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The use of plastics has remarkably increased during the last 50 years. Due to its multi-use possibilities, durability and low cost the use of plastics is expected to double within the next twenty years. Despite the many benefits of plastics, the negative impacts to the environment, such as marine plastic, have raised the public attention and accelerated the demand for legislative action. To respond to these challenges, the European Commission (EC) engaged in developing a set of policies, such as the action plan for circular economy in 2015 which aimed to reduce the use of plastics and to enhance the recycling and reusing of plastics already in circulation. In 2018 the EC introduced a set of revised directives regarding the extended producer responsibility (EPR). I wanted to understand how this revised EPR scheme would be implemented into national legislation, and how it would be confronted by organisations such as ones dealing with wood products. Additionally, I also mapped the potential managerial implications for stakeholders resulting from the implementation of the scheme. I operationalised Primmer's (2011) framework of institutional adaptation which builds on two complementary strands of literature; policy implementation and organisational adaptation. The two focal research questions that derived from the framework were: How did the involved actors recognise and perceive the challenges in 1) implementing and 2) adapting to the extension to the existing EPR scheme in the use of plastic packaging of wood products? The data were collected by conducting seven semi-structured interviews with representatives of stakeholder organisations. The insights were explored and reported from these data via the means of qualitative content analysis. Considering the complexity of policy and the diversity of organisations across Finland – and the European Union, legislators are in a rush to implement the new requirements of the directives into the national legislation. Based on my studies the involved actors recognised the various challenges in the implementation and adaptation processes. There were also expectations that the policy will drive learning and innovation among organisations. Currently, legislation is the major driving factor in the development of plastic recycling and EPR schemes can be a robust legislative tool in attaining the targets, when developed and maintained accordingly.

Globalization encourages migration all over the world. Dietary acculturation, the process of adopting the dietary practices of the host country, has become an interesting issue in community nutrition and nutritional anthropology. This is the first study on Chinese immigrants and dietary acculturation in Finland. In this study, the Koctürk model is used as conceptual framework which had showed its usefulness to structure the various foods and changes which may occur. The thesis aims to investigate whether any changes take place in the food habits of Chinese students after migration to Helsinki, Finland, as well as the food habits which are still maintained after migration. If changes or stability occur, the factors that are associated with the changes or stability are analyzed. Data were collected from 16 Chinese students who study in university of Helsinki, and have been staying in Finland at least 6 months. A tape-recorded interview was arranged, which included a questionnaire and an interview with semi-structured questions. After migration, several changes appeared in meal pattern, food choices and food preparation methods among Chinese students. Breakfast seemed to be the first meal to be “Westernized”. In accordance with the Koctürk model, the cultural importance of breakfast and lunch has diminished, and dinner became the most important meal. Weekend diet became more traditional than weekdays’ diet. Chinese participants still keep festival diet in certain Chinese festivals, and they also tried some typical Finnish festival foods, especially mämmi and glögi. Food choices changed among all food groups—staple, complementary and accessory foods. Of 37 foods listed on the questionnaire, the consumption frequency increased significantly for 14 Finnish style foods and decreased significantly for 7 Chinese style foods. The Chinese students also prepared foods more often by methods such as baking, adding cheese or butter/margarine to foods. The interviews revealed various factors influencing changes and stability: Chinese cultural beliefs, attitude, taste preference, stress, social relations, food cost, convenience, availability and perceived freshness of foods.

The shelf life of ESL milks and pasteurized creams is limited by bacterial spoilage. The current state of knowledge with respect to the diversity and activities of the spoilage microflora in these products is still limited. To date, studies on this subject are yet to be carried out in Finland. The aim of this study was to characterize the spoilage microorganisms found in ESL milks and pasteurised creams mainly produced in Finland. 10 ESL milks and 8 pasteurised cream packages were obtained from the Finnish retail market and were stored at 8?C, 15?C, room temperature and 30?C, respectively, until expiration. Total bacterial counts were evaluated and 31 bacterial isolates were selected. An attempt was made to characterize the bacterial isolates using rpoB and 16S rRNA partial gene sequence analyses. Psychrotrophic, spore-forming and spoilage features were also determined. Strain diversity was determined by rep-PCR profiling. Five selected isolates were subjected to metabolic profiling using the API50 CHB test. When growth was detected, total bacterial counts ranged from 2.67 to 3.74 and 2.54 to 8.98 log units in cream and ESL milk samples, respectively. Both ESL milks and cream samples were dominated by heat-resistant, spore-forming species of the Bacillus genera: the isolates were related to B. licheniformis. B. weihenstephanensis, B. safensis, B. kochii, B. pumilus, B. subtilis and B. anthracis. Intraspecies and intrasample strain diversity was very high. Isolates mainly displayed proteolytic and lipolytic activity. Psychrotrophic activity was high in the cream samples while most ESL milk isolates were mostly mesophilic. Tested isolates were non-lactose fermenters and had varying metabolic profiles. The study revealed that Finnish ESL milks and pasteurized creams are prone to spoilage by species of the Bacillus genera that have potential for lipolytic and proteolytic spoilage and some of which are considered toxigenic. There is wide intraspecies strain diversity responsible for the wide variability in metabolic, psychrotrophic and spoilage features. Phenotypic tests based on API50 CHB cannot be relied upon to make conclusions on taxonomy. Combining various methods is important for the comprehensive characterization of the bacterial isolates. Strategies to eliminate bacterial species of the Bacillus genera are important to prolong shelf life. However, intervention should be strain-specific to be effective. Large scale studies are required to confirm findings from this study.

Efficient utilization of renewable plant and crop based biomass is one of the main areas of study in industrial biotechnology. Roughly 20-30 % of all plant biomass consists of hemicellulose, a polymer composed of six carbon (hexose) and five carbon (pentose) sugars. This proves a challenge in utilizing all available plant biomass efficiently, since many micro-organisms, which for instance readily ferment glucose (a hexose) into ethanol, will not do so for pentose sugars. Alternative pathways for utilization of pentose sugars are being looked for. Characterization of the Caulobacter crescentus D-xylonate dehydratase (Cc XyDHT) was the aim of this study. Cc XyDHT belongs to the ILVD/EDD protein family, whose members incorporate an iron-sulfur (FeS) cluster into their protein structure coordinated by two to four cysteine amino acids. Removal of the cysteine ligands should disrupt the incorporation of the FeS cluster, and thus presumably the activity of the enzyme. Three cysteines in Cc XyDHT which could act as ligands for the FeS cluster had previously been identified by sequence alignment among the ILVD/EDD family. Accordingly, four single cysteine-to-serine mutants of the Cc XyDHT as expression plasmid constructs were designed, with a non-conserved cysteine residue selected as a control. The wild type and mutated Cc XyDHTs were produced in Escherichia coli and purified with affinity chromatography using Strep-tag. SDS-page and Western blotting with an anti-Strep-tag antibody were used to confirm that the expressed proteins were Cc XyDHTs. The mutations’ effects on the protein fold and to the presence of the FeS cluster were investigated with UV and circular dichroism spectroscopy. Cc XyDHT catalyzes the conversion of D-xylonate into 2-keto-3-deoxy-xylonate. A colorimetric assay using thiobarbituric acid (TBA) as the reactant was used to measure enzymatic activity. The pH and temperature optima, substrate specificity, and enzyme kinetics of the wild type Cc XyDHT were determined. For the two best substrates, kcat/Km = 1220 mM/min for D-xylonate and kcat/Km = 1160 mM/min for D-gluconate were found. All three conserved mutations were found to reduce enzymatic activity more than 99 % with these substrates. Depending on which cysteine was disrupted, differences in the kinetic constants between D-xylonate and D-gluconate were found. The non-conserved mutation reduced activity by approx. 40 %. The spectroscopic results indicate that the three conserved mutants lacked the FeS cluster, while the non-conserved mutant still incorporated it. It can be concluded that the three conserved cysteines are involved in coordinating the FeS cluster, which itself plays an important role in the catalytic activity of Cc XyDHT. These findings should be helpful for follow-up studies in biotechnological application of pentose sugars acids and their derivatives.

Cell walls (CWs) are the safeguards of plant cells and have a crucial role in controlling cell integrity and plant-environment interactions. Perception of cell wall damage (CWD) results in activation of signaling pathways leading to activation of plant defiance responses. Previous research utilizing murus1 GDP-L-fucose deficient mutant has shown the role of GDP-L-fucose biosynthesis in controlling the structure and integrity of the CWs. Here, we investigated whether phenotypical consequences provoked by the lack of GDP-L-fucose, observed in mutants lacking MURUS1 can be suppressed by blocking signal transduction pathways involving THESEUS1 (THE1) receptor-like kinase or WALL-ASSOCIATED KINASE 2 (WAK2)- MAP KINASE 6 (MPK6) signaling module involved in CWD responses. For this, mur1 the1 and mur1 mpk6 double mutants were generated and analyzed. We found that the phenotypes of double mutants closely resemble that of mur1 mutants, indicating that the morphological consequences of GDP-L-fucose deficiency develop independently of THE1 and MPK6 signaling. Further, we aimed to investigate whether GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductases GER1 and GER2 controlling the final step of GDP-L-fucose synthesis are functionally redundant. Due to the inability of generating ger1 ger2 double mutants, we have applied the artificial microRNA (amiRNA) strategy to generate ger1 ger2 amiRNA knockdown plants. We found that these knockdown plants exhibit severe developmental growth defects similar to those observed in plants impaired in import of GDP-L-fucose into the Golgi apparatus and these growth defects exceeded those observed in plants lacking MUR1. Therefore, we have concluded that GER1 and GER2 are functionally redundant.

Despite their immobile nature, their ability for adaptation allows plants to face harmful conditions from the environment to successfully survive and reproduce. Plant cells sense and integrate signals from the environment and activate response mechanisms. Participants in these mechanisms are the receptor-like protein kinases (RLKs) and a subgroup of RLKs, the cysteine-rich receptor-like kinases (CRKs). Members of this family have been associated with functions related to environmental stress responses in plants. CRK2 is one interesting member of the CRK clade of RLKs. While roles of CRK2 in the response to biotic and abiotic stimuli have been recently described, many aspects of the diverse functions of CRK2 remain elusive. The reduced size of the crk2 mutant suggests that developmental processes are affected by the absence of the protein. One of the objectives of this work was to analyse potential reasons for the smaller size of crk2. The difference in plant size could be due to a reduced number of cells. Results from the analysis of young cotyledons showed that the smaller plant size is not due to a reduced cell number in leaves when compared to Arabidopsis thaliana (Arabidopsis) ecotype Columbia (Col-0). Another way to understand the processes in which a protein is involved is to target possible interaction partners. Therefore, genotyping and analysis of growth phenotypes of T-DNA insertion mutant lines for candidate interaction partners for CRK2 was performed. The results revealed smaller phenotype for a nitrate transporter (NRT1.7) mutant in fresh weight and rosette area whereas for a protein kinase (QSK1) mutant, higher fresh weight but reduced rosette area was observed compared to Col-0. Generation of constructs for fusion protein expression and purification revealed the possibility of expressing tagged cytoplasmic regions of these proteins for further analysis of protein-protein interaction through kinase assays due to the kinase activity of CRK2. Generation of fluorescent-tagged proteins from the candidate interaction partners allowed for localization studies via confocal microscopy to determine the co-localization to the plasma membrane of these proteins with CRK2, which is located to plasma membrane under standard growth conditions. The co-localization results suggest that the proteins NRT1.7 and QSK1 colocalize with CRK2, which is a step forward in the verification of their possible interaction in planta. The smaller size of the nrt1.7 and qsk1 mutants indicates that the lack of these proteins affects plant development.

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.

Aspergillus niger is a well-studied filamentous ascomycete fungus and one of the most used fungal species in various biotechnological sectors. Recently the research focus on A. niger has shifted towards production of biochemicals and biomaterials from plant biomass residues. However, there are still many aspects of the plant biomass conversion process by A. niger that are not known in detail, including its sugar uptake systems. Sugar uptake in fungi relies on sugar transporter (ST) proteins that control the uptake of different sugar molecules. Fungal STs are abundant and diverse in their function, which is why many of them remain uncharacterized. Identification of fungal STs for substrates like xylose can improve the industrial production of lignocellulose-based bioproducts by e.g. improving the substrate uptake rate in fungal cell factories. In this thesis, four putative xylose STs from A. niger, XltD, XltE, XltG, and XltH, were characterized both physiologically and functionally in A. niger and Saccharomyces cerevisiae, respectively, together with two previously functionally characterized xylose transporters, XltA and XltB. In addition, a fifth putative xylose transporter XltF was characterized functionally in S. cerevisiae. For the physiological characterization, A. niger 6Δxlt strain was generated by deleting xltA, xltB, xltD, xltE, xltG, and xltH genes using CRISPR/Cas9 methodology. The physiological analysis of the 6Δxlt strain revealed the presence of additional xylose transporters in the A. niger genome, which still remain to be discovered. The functional characterization of the putative A. niger xylose STs was carried out by creating six recombinant S. cerevisiae IMK1010 strains producing the STs fused with a green fluorescent protein. Functional characterization confirmed the correct localization of the STs within plasma membrane, except for XltG which accumulated inside the yeast cells. This indicated potential localization of XltG within endoplasmic reticulum and a putative role in intracellular sugar transport. Growth assays of the recombinant yeast strains demonstrated variable affinities of the A. niger STs for hexoses. The results showed XltF not to be a xylose ST, but instead a hexose ST with low-affinity for fructose and dual-affinity for glucose. The STs were further tested for their substrate specificity and affinity for xylose in a competitive assay between xylose and glucose. XltE displayed a preference for xylose over glucose identifying it as a new low-affinity xylose transporter. Although further research is needed to elucidate the roles of the studied A. niger STs, XltE is a promising candidate for enhancing xylose uptake in fungal cell factories.