Browsing by study line "ei opintosuuntaa"

The genetic and morphological diversity of viruses and more specifically membrane-containing bacteriophages (phages) with single-stranded DNA (ssDNA) genomes is largely unexplored. It can be difficult to detect evolutionary relationships of viruses using solely sequence-based methods due to their rapid sequence evolution. However, more distant evolutionary connections of viruses have been observed based on structure data. Here we introduce an icosahedral tailless ssDNA phage, Cellulophaga phage phi48:2, isolated from the Baltic Sea that has not been assigned to any virus family or taxa. Phage phi48:2 has been previously linked to the family Finnlakeviridae whose members are icosahedral, internal membrane-containing phages with circular ssDNA genomes. However, the presence of lipids in phi48:2 virion has not been studied. In this study, different buffer conditions were tested for infectivity and stability of phi48:2 allowing us to optimize the purification of the phage particles by rate zonal and equilibrium ultracentrifugation in sucrose. Solvent tests in chloroform and ether, as well as low buoyant density of the virion suggested the presence of lipids in the phi48:2 virion. Analysis of the phi48:2 lipids extracted from highly purified virions by thin-layer chromatography revealed that phi48:2 is a membrane-containing phage and acquires its lipids unselectively from its host bacterium Cellulophaga baltica. Lastly, cryogenic electron microscopy of the purified virions also proposed that lipids form a membrane vesicle under the capsid. Altogether our results show that phi48:2 is an icosahedral membrane-containing phage, thus connecting it further with FLiP, which is the sole member of family Finnlakeviridae. Moreover, FLiP and phi48:2 virions are both ~60 nm in diameter and showed some similarity in their major capsid protein sequences (~21% amino acid identity). To conclude, even though phi48:2 and FLiP share various similarities they cannot be placed within the same family due to the low similarity in their genome sequences. However, for now we can assume they are possible distant relatives. The diversity and abundancy of membrane-containing ssDNA phages is gradually starting to uncover and through their characterization and classification we might consequently understand better their significance in microbial ecology.

In recent years, exceptionally large bacteriophages with genome sizes over 500 kilobase pairs (kbp), called megaphages, have been discovered from sequence data, but no previous publications discussing megaphage isolates have been published. In 2011, a phage infecting a Flavobacterium strain was isolated from the Kymijoki river. The phage, named FKy-1, was determined to have a genome size of 643 kbp, based on yet unpublished results, making it the first described megaphage isolate. In this study, we focused on characterizing megaphage FKy-1, by observing the virus morphology, determining the type and length of its life cycle, and measuring its stability in different temperatures and conditions. Purification of the phage by precipitation and ultracentrifugation in a sucrose density gradient resulted in separation of both virion and phage subcomplexes. Based on transmission electron microscopy and cryogenic electron microscopy, FKy-1 was observed to have typical myovirus morphology, with a large icosahedral head of around 160 nm in diameter, and a tail of around 180 nm in length. Molecular masses of the major proteins present in the virion and phage subcomplexes were estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 50-70 kDa for the major capsid protein, 60-70 kDa and 150-200 kDa for the major tail proteins. Digestion attempts with restriction endonucleases proved unsuccessful, indicating possible phage genome modifications or other defensive mechanisms. The phage was determined to have a lytic life cycle which takes over 3 h to cause cell lysis, resulting in the release of around 10 progenies per infected host cell. The phage proved to be quite stable, with minimal impact on infectivity measured at a temperature range of -20 °C to +40 °C, and in minimal buffer conditions. In summary, we proved that the purification method used here is well suited for megaphages, and that FKy-1 is of myovirus morphology, produces a low number of progenies per host, and is relatively stable. As no other publications regarding megaphages exist, this study acts as a good basis for future research regarding megaphage morphology, infection cycle and stability.

Chloroplasts are essential plant photosynthetic organelles evolved from a prokaryotic endosymbiont many years ago. A vast majority of chloroplast proteins are encoded in the nucleus and then imported post-translationally by multiprotein translocases located in the membrane of the organelle. It was identified that outer envelope membrane (OEM) components are subject to ubiquitin-proteasomal degradation, governed by a recently established proteolytic system called CHLORAD (chloroplast-associated degradation). It has been suggested that this machinery is involved in regulation of plastid biogenesis and stress tolerance in plants by protein import regulation and remodelling of the organellar proteome. In this study, to further investigate factors involved in chloroplast protein import regulation, we aimed to characterize two putative regulators SKIP6, an F-box/kelch repeat protein, and ASK1, a component of CUL1-based SCF E3 ligase, identified by tandem affinity purification of TOC components and SP1. We performed physiological analyses on skip6-1 and ask1-1 single mutant Arabidopsis plants to identify whether these factors are required for degradation of OEM translocase components (TOC machinery). To identify an association of these factors with the TOC machinery and CHLORAD components, we employed subcellular localization and co-immunoprecipitation (IP) assays in protoplasts. Double mutant sp1 ppi1 and sp2 ppi1 plants were previously shown to specifically supress an atToc33 mutation (specific suppression of ppi1 chlorosis phenotype), resulting in greener and larger plants. Following, for second-site specific suppressor analyses of the atToc33 mutation, we generated ask1-1 ppi1 double mutant plants and provided their initial characterization. As the CHLORAD system was shown to be vital for plant development and to contribute to stress tolerance, therefore, in this study the involvement of SKIP6 in stress tolerance in mutant plants was analysed by implementing osmotic and salt stresses. Physiological analyses revealed an early-senescence phenotype in the skip6-1 single mutant plants, which could be attributed to degradation of TOC components and subsequent decrease in chlorophyll level. Interestingly, an opposite effect was observed after dark treatment, in which SKIP6 knockout mutants remained greener with higher abundance of TOC proteins and chlorophyll level in comparison to wild-type plants. Stress-induced experiments did not show the involvement of SKIP6 in stress tolerance at early developmental stages. Subcellular localization and co-IP experiments revealed cytosolic localization of SKIP6 and its physical interaction with the TOC machinery, respectively. Obtained double mutant ask1 ppi1 plants presented male sterility as well as growth suppression followed by greener leaves at late developmental stages. In summary, our results provide initial characterization of unknown SKIP6 protein suggesting its involvement as a component of SCF E3 ligase (CUL1-ASK1-SKIP6) in the reorganization of the TOC machinery and CHLORAD components at early and late developmental stages, respectively. These initial data represent one of the first steps towards broadening our knowledge on the regulatory network of chloroplast biogenesis in plants, as well as important advance in the development of new strategies for crop improvement.

Abstract Faculty: Faculty of Agriculture and Forestry* and Faculty of Medicine *coordination Degree programme: Master′s program in Microbiology and Microbial Biotechnology Author: Johanna Potila Title: Characterization of potentially therapeutic bacteria from a healthy fecal donor. Level: Master′s thesis Month and year: August 2023 Number of pages: 40 Keywords: Clostridioides difficile, dysbiosis, FMT, next-generation probiotics, adhesion, anti-inflammatory Supervisors: PhD Kaisa Hiippala, PhD, Docent Reetta Satokari and PhD Pauliina Lankinen Where deposited: E-thesis University of Helsinki Abstract: Recurrent Clostridioides difficile infection (rCDI) is a healthcare-associated infection related to antibiotic use, that causes significant morbidity and mortality. Fecal microbiota transplantation (FMT) is the most effective treatment for rCDI and it is successful in nearly 90% of patients. However, there are some risks related to FMT use such as the potential risk of transferring pathogens or other phenotypes despite donor screening. Defined bacterial mixtures consisting of endogenous commensal gut microbes with beneficial properties could be used instead of FMT to mitigate the risks and improve the availability of the treatment. 12 bacterial strains previously isolated from a healthy fecal donor were characterized in this study. At first, oxygen tolerance and culturability of the isolates in several different media were examined. The second aim was to investigate if these isolates are safe for bacteriotherapeutic use by testing hemolytic properties, antibiotic susceptibilities and proinflammatory properties. The third objective was to investigate potential beneficial properties such as adherence of the isolates to mucus and epithelial cell lines and anti-inflammatory effects on epithelial cells. Caco-2 and HT-29 cell lines were used as a model of intestinal epithelial cells. Growth was abundant on standard brain heart infusion (BHI) medium supplemented with 0,5% yeast extract and more than half of the isolates tolerated the 4-hour oxygen exposure. These results suggest that many of the strains have good production characteristics. All 12 isolates were non-hemolytic and most of them were susceptible to many commonly used anti-microbials such as amoxycillin/clavulanic acid and piperacillin/tazobactam. Low induction of interleukin-8 (IL-8) release from HT-29 cells was observed for all the isolates which indicates no pro-inflammatory effect. These safety tests suggest that the isolates are safe for therapeutical use. Adhesion to mucus and intestinal epithelial cells (HT-29, Caco-2) was low to moderate (2-7%), which can potentially promote their colonization in the gut. No attenuation of Escherichia coli lipopolysaccharide (LPS)-induced IL-8 release from HT-29 cells was observed, which indicates that characterized strains do not have anti-inflammatory effects on epithelial cells. However, it is likely that they have some other important roles in the gut e.g., in cross-feeding networks and can thus help with restoration of a healthy, diverse gut ecosystem. In conclusion, the characterized isolates could be suitable for bacteriotherapeutic use in the treatment of rCDI.

The coronavirus disease 19 (COVID-19) pandemic currently poses a challenge to the healthcare system and global public health. The upsurge of new SARS-CoV-2 variants, the uneven vaccine distribution worldwide, and the documented reinfections raise a concern about the protective immunity of COVID-19 recoverees. In this context, reliable methods for the detection of SARS-CoV-2 neutralizing antibodies are needed. Considering the methodological complexity and limitations of traditional virus neutralization tests, surrogate enzyme-linked immunosorbent assays (sELISA) constitute a promising alternative allowing high-throughput testing. However, there is still a need of assessing the specificity and sensibility of these assays so that they can be clinically applied. In this thesis, two goals were pursued; the detection of neutralizing antibodies in COVID-19 recoverees plasma samples using an in-house microneutralization assay and the comparison of these results with those obtained with two sELISA; SARS-CoV-NeutraLISA surrogate neutralization (Euroimmun) and cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript). The SARS-CoV-2 microneutralization assay was performed with VERO E6 cells and the Fin-1 strain of the SARS-CoV-2 virus. The plasma samples were provided by the Helsinki University Hospital and were previously screened with commercial IgG-ELISA targeting the anti-SARS-CoV-2 spike subunit 1 (Euroimmun) and nucleocapsid (Abbott) proteins. A total of 111 samples were tested, 74% of them presented a detectable NAb titer with at least two of the methods. The neutralizing antibody titer obtained with the microneutralization assays resulted in an overall proportion of positives lower than expected. Therefore, the in-house microneutralization assay needs further optimization or a different neutralization assay should be selected instead for future analysis. The combined data from the three tests was used to determine the sensitivity (99%, 83%, 81%) and specificity (72%, 100%, 100%) of cPass, Neutralisa and microneutralization assays respectively. This data suggests the use of cPass (GenScript) in primary screenings, in combination to Neutralisa (Euroimmun) to confirm secondary tests.

Semliki Forest virus (SFV) is an enveloped virus with positive-sense single-stranded RNA genome that encodes nine proteins, of which four non-structural proteins, nsP1-4, form the replication/transcription complex (RTC) along with several host proteins, which play an important role in the replication of the virus. To establish the interactome of SFV RTC, a promiscuous biotin ligase capable of biotinylating proximal endogenous proteins in the presence of exogenous biotin was genetically fused to nsP3. After establishing the stability, kinetics and functionality of this virus, BHK-21 cells were infected with this mutant SFV at multiplicity of infection of 50 plaque forming units per cell. At an early time point of 2.5 hours post infection, 50 μM biotin was added to medium for 15 minutes. Cells were lysed, and biotinylated proteins were enriched with streptavidin beads, and analyzed through tandem mass-spectrometry. We were able to identify several key host protein interactions, some of which were already established before, but also a several new ones. Many of the host proteins detected were involved with the formation of stress granules, including G3BP’s, or contained a SH3-binding domain (SRC homology 3) like CD2AP, SH3KBP1 and BIN1, and some of them also had RNA binding motifs. In future, we wish to study the role of these identified host proteins in the replication of SFV through gene silencing as well as their co-localization with the RTC and nsP3 with the help of Immunofluorescence.

Preserving viral nucleic acids is of outmost importance to capture the viral diversity in metagenomic studies. In my master’s thesis, I compare viromes of genus Culex mosquitos stored in two different virus storage media and empty tubes. The mosquitos were collected from Kalajärvi in Espoo, Finland in the summer of 2020 as larvae and were grown to adults in laboratory conditions. Eight pools of five female mosquitos each were stored in each of the two media as well as empty tubes and the samples were homogenized The homogenates were filtered, and the RNA was extracted from them with TRIzol reagent. RNA was then reverse-transcribed to cDNA and amplified with a whole transcriptome amplification kit. The PCR product was prepared with a library preparation kit for sequencing with Illumina Next Generation Sequencer. The resulting reads were processed with a bioinformatic pipeline for identifying viruses from metagenomic sequence data. The results show a clear difference in virus species distribution by storage media. We identified 34 virus species from at least 13 families. Samples stored in ∑-Virocult had the highest yield of viral reads (70.40% of all reads from the pools) as well as the widest variety of mosquito species (n=26). Samples stored in empty tubes had the second most mosquito species (n=10) but the lowest viral read yield (1.25%). RNAlater stored samples had the least virus species (n=7) but a higher viral read percentage than those stored in empty tubes (3.26%). The results indicate the importance of choice of storage media. Since ∑-Virocult had the highest amount of reads and the widest variety, it might be the most useful storage media for our purposes. However, some viruses were found in other samples but not in ∑-Virocult stored samples, indicating a need of different storages conditions for different viruses. It is also important to be consistent in the use of media as it may affect virome results. More work needs to be done to assess if these results are true for other mosquito species as well.

Punkkivälitteiset patogeenit (PVP) ovat yleisimpiä zoonoottisten tartuntojen aiheuttajia Suomessa. Tautitaakkaan vaikuttavat erityisesti Borrelia burgdorferi sensu lato -bakteerien (BBSL) ja puutiaisaivokuumeviruksen (TBEV) aiheuttamat tartunnat. Molemmat ovat lisääntyneet Suomessa viime vuosikymmeninä. Tapausten lisääntymisen voi pääosin laskea taudinaiheuttajien päävektorien, puutiaisen (Ixodes ricinus) ja taigapunkin (I. persulcatus), levittäytymisen ansioksi. Ulkoloisina punkit välittävät tauteja useisiin selkärankaisiin ruokinnan yhteydessä. Ankarat talvet ovat rajoittaneet punkkien levittäytymistä pohjoiseen, mutta ilmastonmuutoksen aiheuttamat leudommat talvikelit ovat edistäneet valloitusretkeä uusille alueille. Vaikka sopiva ilmasto on olennaista punkkien esiintyvyydelle, vaikuttaa punkkien isäntälajiston rakenne mitä luultavimmin punkkien ja PVP:ien yleisyyteen paikallisella tasolla. Tässä tutkielmassa punkkeja kerättiin 6 aitausalueelta ja niiden ympäristöstä. Samoilta alueilta määritettiin lisäksi piennisäkkäiden ja hirvieläinten määrät. Koska aitaukset ja niiden ympäristöt luultavasti edustavat samankaltaista ilmastoa, pyrittiin tutkielmassa tutkia, kuinka punkki-isäntiin liittyvät tekijät vaikuttavat punkkien ja PVP:ien esiintyvyyteen. TBEV-positiivisen poolin sekä TBEV-vasta-aineita kantavien piennisäkkäiden perusteella Hangossa varmistettiin mahdollisesti uusi TBEV pesäke. Aitausten, piennisäkäsmäärien ja hirvieläintiheyksien pohjilta luotiin yleistettyjä lineaarisia sekamalleja (GLMM) selittämään punkkien ja PVP:ien esiintyvyyttä tutkimusalueilla. TBEV jätettiin mallinnuksen ulkopuolelle pienen otoskoon takia. Hirvieläintiheydet olivat erittäin merkitsevä punkkien esiintyvyyteen vaikuttava tekijä. BBSL:n yleisyyteen ei yksikään tekijä vaikuttanut merkitsevällä tasolla – mahdollisena syynä piennisäkäspopulaatioiden muutoksen viiveellinen heijastuminen kerättyjen punkkien tartunta-asteeseen.

Take away food has increased in popularity in the past years. However, there are not many cardboard-based take away packaging options for restauranteurs. Plastic materials such as expanded polystyrene are most commonly used for take away packaging, but the single-use plastics directive by the European Commission has added polystyrene as one of the materials to be banned by 2021. Additionally, consumers are also becoming more educated on material sustainability, which brings added pressure and opportunity for developing new alternatives to the market. This case study implemented customer-dominant logic (CDL) to the design process of a cardboard-based take away package. In CDL, value-creation is perceived as a personal, subjective and holistic process, where the customer is in the center of the value formation process. The aim was to create a CDL based design framework and test whether it resulted in a cardboard-based take away package which created value for the consumer. The case study also aimed to uncover the factors of take away packaging which contributed to the value creation for customers. The results were collected through three consumer studies, which used qualitative methods such as responsive interviews and the Value Toolkit®. It was concluded that cardboard as a material was seen as renewable and easy-to-recycle. The cardboard-based package was successfully designed, as it was rated highest in comparison to a polystyrene-based package and a compostable bagasse package in the final consumer study. It was discovered that out of the four value types: performance, experience, status value, and responsibility, consumers thought performance was the most important in take away packaging. The CDL based framework for package design was successful. The framework can further be studied with collateral case studies, where one design team uses the CDL based framework and the other uses a more traditional approach to design.

Emerging research suggests that bacteriophages (phages) may exhibit alternative infection strategies that deviate from the preconceived lytic or lysogenic life cycles. Carrier cell infection is an alternative phage life cycle where complete virus particles are formed and remain within host cells, without cell lysis or integration into the host genome. Phage Φ6 (Φ6), the type member of the double-stranded RNA (dsRNA) virus family Cystoviridae, is a lytic phage that can also establish a carrier cell within its plant pathogenic host, Pseudomonas syringae pathovar (pv.) phaseolicola strain HB10Y (HB10Y). This thesis contributes to current limited knowledge and provides an insight on the underlying mechanisms of the Φ6 carrier cell infection. This study has agricultural and ecological relevance and may contribute to future plant therapeutic options. Synthetic carrier cell lines harboring Φ6 tri-segmented genome or Φ6 genomic constructs in which the coding regions in the S- and/or M- segments were replaced by heterologous sequences from tobacco mosaic virus (TMV) were created using a reverse genetics method. Spontaneous Φ6 carrier cell lines were also isolated from HB10Y after exposure of the host to excess phage. Spontaneous carrier cells were not stable, but rather occasionally released phage into liquid culture. Synthetic carrier cell lines were subjected to secondary phage infection and were found to be less susceptible than wild type (WT) to Φ6 but not Φ8, a more distant member of Cystoviridae. Studies suggest that carrier cell resistance to secondary infection (superinfection exclusion) is exhibited through the Φ6 S-segment gene 8. To test how temperature affects the stability of Φ6 carrier cells, spontaneous carrier cell line culture was incubated at RT and 30°C, and phage productivity was compared. Elevated temperature induced carrier cell stability. Comparison of the growth curves between Φ6 synthetic and spontaneous carrier cell lines and their respective WT strains showed that Φ6 carrier cell infection does not greatly affect host growth.