessel, and the metabolic stages were separated by inoculating microorganisms sequentially with a 24 h time interval between each inoculation. Creating cocktail blends from individual fermentates also produced bioflavoured, fruity, aromatic, low alcohol malt beverages. This method was time-efficient with maximum ease of operation. The resulting beverages from these different fermentation systems were novel and had fruity flavours and aroma from the metabolites synthesized by organisms S. suaveolens, L. plantarum and S. cerevisiae. Thus, bio-flavoured, low-alcohol, malt beverages with signature fruity flavour and aroma were created at VTT. For the first time S. suaveolens was used in combination with L. plantarum and S. cerevisiae for beverage production using novel three-microbe fermentation systems to control complex fermentation.
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(2023)Phage lysins are enzymes, which hydrolyze peptidoglycan on bacterial cell wall. Most phage lysins have a modular structure, where N-terminal enzymatically active domain is linked to a C-terminal cell binding domain (CBD). Wild-type Lactococcus lactis LAC460 secretes three prophage lysins, LysL, LysP, and LysT, of which LysL and LysP act as antimicrobial agents killing other closely related bacteria. Of these lysins, only LysL have been shown to contain a CBD in its C-terminus. The aim of this study was to characterize the LysP and LysT enzyme activities by producing them in a heterologous Lactococcus cremoris strain, and to investigate the existence of CBDs in their C-termini. Antimicrobial activities were studied against indicator cells on agar plates, in cell suspensions, and in zymographic gel. Putative CBDs were fused with green fluorescent protein (GFP) to measure fluorescence after binding the fusion proteins onto indicator cells. It was revealed that LysP and LysT antimicrobial activities were detected from heterologous producers’ cell-free supernatants and cell lysates. The lysins were shown to function independently without the need of other phage proteins, and their unusual slow migration in zymogram gel was confirmed. L. cremoris production of the GFP-CBD fusion proteins resulted in low fluorescence, which was insufficient for detecting the proteins’ binding onto indicator cells. In conclusion, heterologous expression and characterization of LysP and LysT were successful, while the existence of CBDs remained to be elucidated.
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(2023)Continuous exposure to ultraviolet radiation can cause detrimental health effects in humans, including erythema, hyperpigmentation, photo-aging, and skin cancer. Sunscreens provide important protection of skin against the harmful effects of ultraviolet radiation. However, chemical and physical sunblock filters can be harmful for both human beings and the environment. There is a need for natural alternatives to commercial filters currently used in sunscreens. Mycosporine-like amino acids (MAAs) are natural microbial sunscreens produced mainly by marine organisms to protect themselves from ultraviolet radiation. There has been over three decades of interest in these natural compounds for applications in the cosmetic industry. However, low production levels in nature have hindered large-scale processes for industrial applications. Here, we demonstrates the successful expression of a MAA biosynthetic pathway from cyanobacteria in a metabolically engineered strain of Escherichia coli. Heterologous expression of the artificial codon-optimized MAA biosynthetic pathway resulted in high-level production of porphyra-334 and shinorine. These two MAAs have exceptional high molar extinction coefficients and are found in a number of commercial sunscreen formulations that rely on algal extracts. The expression of a gene encoding an ABC-transporter, which is associated with cryptic MAA biosynthetic gene clusters in cyanobacteria, in Escherichia coli resulted in the effective transport of both porphyra-334 and shinorine outside the cell. Together, these two advances improve the possibility of biotechnological production of these microbial sunscreens in industrial microbial hosts.
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(2022)S. aureus infections are prominent worldwide, and with the rapid increase in antimicrobial resistant variants such as methicillin-resistant MRSA, the need for new treatment alternatives is imminent (Monaco et al., 2017). Lytic bacteriophages are continually evolving new methods for the destruction of bacterial cells while avoiding their defence mechanisms. Screening hypothetical proteins of unknown function (HPUFs) from bacteriophages for toxic activity against bacteria may provide new and potentially life-saving approaches to combat bacterial infections (Liu et al., 2004, Singh et al., 2019). The Stab21 phage of Staphylococcus is a recently described lytic phage with over 85 % of its open reading frames annotated as HPUFs (Oduor et al., 2019). The successful identification of potentially toxic gene products could facilitate the discovery of novel bacterial targets for the development of new antimicrobials. It could also provide treatment options to multi-drug resistant S. aureus caused infections where no effective drugs are currently available. To reduce unnecessary screening of phage particle associated yet poorly annotated proteins, total proteins of phage particle were previously identified by LC-MS. Similar studies have previously been performed with Yersinia phage fR1-RT and Klebsiella phage fHe-Kpn01, where a handful of toxic proteins were discovered (Mohanraj et al., 2019, Spruit et al., 2020). To accelerate the screening process, a next-generation sequencing (NGS) high-throughput screening method was further developed by Kasurinen et al. (2021). In this study, 96 true HPUFs were selected and screened for their bactericidal activity in E. coli using the NGS-based approach. Fourteen potentially bacteriotoxic Stab-21 gene products were identified through toxicity screening in E. coli. Of these, three had a particularly low ratio of isolated plasmid after transformation while having a significant number of reads over each joint sequence, indicating their potentially high toxicity. The three most promising candidates were the gene products of g008, g081c and g175 of the Stab21 bacteriophage.
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(2023)Enterovirukset ovat tärkeitä patogeenejä, jotka kuuluvat Picornaviridae-heimoon. Enterovirus-sukuun kuuluu lukuisia tärkeitä ihmisen patogeenejä, kuten poliovirus, enterovirus 71 ja coxsackievirukset. Yhdisteet, jotka sitoutuvat viruksen rakenteeseen ja estävät viruspartikkelin laajenemisen ovat tutkituimpia enterovirusinfektiota vastaan tarkoitettuja antiviraaleja Testasin kahden yhdisteen, CL213:n ja epigallokatekiinigallaatin (EGCG) inhibitorista aktiivisuutta coxsackievirus A9 (CVA9) -infektiota vastaan. Solukulttuureihin perustuvilla menetelmillä määritettiin molempien yhdisteiden inhibitorisen aktiivisuus CVA9-infektiota vastaan. Lisäksi aktiivisemman CL213:n vaikutus CVA9:n lämpöstabiilisuuteen määritettiin. Jotta varmistettaisiin, sitoutuuko yhdiste kapsidiin, viruksen rakenne kompleksissa yhdisteen CL213 kanssa selvitettiin cryo-elektronimikroskopian avulla. Tulokset osoittivat, että CL213 on voimakas CVA9-infektioinhibiittori in vitro ja se lisää viruksen lämpöstabiilisuutta. CVA9:n rakenne kompleksissa CL213:n kanssa selvitettiin 2.5 Ångströmin tarkkuuteen kryoelektronimikroskopialla. Vaikka lämpöstabiilisuustestauksen tulokset viittaavatkin siihen, että yhdiste sitoutuu viruksen rakenteeseen ja viruksen rakenteen ikosaedrisellä rekonstruktiolla (icosahedral reconstruction) oli korkea resoluutio, yhdisteeseen sopivaa tiheyttä (engl. density) ei löydetty viruksen rekonstruktiosta. Tämän vuoksi yhdisteen tarkka toimintamekanismi jää epäselväksi. Yllättävä tulos tutkimuksissa oli se, että EGCG, jonka on aiemmin osoitettu inhiboivan CVA9-infektiota, ei ollut lainkaan aktiivinen näissä antiviraalisen aktiivisuuden määrityksissä. Todennäköisesti ilmiö johtuu siitä, että EGCG:n toiminta perustuu puhdistetun viruksen saostamiseen ja aggregointiin liuoksesta, mutta yhdisteen aktiivisuus katoaa, kun virus on solujen kasvatusliuoksessa. Koska kumpikaan yhdiste ei tulosten perusteella sitoutunut viruksen rakenteeseen, CVA9:n ja enterovirus 71:n VP1 proteiinin hydrofobisten taskujen (engl. VP1-hydrophobic pocket) rakenteita verrattiin, jotta voitaisiin selvittää ovatko yhdisteet, joilla on nanomolaariset IC50 arvot, hyviä lähtökohtia yhdisteiden jatkokehitykselle. Vaikka molempien virusten rakenteista löytyy kyseinen hydrofobinen tasku, suurin osa yhdisteiden sitoutumiseen vaadittavista sivuketjuista eivät ole konservoituneita. Tehokkaiden antiviraalisten yhdisteiden kehitys enterovirusinfektioita vastaan on siis edelleen merkittävä haaste.
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(2023)Semliki Forest virus (SFV) is a well-studied model virus of medically important mosquito-borne alphaviruses, like chikungunya virus and Sindbis virus. SFV replicates within membrane invaginations called spherules at the host plasma membrane, facilitated by the virus-encoded nsP1 protein. The objectives of this MSc thesis were to identify candidate host proteins interacting with nsP1 based on previous proteomics work and develop a screening workflow to identify stable nsP1 interactors. The overarching aim is to improve our understanding of the roles of host proteins in spherule formation and replication complex functions. The screening workflow involved knockdown assays to assess the antiviral and proviral effects of potential host interactors, followed by cell viability assays for toxicity assessment. Selected promising hits were further investigated for protein interaction with nsP1, which required cloning for mammalian expression and tagging with Myc epitope. Knockdowns resulted in several statistically significantly proviral and antiviral host factors, and all the knockdowns were non-toxic to the cells. Among the significant proviral hits, four promising candidate host proteins were cloned and expressed. Pull-down assays did not reveal stable interactions, suggesting transient or indirect interactions between these host proteins and nsP1. The lack of co-localisation with SFV replication complex supported this finding. This work sheds light on possible transient or indirect interactors of nsP1. The screening workflow effectively identified protein-protein interactions and can be applied to screen additional proteins. Future studies should employ methods suitable for studying transient interactors to gain further insights. This would enhance our understanding of key host proteins in SFV spherule formation and replication, potentially leading to novel antiviral therapies targeting alphavirus replication.
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Isolation and characterization of new bacteriophages against clinical Klebsiella pneumoniae strains (2023)The objective of this thesis was to isolate and characterize new bacteriophages (phages) against clinical Klebsiella pneumoniae strains for phage therapy. K. pneumoniae is causing an emerging threat to global health due to its broad antibiotic resistance profile and hypervirulent strains. New treatment options are urgently needed to defeat the crisis. Phage therapy could provide one option to treat multiresistant K. pneumoniae infections. In this thesis, five new phages were isolated and characterized from Finnish wastewater and Georgian river water against two clinical K. pneumoniae strains. The three phages from Georgian river water, fMtkKpn01, fMtkKpn03, and fMtkKpn04, resembled Drulisviruses based on phylogenetic analysis. The two phages from Finnish wastewater, fJoKpn03 and fJoKpn05 were phylogenetically distinct. fJoKpn03 couldn’t be classified. fJoKpn05 resembled Weberviruses. Based on sequence analysis, none of the phage genomes included any harmful genes that would prevent their use in phage therapy. All phages demonstrated a 6-hour total inhibition to host bacterial growth. Their host range was determined to be narrow, only infecting their respective host strains from the 80 bacterial strains tested. All the phages tolerated high pH well. fJoKpn03 was the only one tolerating very low pH. All phages showed a synergistic effect on the inhibition of bacterial growth when applied together with piperacillin. In conclusion, all five phages proved potential for phage therapy. They demonstrated inhibitory action against K. pneumoniae strains with capsule types against which there previously were no phages in our collection. Due to their narrow host range, they could be suited for personalized phage therapy or used in combination therapy with antibiotics to increase efficacy and duration of action. fJoKpn03 could provide an opportunity for oral administration due to its broad pH stability profile.
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(2021)Global warming affects permafrost in the Arctic regions, where melting organic carbon storages will increasingly contribute to the emission of greenhouse gases. Little is known about tundra soil microbial communities, but Acidobacteria and viruses seem to have important roles there. Here, for the first time, we isolated five Acidobacteria infecting viruses from Kilpisjärvi tundra soils using host strains previously isolated from the same area. Three viruses were isolated on Edaphobacter sp. X5P2, one on Edaphobacter sp. M8UP27, and one on Granulicella sp. X4BP1. The viruses had circular double-stranded DNA genomes 63,196–308,711 bp in length and 51–58% GC content. From 108 to 348 putative ORFs were predicted, 54–72% of which were sequences unique to each virus. Annotations indicated that all five phages most likely have tailed virions. The diversity of viruses present in the studied soils was estimated with the metagenome analysis. Only 0.1% (627) of all assembled metagenomic contigs were phage-positive. The gene-sharing network analysis showed approximately genus-level clustering between the virus isolates and a few metagenomic viral contigs, but overall, all (except one) viral contigs clustered only with each other, not with any known viruses from the NCBI database. No taxonomical assignments could be done for the metagenomic viral contigs, highlighting overall undersampling of soil viruses. Further detailed studies on virus-host interactions are needed to understand the impact of viruses on host abundance and metabolism in Arctic soils, as well as the microbial input into biogeochemical cycles.
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(2022)Sorghum (Sorghum bicolor) and amaranth (Amaranthus spp.) are gluten-free cultivated crops native to Africa, and thanks to their drought-tolerating capabilities and adaptive nature, they provide an energy source in areas where cultivation of wheat is not possible. There is a need to exploit these indigenous crops and develop food products with better technological and nutritional properties to battle malnutrition on the continent. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) have shown promising properties in improving the technological quality of gluten-free foods. The aim of this project was to identify naturally occurring LAB and yeasts from red sorghum grain, amaranth grain and amaranth leaf flour and from spontaneously fermented sourdoughs made from these flours. Possible EPS-producing LAB were of special interest. The sourdough pH and total titratable acidity were recorded daily, and microbial densities were calculated on selective media. Isolates were selected for sequencing based on morphology and biochemical properties. Twenty seven bacterial isolates, twelve of which produced EPS, were identified by partial 16S rRNA and pheS gene sequencing, and seven yeast isolates were identified by sequencing the variable D1/D2 region of the large subunit rDNA. The identified LAB belonged to five genera: Enterococcus, Lactiplantibacillus, Leuconostoc, Pediococcus and Weissella. The yeasts belonged to the genera Meyerozyma, Pichia and Rhodotorula. In general, many studies focusing on sourdough microbial communities usually concentrate on LAB, with little information on the yeasts currently being available. To the best of the author’s knowledge, this is the first report on sourdough made from amaranth leaves and its native microbial content, with 9/12 isolates producing EPS. This provides an excellent starting point for further study of the technological side of creating a gluten-free baked product using starter bacteria native to the flour itself.
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(2021)The aim of this master’s thesis was to examine university teachers’ professional vision and misconceptions from the perspective of the role of students’ prior knowledge in learning. We also examined how participants’ professional vision and concepts changed during the pedagogical course. University students can also have a lot of misconceptions which differ from scientific view. Those misconceptions can make learning harder and even hinder it. Teachers should recognise these misconceptions and they should be able to support students’ conceptual change in their teaching. Participants (N=73) were life science university teachers. They were selected to this study because they participated in two university pedagogical courses with the same content. Participants’ professional vision, conceptions and beliefs were investigated with a video annotation and two questionnaires. Study is quasi-experimental research with pretest-posttest design. Video annotation and one of the questionnaires were tested before and after the pedagogical course. The delayed questionnaire was collected six months after the course. Purpose of the delayed questionnaire was to know if the teachers have been using the things they learn in a course in their own teaching. At the analysis phase participants were divided in to three groups according to their previous teaching experience and pedagogical courses (novices, experienced teachers, and most experienced teachers). Then we were able to compare these three groups and examine if the teaching experience had any effect on the answers. This study utilized a mixed methods approach and analysis was made with both quantitative and qualitative methods. The results show that pedagogical course changed teachers’ concepts considering learning and teaching. All participants’ groups got better scores in professional vision after the pedagogical course despite their previous teaching experience or pedagogical courses. Novices got lowest scores in the pretest which was expected because they didn’t have any previous experience. Their answers changed significantly in all research aspects. Experienced and most experienced teachers also got better scores in posttest especially in professional vision. Developed professional vision was related to more constructivist beliefs of learning. These findings support previous studies that even short pedagogical course can change teachers’ beliefs and concepts about teaching and learning.
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(2022)Common sexually transmitted pathogens like human papillomavirus (HPV) and Chlamydia trachomatis have profound effects on sexual health of women ranging from acute genital tract infections to cancer and infertility. These infections are often asymptomatic, increasing the risk of being left untreated and thus increasing the probability of complications. Most cervical cancer cases are caused by persistent HPV infection and would be preventable by timely HPV detection. Cervical cancer screening using HPV nucleic acid detection has proven efficient and is used in many European countries, including Finland. Chlamydia trachomatis and Neisseria gonorrhoeae infections can lead to upper genital tract infection and increased risk of ectopic pregnancy and infertility. C. trachomatis infections are also associated to increased risk of epithelial ovarian cancer. Due to lack of national screening program and often asymptomatic nature of these infections, many of them are left untreated. In order to streamline cervical cancer screening and to include C. trachomatis and N. gonorrhoeae analysis in the same package with HPV, a liquid-based cytology sample medium, BD SurePath™ was validated for use with cobas® 4800 HPV and CT/NG nucleic acid detection tests. Work presented here shows that HPV and CT/NG tests and cytological analysis can all be done from a single sample in an efficient, reliable and cost-beneficial way. Based on the results, Vita Laboratoriot will be able to offer the package analysis to their customers.
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(2023)Lignocellulosic biomass is an abundant and sustainable resource to produce valuable products through biorefinery processes. However, the challenge of lignin degradation remains complex. My thesis aimed to investigate the potential of Pleurotus ostreatus, a white rot fungus, for use in biorefinery applications. This study investigated the capability of P. ostreatus to grow and consume technical lignin (organosolv) with great potential for utilization in lignocellulose biorefineries. To further enhance its bioconversion potential, the study also explored the metabolic engineering of P. ostreatus to produce protocatechuate. Additionally, the P. ostreatus PC9 monokaryon strain was found to be suitable for genetic engineering using the CRISPR Cas9 system; however, the inability to disrupt the KU80 gene may have contributed to the lower efficiency of gene targeting observed in this study. The lignin degradation potential of P. ostreatus secretomes was investigated by measuring enzyme activities and using model compounds. The use of P. ostreatus secretomes has shown potential for improving lignin degradation in co-culture with an engineered Aspergillus niger strain which accumulates the protocatechuate, and the addition of nucleophilic thiols, such as L-cysteine, showed promise in promoting lignin bioconversion.
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(2023)The emergence of antibiotic resistance is a growing concern globally. The horizontal spread of antimicrobial resistance genes (ARG) causes multi drug resistant strains that can be harmful to human- and animal health. This risk must be considered when new bacterial strains are used in the plant protection industry, therefore this master’s thesis presents a new bioinformatical method to evaluate the potential of ARG to horizontally transfer to another bacteria. This thesis will walk through analyses that can be used to hunt for ARGs and mobile genetic elements (MGE) in bacterial whole genome sequence data. Also, this thesis presents a new computational analysis tool called MGEradar that reveals MGEs that are linked to a specific ARG. Also, this thesis presents a simple metod for studying the prevalence of an ARG among other bacterial strains of the suspect species. MGEradar, the prevalence analysis and the bioinformatical pipeline can be helpful tools to evaluate the intrinsicness and mobility of an ARG. For the evaluation of the bioinformatical method, the genomes of Escherichia coli and Bacillus thuringiensis are examined to determine the MGEs associated with two ARGs, mcr-1 and fosB.
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(2023)The use of plastics has increased globally and more and more of it ends up in the environment. Microbes can be used to produce polyhydroxyalkanoates (PHA), a biodegradable plastic substrate. Instead of nonrenewable fossil raw materials, such as renewable sewage sludge can be used as a carbon source for polyhydroxyalkanoates. The aim of this work was to investigate the microbial diversity and metabolism of the first hydrolysis step of the three-step polyhydroxyalkanoate (PHA) production process. In the first step, organic polymers, such as carbohydrates, lipids and proteins are hydrolyzed into monomers, which are then converted into short-chain volatile fatty acids (VFA). The volatile fatty acids are used in the third step as a substrate in microbial PHA production. In the second step, the polyhydroxyalkanoates accumulating microbes are enriched, and used in the third step for PHA synthesis. In this work, different types of sludges were used as a carbon source. Sludges were sludge for biogas production, sludge after biogas production and sludge after nitrogen removal in stripping. The concentration of volatile fatty acids, cellulose and lignin was determined in the bioreactors. Volatile fatty acids were determined by gas chromatography. Cellulose and lignin were determined after hydrolysis by filtering and drying the samples. The nucleotide sequence of the 16S rRNA gene was determined from pure materials. Shotgun metagenome sequencing was performed on bioreactor DNA samples to sequence the entire microbial genomes. Hydrolysis bioreactors were maintained for 12 days. Microbes did not degrade cellulose and lignin well. The best volatile fatty acids yields were obtained from the sludge for biogas production (172 mg/g organic matter ± 6.25). Proteobacteria and Firmicutes were the major phyla in the bioreactors, and microbial genera differed greatly between bioreactors. Microbial genes coding for carbohydrate and protein metabolism were predominant in the bioreactors.
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(2023)Microbiology is included in the subject of biology in lower secondary school as part of the National Core Curriculum for Basic Education (POPS). Microbiology teaching has traditionally relied on inquiry-based methods and practical work, where inquiry is highlighted in POPS. In this study, a survey of teachers investigated microbiology teaching and inquiry-based methods in lower secondary school. The study focused on these areas: 1. What groups of microbes are mentioned and in what biological contexts? 2. What methods are used in teaching microbiology? 3. How much are inquiry-based methods used and does the amount vary regionally? 4. What type of inquiry-based teaching is used? and 5. What are the potential limitations in delivering inquiry-based teaching? The data were collected by an online survey for biology teachers in lower secondary schools around Finland and 36 responses were received. The data were analyzed mainly by qualitative content analysis which was supported by quantitative and regional analysis. According to the results, the coverage of microbial diversity is wide in biology teaching but Archea may receive less attention. Microbiology is taught well in many biological contexts but least in the context of evolution and development of life. The teaching methods are diverse, and many different practical activities are carried out. Inquiry-based methods were utilized by 97.2% of teachers and the amount does not vary regionally. Structured inquiry is used the most in microbiology teaching and the majority of the teachers found inquiry-based methods valuable. The amount of inquiry is limited by a lack of time, size and heterogeneity of the student groups, lack of equipment and workspace. The results indicated that the teaching of microbiology in lower secondary school is diverse and inquiry-based methods are common, but limitations were expressed. Solutions in response to these concerns could be, for instance, virtual activities, improved learning materials and more collaboration between universities and schools which is now poor. There are so far no other published studies about this in Finland. In the future, it would be interesting to study further how inquiry-based methods and the teacher’s self-efficacy and own training in these areas affect the outcomes of microbiology education. Such research could then be used to improve teacher education to address the limitations presented in this thesis.
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