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The signal recognition particle (SRP) targets newly synthesized secretory and membrane proteins from the cytosol to the translocon complex on the endoplasmic reticulum membrane. This highly specific co-translational protein targeting is essential for proteostasis by preventing the accumulation of proteins in the cytosol and the mistargeting of proteins. Defects in the SRP68 and SRP72 subunits of eukaryotic SRP have been linked to various inflammatory muscle diseases such as myopathy and myositis. The full role of these subunits in protein targeting and regulation of targeting is unknown. Previously the yeast SRP72 subunit has been degraded using an auxin-inducible degron (AID) system to explore the effect of depletion on protein targeting and cell viability, but the mammalian SRP72-AID has not yet been studied. The aim of this study was to deplete the mammalian SRP68 and SRP72 subunits using the AID system. This study revealed that in the case of SRP68-AID, approximately 65% of the protein is degraded after 2 hours. Respectively, 75% of SRP72-AID degrades after 2 hours and 85% after 4 hours. However, complete depletion of subunits was not achieved during 24 hours of auxin treatment. Quantification of depletion also showed that the strongest decrease in SRP occurs during the first 2 hours. This study demonstrated that mammalian SRP subunits can be depleted using the AID system, providing a good basis for further research to examine the effect of subunit depletion on protein targeting. This may help to solve the mechanisms of diseases associated with SRP68 and SRP72 defects and to develop therapeutics for them.

Tiivistelmä – Referat – Abstract Mental disorders are among the leading causes of global disease burden and years lived with disability. Their pathogenesis is poorly understood and there are enormous challenges in the development of biomarkers to aid in diagnosis and more effective therapeutic options. It has been documented that the microbiota-gut-brain axis shows alterations in mental disorders such as anxiety, depression, autism spectrum disorders, bipolar disorder and schizophrenia. Here we study the gut microbiota of individuals with axis I mental disorders and their unaffected siblings by 16S RNA gene amplicon sequencing. In the Central Valley of Costa Rica, a total of 37 participants were recruited and diagnosed using a Best Estimate Diagnosis protocol. For each of the individuals diagnosed with a mental disorder a healthy sibling was selected after matching by age and gender. A total of 13 pairs of 26 siblings, affected and unaffected, was used for the analysis. In a subsequent analysis, individuals were also divided into the three categories of “unaffected” (UA), “affected without psychosis” (AA) and “affected with psychosis” (AP). They underwent clinical assessments about their habits and diet and about resilience (Connor-Davidson Resilience Scale), current status (SADS-C) and disability (WHODAS 2.0). Their fecal samples were collected freshly and stored at -80°C. DNA was extracted, libraries constructed by PCR and subjected for Illumina MiSeq 300 paired-end 16S RNA amplicon sequencing for analysis of the gut microbiota. The sequencing data were analyzed using the R packages mare and vegan for gut microbiota composition, diversity and richness, taking into account the identified confounders. All participants were of Hispanic ethnicity, residents of the San José Greater Metropolitan Area, adults and 69% of them were women. Affected individuals had major depression, bipolar affective disorder, psychosis non-otherwise specified or schizoaffective disorder. Based on beta-diversity analysis as a measure of the community-level microbiota variation, it was found that the use of levothyroxine (R2=0.08, p=0.005) and of irbesartan (R2=0.068 ,p=0.001) had a significant impact on the microbiota composition and hence the use of these drugs was included as confounder in further analyses. Several statistically significant differences in the relative abundance of intestinal bacteria were identified: Differences were found in the relative abundance of bacterial families Peptostreptococcaceae, Ruminococcaceae, Porphyromonadaceae, and in bacterial genera Pseudomonas, Barnesiella, Odoribacter, Paludibacter, Lactococcus, Clostridium, Acidaminococcus and Haemophilus. Our results indicate that affected individuals have more pro-inflammatory Proteobacteria (Pseudomonas) and less bacteria associated to healthy phenotype, such as Barnesiella and Ruminococcaceae, the former being dose-dependently depleted in AP and AA compared to UA. Furthermore, we documented decreased bacterial richness among affected participants while no significant differences were detected in alpha diversity. Our study identified significant differences in the microbiota of individuals affected by mental illness when comparing to their healthy siblings. The results may have important implications for the holistic understanding of mental health and its diagnosis and therapeutics. Larger studies to confirm these findings would be justified.

In visual detection, thresholds for light increments are higher than thresholds for light decrements. This asymmetry has been often ascribed to the differential processing of ON and OFF pathways in the retina, as ON and OFF retinal ganglion cells have been found to respond to increments and decrements, respectively. In this study, the performance of human participants in detecting spatially restricted (diameter 1.17 degrees of visual angle) and unrestricted increments and decrements was measured using a two-interval forced choice task. Background light intensities ranged from darkness through scotopic to low photopic levels. The detection threshold asymmetry found in earlier experiments was replicated with local stimuli. In contrast, however, the asymmetry between increment and decrement detection thresholds disappeared with fullfield stimuli. An ideal observer model was constructed to evaluate the role of two factors, Poisson variations and dark noise, in determining detection thresholds. Based on the model, these factors are insufficient to account for the increment-decrement asymmetry.

Cytokine release syndrome is a severe systematic inflammatory disease that can be triggered upon pharmaceuticals intake. Evaluating the potential risk levels of novel therapeutics with an optimal assay is therefore essential. In this study, we tried to set up and validate a cytokine release assay from human peripheral blood mononuclear cells (PBMCs) for its application in nonclinical immunotoxicity assessments. Fresh PBMCs were isolated from buffy coats obtained from 11 healthy donors of different characteristics. Freshly isolated PBMCs were treated with LPS, positive control antibodies (anti-CD28, anti-CD3) and their corresponding isotypes (negative control antibodies) in both aqueous and solid formats to assess their abilities to induce cytokine release. Similarly, cryopreserved frozen PBMCs were also incubated with LPS, the positive control antibodies and the negative control antibodies, and compared their cytokine releasing capacities with freshly isolated PBMCs. A nine-cytokine panel (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNFα, IL-12) was screened to select four cytokines (IFNγ, IL-2, IL-6, TNFα) in the following experimental setup. Freshly isolated PBMCs appeared to have higher sensitivity in response to the treatments as shown by the higher level of cytokine release. However, similar trends of cytokine release were observed between freshly isolated and frozen PBMCs in both aqueous and solid assay formats. LPS and anti-CD3 strongly induced cytokine release in all donors. Conversely, anti-CD28 induced cytokine release in some, but not all donors, possibly due to donor specificity. In summary, we have successfully developed and optimized a cytokine release assay, and it can be used to test the potential risk of immune-modulating drug candidate in the preclinical safety studies.

In addition to Chlamydiaceae, eight novel families have been discovered to belong to the phylum Chlamydiae. The eight families are Parachlamydiaceae, Waddliaceae, Criblamydiaceae, Parilichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, Clavichlamydiaceae and Piscichlamydiaceae.These families are phylogenetic relatives to Chlamydiaceae, share the intracellular developmental cycle and are widely distributed in nature and are therefore referred to as environmental Chlamydiae or Chlamydia related bacteria (CRB). CRB have a broad range of potential hosts. All families except Criblamydiaceae cause disease in animals and infect for example fish, arthropods and cattle. Families Parachlamydiaceae, Waddliaceae, Rhabdochlamydiaceae and Simkaniaceae are also shown to cause respiratory disease and adverse pregnancy outcomes in human. Free-living amoebae (FLA) are natural hosts of some CRB. CRB are able to survive and replicate inside of FLA that offers protection and nutrients for CRB. It has been suggested that CRB are transported to new environments inside of FLA. CRB DNA has previously been found on human skin (Hokynar et al. 2016, Hokynar et al. 2018, Tolkki et al. 2018) and in our water distribution system. CRB distributed to our tap- and shower water systems inside of FLA (Thomas and Ashbolt 2011) could be a potential rout of transmission of CRB DNA to human skin. As the diversity and size of the CRB group is large and CRB are very laborious to grow in vitro, it is challenging to detect CRB and to study their pathogenicity. Detection of CRB in clinical and environmental samples is mainly based on PCR methods. A non species-specific PCR method targeting Chlamydiales 16S rRNA (PanChl16S), that in theory amplifies all known CRB, has successfully been used in detection, but post PCR sequencing of the amplicon is required to identify the species. Also, more specific quantitative PCRs have been designed to detect specific families or species of Chlamydiae. However, the volume of clinical specimens available is often limited and allows only few separate analyzes. Due to the challenges identified with detection of CRB, efficient multiplex PCR assays would save time and resources and would be useful tools when detecting CRB DNA. The objective of the work was to explore the possibility of applying multiplexed analyzes to a limited specimen volume effectively. One aim of this thesis was to set up two multiplex PCR assays for detection of seven different CRB and a multiplex PCR for detection of three different FLA. Another aim of the work was to analyze the possibility of CRB to be transported to human skin from our water distribution system inside of FLA. In this thesis we set up two multiplex PCR assays for detection of CRB reference strains P. acanthamoebae, C. sequanensis, S. negevensis, Protochlamydia spp., Rhabdochlamydia spp., W. chondrophila and E. lausannensis. We also set up two PCR assays for detection of three different FLA reference strains: Acanthamoeba spp., Vahlkampfiidae spp., and V. vermiformis. We succeeded in developing two real-time multiplex PCR assays for detection of CRB DNA and two real-time PCR assay for detection of FLA DNA. Variability between replicates for each PCR target was low and the detection limit (100%) for each target ranged from 50-500 control plasmid copies per PCR reaction. The R2-value for each target was ≥0.98 and the reaction efficiency for each target ranged from 82-111%. Samples collected from showerheads (n=18) and water filters (n=2) as well as skin swabs (n=27) were studied with these newly established assays and PanChl16S PCR. The results obtained with the multiplex assays developed in this study were similar to the results obtained with the PanChl16S. CRB DNA was detected in 67% of the showerhead samples, in 100% of the water filter samples and in 31% of the skin swabs. Amoebae DNA was detected in 80% of the showerhead samples. Our results confirm earlier observation that Chlamydiae DNA is frequently observed in human skin swabs and suggest that CRB could be transported to human skin from our water distribution system inside of FLA.

Trichoderma reesei, an anamorph of Hypocrea jecorina, is a filamentous fungus widely used for producing industrial enzymes. T. reesei is used for both endogenous and heterogenous protein production. The optimization of the production conditions and the effects of extracellular agents to T. reesei s production and secretion capacity are crucial for economically sustainable biotechnical production. The available carbon sources, most commonly different types of sugars, have a significant effect on the production and secretion of enzymes by T. reesei. Genetic modification of the pathways through which the fungi recognizes extracellular signals could bring advancements to industrial enzyme production. Because of T. reesei s potential and use as a production strain, the species is an interesting platform for genetic modifications that would enhance the production capacities. With the current methods the genome editing of T. reesei is however slow, and introducing multiple mutations to a single strain can take years. The aim of this study is to optimize the fairly new CRISPR/Cas9 genome editing system for use in T. reesei. In the CRISPR/Cas9 method, a catalytically active Cas9 enzyme is bound to a specific locus of the genome, guided by a guide RNA and the Watson-Crick base pairing principle. Once in the RNA-guided locus, Cas9 introduces a double stranded break in the DNA, which can be repaired by the cells endogenous non-homologous end joining pathways. This repair is error prone and produces mutations to site of the double stranded break. A donor DNA is often introduced together with the Cas9 and guide RNA. This donor DNA includes sequence homology to the site of interest and allows for the use of the cells homologous repair pathways. In this case, the mutation can be better controlled, and for example the risk of chromosomal mutations is reduced. Currently the CRISPR/Cas9 system is widely used in mammalian cell studies and up to 100% mutation frequencies have been reported in yeast cells. In this study the method is optimized for use in T. reesei. To our best knowledge, the research community has not found an organism in which CRISPR/Cas9 would not function. The question mainly lies on what type of set up and component introduction is suitable for each cell type and research purpose. In this thesis, three putative and one already published genes believed to be involved in hexose sugar sensing will be deleted from a T. reesei production strain with the help of CRISPR/Cas9. The effect of these deletions will be assessed through studying the secretion and activity of endogenous cellulases with enzymatic assays. One sugar transporter that may play a part in glucose sensing was identified in this study. The deletion of this transporter caused a decrease in cellulase production and/or secretion. The three other transporters or sensors did not have a significant effect on cellulase production in spent grain extract and lactose or glucose media. It s possible that these genes are involved in the uptake and use of other carbon sources. The continuous expression of the CRISPR/Cas9 system in T. reesei proved difficult. In the continuous expression method at least one of the CRISPR/Cas9 components, the Cas9 protein or the guide RNA, is produced in the cells in vivo. Neither was achieved in this study. Instead, a fully synthetic method in which the Cas9 is transformed into the cells as a protein along with an in vitro produced guide RNA was set up and produced up to 1000 × higher mutation frequencies when compared to the traditional transformation method used for T. reesei. This study also demonstrates a simultaneous deletion of two genes in T. reesei. To the best of our knowledge, multiple simultaneous gene modifications have never been achieved in T. reesei.

The Baltic ringed (Pusa hispida botnica) and grey seal (Halichoerus grypus) populations have experienced dramatic changes in their abundances since the early 20th century, when their populations were much larger than today but since then have declined due to over exploitation and reproductive challenges linked to environmental pollutants. Both populations have however, begun to recover, and their numbers have increased since the 1970s. This increase has led to more seal-inflicted damages to coastal fisheries resulting in the demand to control their populations. In Finland, fishermen have reported significant economic losses, and many consider seals as the main threat to their livelihood. However, our knowledge on the diet composition and foraging behaviour of Baltic ringed and grey seals in Finnish sea area is lacking. In order to achieve sustainable seal management, more information on their diet is thus needed. Therefore, to shed light on the diet composition of Baltic seals in Finland, I examined the stomach contents from 156 ringed and 73 grey seals collected in 2017 across the Finnish sea area. Furthermore, I analysed dietary differences between demographic factors (i.e. age and sex), and seals from different geographic regions. A total of 15 prey taxa, of which 13 fish species or groups were identified. Ringed seal diet was dominated by benthic isopod Saduria entomon that was recovered from over half of the stomachs. In addition to Saduria entomon, herring (Clupea harengus) were the most important fish species consumed. Other important prey were gobies (Gobiidae), smelt (Osmerus eperlanus) and common whitefish (Coregonus lavaretus). In terms of biomass, common whitefish became the most important prey whereas in numbers gobies dominated the diet. For grey seals, herring were the most common and numerous prey consumed that made up most of their diet. Other common species were sprat (Sprattus sprattus) and smelt. Other prey did not contribute substantially to grey seal diet. Additionally, the results of this study showed differences in diet composition between seals of different age and sex.

Cyanobacteria are an important part of the phytoplankton community and aquatic ecosystems. Cyanobacteria can form large mass occurrences, i.e. blooms, which can be toxic or cause other harm. Research and monitoring of cyanobacteria has been based on microscopy analysis. However, molecular-based methods, such as 16S rRNA sequencing are replacing microscopy analyses in the near future. The Finnish Environment Institute has stated that molecular methods are part of environmental monitoring before 2030. In this Master’s thesis the aim was to determine whether conventional microscopy analyses and 16S rRNA sequencing differ when comparing nano- and micro-sized cyanobacteria. The material was collected from a laboratory experiment of the Finnish Environment Institute’s (SYKE) MiDAS project, which was conducted in the summer of 2020. The results of the microscopy and 16S rRNA analyses differed from each other. The relative abundances of the cyanobacteria genera differed between sample types. Microscopy analyses estimated that the alpha diversity was higher compared to the results of the sequencing analyses. The main reason for the difference between the types of analyses was due to the differences in cyanobacteria belonging to the order of Synechococcales. Some of the Synechococcales species were observed only by the sequencing analyses, e.g. Snowella and some of the Synechococcales species were only observed by the microscopy analyses, e.g. Romeria and Woronichinia. It was observed that both methods are prone to identification errors. The differences between the 16S rRNA sequencing and the microscopy analyses are vastly different. It may affect on the review of long-term data of the phytoplankton community. Therefore, it is important to examine the differences between the types of analyses. Studying the dissimilarities between the types of analyses should be focused on the research of the small cell-sized colonial cyanobacteria, i.e. the species of Chroococcales and Synechococcales.

Digitaalisia teknologioita hyödynnetään lisääntyvästi kansalaisosallistumisen vahvistamisessa sekä vahvan ja osallistuvan demokratian toteuttamisessa. Tämän niin sanotun e-osallistumisen tai digitaalisen osallistumisen päämääränä on aktivoida kansalaisia ja madaltaa osallistumiskynnystä sekä rohkaista heitä keskusteluun julkisen hallinnon kanssa. Teknologiset ratkaisut lupaavat tiiviimmän ja reaaliaikaisenkin keskusteluyhteyden hallinnon ja kansalaisten välillä. E-osallistuminen koetaan myös ratkaisuna maaseuduille tyypillisten niukkenevien resurssien ja pitkien etäisyyksien haasteisiin. Kiinnostus e-osallistumiseen on tutkimusten mukaan kuitenkin vähäistä. Osallistumista tukevia teknologisia ratkaisuja olisi tarjolla, mutta niiden hyödyntämiseen ei olla innostuttu kunnissa. Tässä tutkimuksessa kartoitetaan, kuinka kuntaorganisaation viranhaltijat kokevat kuntalaisten osallistumisen sekä millaisia haasteita ja mahdollisuuksia he liittävät e-osallistumisen teknologisiin ratkaisuihin. Tutkimusaineisto koostuu 11 teemahaastattelusta, jotka kerättiin Mikkelin kaupungin eri palvelualueita edustavilta viranhaltijoilta. Haastattelupuhe on sosiaalisesti rakentunutta ja aineistoa tarkasteltiin teoriasidonnaisesti kehysanalyyttisella otteella. Analyysissa selvitettiin keskeiset osallistumiseen liittyvät kehystämistavat viranhaltijoiden näkökulmasta. Haastateltavat puhuivat osallistumisesta velvollisuuksien kehyksessä ja käytäntöjen kehyksessä. Vaikka osallistumista pidettiin tärkeänä, ristivetoa esiintyi velvollisuuksien (kuntalaki ja kuntalaisten osallistumismahdollisuudet) ja käytäntöjen (nykyiset toimintakulttuurit ja niukat resurssit) välillä. Edustuksellisen päätöksenteon ja kuntalaisten osallistumisen suhde osoittautui kitkaiseksi. Myös kuntalaisten näkökulmaa ja mielekkäitä osallistumisen tapoja olisi tulosten mukaan kehitettävä. Lisäksi kuntaorganisaatioiden nykyiset toimintakulttuurit kaipaavat osallistumisen huomioon ottavaa uudistamista ja riittävän resursoinnin varmistamisen.

Macrozooplankton is an understudied size class of plankton in the Arctic, but even though species composition is similar around the Svalbard archipelago, the relative composition can act as a proxy for climate change. This study investigated if the fjords around Svalbard are similar in species composition and if any differences could be explained. 11 fjords were sampled with a focus on common and indicator species in terms of abundance, relative composition and length distribution. I found low presence of T.libellula and high abundances of T.abyssorum and T.inermis. T.abyssorum was the only species whos abundance was significantly correlated to water mass (Atlantic water), but T.libellula, T.abyssorum, T.inermis, T.longicaudata and A.digitale all showed significant differences in length distribution. This study provides further understanding of species composition in the different high Arctic fjords around Svalbard.