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Both microplastics (MPs, size 0.1 to 5 mm) and pharmaceuticals and personal care products (PPCPs) are ubiquitous in aquatic environments globally. Polystyrene microplastic (PS-MP) and diclofenac (DCF) are among the most widespread and commonly occurring MPs and PPCPs in the environment. Exposure to these chemicals in single has been reported to cause oxidative stress, immobilization, changes in growth and reproduction and even mortality in Daphnia magna. Moreover, MPs have the ability to act as a vector for PPCPs, and several laboratory studies have reported that PS-MP is able to absorb DCF. However, the combined toxicity of MPs and PPCPs remains largely unknown, especially as is the case for the combined toxicity of PS-MP and DCF in D. magna. In this study, D. magna was exposed to PS-MP and DCF, both in single and combined, at a concentration of 0.5 mg/L each. The aim was to assess the combined toxicity of the two, and determine whether they had a synergistic, additive, or antagonistic effect in D. magna. Whole organism endpoints monitored included mortality, immobilization, and moulting. Physiological endpoints monitored focused on oxidative stress and antioxidant defence, including reactive oxygen species (ROS), superoxidase dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). The results indicated that both PS-MP and DCF in single were able to significantly alter D. magna antioxidant and biotransformation enzyme activities by inhibition of SOD and GST, while there were no observed impacts on the monitored whole organism endpoints or ROS and CAT. However, similar significant inhibition of SOD and GST was not observed in the combined exposure, which indicates that the combined toxicity of PS-MP and DCF in D. magna was most likely antagonistic. These results highlight that the combined toxicity of MPs and PPCPs is a complex topic that still needs further investigation to fully understand the complex interactions between MPs and PPCPs.

Tiivistelmä – Referat – Abstract ROS or Reactive Oxygen Species can be found throughout all living organisms on the planet. Without ROS, processes, which are essential for the sustainment of most living organisms, such as respiration would not be possible. On the other hand, uncontrolled ROS generation can cause severe damage to the cellular structure. The family of ROS includes multiple compounds, which share a common trait of high chemical activity. ROS can be produced on demand by specific enzymes which are localized within cellular structures, such as membranes. One group of enzymes is called NADPH (Nicotinamide adenine dinucleotide phosphate) oxidases. These enzymes possess common structure which is composed of transmembrane region with multiple loop helixes and usually two or more terminal motifs, which are devised into regulatory EF-hand motifs and catalytic motifs. NADPH oxidases are essential ROS producers and can be found throughout most clades of living organism and are widely represented in different cellular compartments and distributed across different tissues in multicellular organisms. As an example, Nox family of NADPH oxidases can be found in human tissues and immune cells. Another common group of NADPH oxidases is respiratory burst oxidase homologues (RBOH) can be found in plants. Members of this group play important role in plant immune defense against pathogens. One example is AtRBOHD, which is expressed in Arabidopsis genus of plants. Upon activation, these enzymes are known to produce hydrogen peroxide (H2O2) as mean of antibacterial defense. These host defense mechanisms are known to be driven by different signaling molecules. It has been determined that in some examples of NADPH Oxidases, including Nox5 and RBOHD, the state of activation can be induced through the effects of Ca2+ ions. Moreover, it has been determined, that ROS-producing state of these NADPH oxidases is achieved through change of conformation. This change in conformation is attributed to the different modes of interaction of motifs of oxidases, which are dependent on concentration of bivalent cation Ca2+. Previous research regarding intramolecular interactions within specific NADPH oxidase- Nox5β has been performed by multiple research teams and different sources appear to contradict each other on the exact mode of interaction of Nox5β EF-hand upon presence of Ca2+. Therefore the exact interaction model of terminals of Nox5β is unclear. In addition, the effect of presence of Ca2+ on the interaction terminals in another representative of NADPH oxidases- AtRBOHD, which possess highly analogous molecular structure of catalytic C-terminus to Nox5β, has never been thoroughly studied, as well as interactive cross-compatibility of the C and N terminals from these two distinct species of NADPH oxidases. The objectives of this research are to analyze intramolecular interactions of N- and C- terminals in Arabidopsis RBOHD and Human Nox5β upon presence of ionic calcium, compare Ca2+-induced terminals interactions in said oxidases and to establish possible cross-compatibility of terminals in these two distinct NADPH oxidase species. Practical aspects of this research included cloning the C- and N- cytoplasmic regions of Nox5β and AtRBOHD into bacterial expression vectors utilizing the PIPE cloning method, heterologous production of epitope-tagged tails of NOX5β and RBOHD in E. Coli BL21 and finally in-vitro pull-down assays to analyse the interactions of the tails upon the presence of Ca2+ as well as interactive cross-compatibility of these tails. By utilizing methods mentioned above, this research has demonstrated that interactions of terminals motifs both in Nox5β and AtRBOHD are possible even in calcium-deprived environment, which was achieved through use calcium-binding agent (EDTA) and the effect of calcium on interactions of terminals both in RBOHD and Nox5β is very limited if not insignificant. This research has also demonstrated that the cross-compatible interactions between terminals of Nox5β and AtRBOHD are possible. Results of this research indicate a strong structural conservation within NADPH oxidases, which indicates similar intramolecular interaction mechanisms within two highly diverged species. These findings may prove to be useful as a background for the future research regarding ROS producing enzymes and evolutional conservation in structures of oxidases.

Suomesta tavataan kaksi majavalajia: alkuperäinen euroopanmajava (Castor fiber) ja vieraslaji amerikanmajava (Castor canadensis). Euroopanmajava metsästettiin sukupuuttoon 1800–luvulla ja viimeinen euroopanmajava ammuttiin tiettävästi Sallan Eniönjoesta vuonna 1868. Vuonna 1935 aloitettiin majavien uudelleenistutukset 17 Norjasta tuodulla euroopanmajavalla. Vuonna 1937 Suomen majavakantaa vahvistettiin seitsemällä Amerikasta tuodulla amerikanmajavalla. Tahallisesta vieraslajin tuomisesta ei kuitenkaan ollut kyse, vaan vasta vuonna 1973 todettiin Castor-suvun koostuvan kahdesta eri lajista. Alueilla, joille istutettiin molempia lajeja, on jäljellä ainoastaan amerikanmajava. Syyt ovat edelleen epäselviä. Molempien lajien kannat ovat edelleen keskittyneet alkuperäisten istutusalueiden läheisyyteen, ja amerikanmajavakanta on muutamia poikkeuksia lukuun ottamatta saanut kasvaa ilman euroopanmajavan kilpailun vaikutusta. Jos lajien elinympäristövaatimukset ovat samat, eivät lajit voi esiintyä rinnakkain. Näin ollen on tärkeää tuntea molempien lajien elinympäristön käyttö mahdollisemman tarkkaan aluekohtaisesti ja ennakoida, johtaako majavalajien kohtaaminen kahden lajin rauhaisaan yhteiseloon vai mahdollisesti euroopanmajavan häviämiseen läntisestä Suomesta. Tutkimuksen tarkoituksena oli vertailla lajien elinympäristön vaatimuksia ja sitä kautta tuoda lisätietoa euroopanmajavan suojeluun. Tutkimusalueeksi valikoitui keskinen Pirkanmaan alue, missä lajien välinen etäisyys on ainoastaan 11 kilometriä linnuntietä. Aineisto koostuu vuoden 2013 Luonnonvarakeskuksen koordinoimista valtakunnallisten majavalaskentojen pesätiedoista sekä erilaisista paikkatietoaineistoista. Elinympäristönkäytön mallin muuttujat on tuotettu CORINE Land Cover 2012 (CLC2012), vuoden 2011 Valtakunnallisen Metsien Inventoinnin (VMI) ja Maanmittauslaitoksen maastotietokannan digitaalisiin paikkatietoaineistojen avulla. Alueelle luotiin 60 satunnaispistettä, jotka kuvaavat tarjolla olevaa elinympäristöä. Lajien elinympäristöjen käyttöä analysoitiin kahden logistisen regressioanalyysimallin avulla, joista ensimmäisessä tarkastellaan ydinaluetta (50m) ja toisessa elinpiiriä (1km). Analyyseissä verrattiin lajeja keskenään sekä satunnaispisteisiin. Ennen regressioanalyysejä tarkasteltiin muuttujien kolineaarisuutta Variance Inflation Factor:in (VIF) avulla, jonka jälkeen tarkasteltiin aineiston yhteensopivuuttaa mallin kanssa Akaiken informaatiokriteerin (AIC) avulla. Pienin AIC ilmaisee parhaan mallin korkeimman selitysasteen ja mallin yksinkertaisuuden (parsimonisuuden) kompromissina. Ydinalueella ei havaittu merkitseviä eroja elinympäristön vaatimuksissa lajien välillä. Majavien elinympäristönkäyttö ei kuitenkaan ollut satunnaista. Kuusen tilavuudella oli positiivinen vaikutus euroopanmajavan esiintymiseen ja lehtipuiden tilavuudella oli positiivinen vaikutus amerikanmajavan esiintymiseen verrattuna tarjolla olevaan ympäristöön. Elinpiirianalyyseissä lajien välillä havaittiin merkitseviä eroja, joissa euroopanmajava esiintyi alueilla, missä kuusen tilavuus ja sekametsän osuus oli suurempi verrattuna amerikanmajavan elinpiiriin. Kasvavalla kuusen tilavuudella, sekametsän osuudella sekä vesistöjen määrällä oli positiivinen vaikutus euroopanmajavan esiintymiseen ja rakennetuilla alueilla negatiivinen vaikutus euroopanmajavan esiintymiseen verrattaessa tarjolla olevaan ympäristöön. Amerikanmajavan elinympäristön käyttö ei poikennut satunnaisesta. Tulokset saattavat heijastaa rantavyöhykkeen metsäsukkession eri vaiheita, mutta on huomattava, että lajien välillä oli merkittäviä eroja. Elinympäristön muuttujat selittivät huomattavasti enemmän euroopanmajavan esiintymistä alueella verrattuna amerikanmajavaan. Toisin sanoen euroopanmajavan elinympäristön vaatimukset ovat spesifimmät/tarkemmat/ominaisemmat. Tulosten avulla on mahdollista ennustaa niin amerikanmajavan leviämistä alueella kuin hyödyntää tietoja euroopanmajavalle tärkeiden elinympäristöjen suojelussa.

While the effects of agricultural land use on biodiversity are beginning to unravel in Europe and North America, Africa remains poorly studied. Biodiversity in a broad sense provides ecosystem functioning and services, whose importance has become obvious to humankind in the quickly changing modern world. In Ghana, the practice of mango farming continues to grow in popularity due to suitable climate and potential source of livelihood. With increased demand and production of mango, natural habitats, namely savannahs, are being converted to plantations. The effects of such habitat conversion on local biodiversity are unknown for most taxa, thus providing an interesting study system for biodiversity research. Given the direct relation between functional diversity and ecosystem services, in this work I compare the taxonomic and functional diversity of ground-living spiders between mango orchards and savannah. I chose spiders (Arthropoda; Araneae) as model organisms as they critically contribute to several ecosystem services, such as biological pest control and nitrogen cycling, while being ubiquitous and abundant, thus easy to collect in the field. Spiders were captured with pitfall traps from six mango and four savannah areas in the Northern region of Ghana. A total of 424 individuals and 53 (morpho)species were identified and counted. Additionally, six morphological and four ecological traits were quantified for each (morpho)species. With these data, I calculated taxonomic richness and evenness, functional richness, dispersion, and evenness, and beta diversity of the different assemblages in the R environment. These metrics were then compared between spiders collected from mango orchards and savannah. Mango orchards showed lower taxonomic and functional evenness than savannah, contrary to all other alpha diversity measures. The two habitat types share many of the same diversity of ground-living spiders, but species and traits are distributed less evenly in mango orchards due to incomplete niche differentiation between species. Both taxonomic and functional beta diversity were significantly different between habitat types indicating that mango orchards sustain a similar richness of species as savannah, but the species composition is different. In conclusion, mango orchards have the potential to conserve some aspects of the original diversity, but species composition and the way species interact are substantially different. We should strive to find the best practices to produce mango without radically changing the natural biodiversity patterns.

Genome wide linkage and association methods are used to map genes affecting traits with genetic predisposition. In this thesis, I compare the methods suitable for quantitative trait mapping in complex, extended pedigrees. As a case study, gene-mapping study of musical aptitude is performed with these methods. Linkage analysis methods are developed for family studies. However, only a few methods are suitable for extended families with a quantitative trait. Three linkage programs were successfully applied for such data in this study. These programs are the SOLAR, JPSGCS and KELVIN. All of these three programs are based on different methods and thus, the same calculations are not repeated. SOLAR is based on the variance components method, JPSGCS on a graphical method and KELVIN on the Bayesian method. Association analysis is also difficult to implement for large pedigrees, because it is best suited for case-control data. Fortunately, methods are extended also for family-based studies. Here, a genomic control method was used to correct for the familial relationships. The method evaluates the relatedness from the whole genome data and the association tests are corrected for the relatedness rates. This method was implemented from the GenAbel program. As a case study, these methods were applied to a musical aptitude study. The musical aptitude is here understood as an ability to perceive the melody, harmony and rhythm of music, and to recognize structures in set of sounds. These abilities were tested with Carl Seashore s tests for pitch and time and Kai Karma's test for auditory structuring. The data consists of 107 pedigrees and 93 sporadic subjects, comprising in total of 915 individuals. Each family includes 2 - 50 individuals. These individuals were genotyped with a SNP chip for over 700,00 SNPs. The linkage analyses revealed several promising loci for the musical aptitude. The best result was located in 4q12 and it was found with all of the three linkage programs. Most of the other results could also be identified with multiple programs, but some differences also occurred. However, none of the findings could be discovered with association analysis, probably due to a too small sample size.

Deforestation is the main threat to biodiversity, ecological integrity and socio-ecological resilience of the of the Amazon biome, one of the most biodiverse places on Earth and home to at least 2 million people. A complex network of diverse protection strategies exists across the Amazon as key component of the global strategy to halt biodiversity loss. Biosphere reserves are a part of this network that aims to create spaces to learn how human communities can develop sustainably, while the protecting the environment, by implementing a zonation system with different degrees of protection We consider that it is necessary to produce relevant efficiency assessments on area-based conservation strategies in this region, and to understand how different protection strategies affect conservation outcomes. We used state-of-the-art matching methods to create a counterfactual deforestation avoidance measure of seven Biosphere reserves the western Amazon: Yasuní, Podocarpus-El Cóndor, Sumaco, Manu, BIOAY, Pilón Lajas and Beni. We obtained diverse efficiency results, some of the studied reserves avoiding large quantities of deforestation to reserves that were attracting deforestation. We found that more strictly protected zones were subjected to significatively lower relative pressure levels and did not have higher deforestation avoidance values. Representativity of the matched treatment area was also lower for these zones, meaning that the matching analysis was more difficult to perform in these areas. These research findings add to growing evidence about the important role of biosphere reserves in buffering against deforestation in one of the world’s most important biodiversity hotspots.

Reptiles have long been studied in search of the mechanisms behind neuronal regeneration. This thesis delves into the regenerative areas of two emerging model species to the field of regenerative research: Pogona vitticeps (bearded dragon) and Pantherophis guttatus (corn snake). This fluorescent immunohistochemical study maps out and compares the constitutive proliferative zones in these two species to better define the focus of future comparative neurodegenerative experiments. A BrdU pulse chase experiment in conjunction with PCNA reveals proliferative zones in the lateral ventricular ependyma of both species. Stem cell niches were found in the ependymal lining adjacent to the medial cortex and dorsal ventricular ridge in both species, however, the nucleus sphericus ependyma was an active proliferative zone only in Pantherophis. Imaging of further markers in this study support the findings of the pulse chase experiment. High levels of the stem cell marker Sox2 was found in lateral ventricular ependymal cells in both species. The glial marker GFAP reveals a highly ordered array of radial glia in the cortical areas of Pogona, which is significantly reduced or absent in Pantherophis. And lastly the neuronal marker HU was found in the same cells that were BrdU positive and had migrated a short distance from the proliferative zones, which shows that the proliferative areas in the lateral ventricular lining do indeed produce neurons. The BrdU and PCNA marked cells were quantified in both species, and a brief comparison between the species showed that Pogona had a significantly higher number and concentration of proliferative cells in the proliferative zones than Pantherophis. Scattered BrdU positive cells that were neither neuronal nor positive for any other marker were also found scattered throughout the parenchyma of Pogona, and these cells remain uncharacterized. Differences between these two species are not surprising, as lizards are known to have better regenerative capabilities than snakes, however, more comparative research between these species is needed to gain further insight into the mechanisms behind their contrasting regenerative capabilities.

Knowledge co-production has become more widespread in sustainability research as it possesses the potential as a tool to provide policymakers with usable knowledge in collaboration with researchers to increase the impact of scientific knowledge. This thesis complements the current literature by providing empirical insights of the perception of Finnish national-level policymakers who have participated in Strategic Research Councils (SRC) projects. SRC is a funding instrument with the aim to produce high-quality research with societal impact in collaboration with relevant stakeholders. The perception of Finnish researchers is already somewhat covered in literature and therefore the material for this thesis was collected by interviewing non-researcher participants in SRC projects. The analysis of the material was conducted as a theory-bound qualitative content analysis. The first coding was conducted with a material-based approach to allow the perception of the interviewees to prevail. The final analysis is conducted with three different modes of interaction obtained from the literature: science-push, policy demand, and iterative modes used as main categories. The analysis provides a standpoint on the research question: How do policymakers perceive the interaction in the co-production process? Methods to include policymakers and their participation in national-level co-production processes are versatile in Finland. The knowledge co-production between researchers and national-level policymakers results in usable knowledge and furthers policy uptake especially when collaboration and participation are genuine and profound. This study concludes that the inclusion of policymakers in the early stage of research, two-way communication, iterative interaction, and trust building are factors related to successful interaction, as the policymakers perceive success. The success factors could also help to overcome barriers of co-production presented in earlier literature.

Spinal muscular atrophy of Jokela type (SMAJ) is an autosomal dominant motor-neuron disease caused by a missense mutation c.197G>T, p.G66V in the gene CHCHD10. Coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is a nuclear-encoded mitochondrial protein located in the intermembrane space (IMS) of mitochondria with an unknown exact function and disease-causing mechanism. In this project, the overarching aim was to correct a heterozygous SMAJ-causing mutation in patient myoblast cells with CRISPR-Cas9 genome editing. The goal was to create a genetically identical, isogenic, cell line to study only the effects of the mutation on cellular phenotype in vitro. Human myoblast cells isolated from patient biopsies provide the most pertinent experimental model to study neuromuscular atrophy-associated mutations in their natural genomic environment. More specific aims included genome editing optimization with myoblast cells, since it is not as widely conducted as with some other cell types, such as iPSCs. CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology-directed repair (HDR) in the genome of patient-derived myoblast cells and correct the mutation. After optimization of electroporation conditions for myoblast cells, guide RNAs were designed and transfected into patient myoblasts. Clonal cell lines were made by utilizing techniques such as fluorescence adjusted cell sorting (FACS) and manual colony picking. The success and precision of genome editing were analyzed by Sanger sequencing, comparing the performance of the different guide RNAs with restriction enzyme analysis and Synthego ICE CRISPR web tool, and screening regions of potential off-target genome editing. A genome-edited myoblast cell line with the CHCHD10 c.197G>T mutation corrected, was successfully generated to provide an isogenic control for the patient myoblast cell line. Optimization of myoblast electroporation was successful and conditions used proved to be effective. Clonal cell line creation proved to be challenging with myoblast cells and work is still needed to improve the viability of single-cell clones after FACS. Nevertheless, the advances taken here regarding myoblast genome editing with CRISPR-Cas9 offer a fertile avenue for future research of myoblasts genome manipulation, myogenic disorders, and the role of CHCHD10 in skeletal muscle and SMAJ. Comparing the CHCHD10 protein level and mRNA expression between patient cells, corrected myoblasts, and differentiated myotubes is an area of future research. Future work also includes measuring the mitochondrial integrated stress response in both cell lines and co-culturing myotubes and iPSC derived motor neurons to study the effects of p.G66V on neuromuscular junction (NMJ) formation.

Tiivistelmä – Referat – Abstract Telomeres are cap shaped structures at the very end part of each chromosome that protect DNA from degradation or unwanted chromosome-chromosome attachments. Telomere lengths show considerable heterogeneity in different cells of the same cell population. Reasons for heterogeneitiy and mechanisms inside cells causing them are not fully understood. In this study, we explored the correlation between telomere length and different gene expressions. First, using FACS technique we sorted each single cell into each well of 96-well plate. Second, we used SYBR green based qPCR for telomere length measurement. Third, we used Illumina-seq for sequencing extracted mRNAs. [6] We found a set of genes that were in strong correlation with telomere length, giving opportunity to explore the biological pathways. We compared pathways between different samples and found strong connections between genes involved in viral cycles and immune system with extracted genes that were in high correlation with telomere. We found heterogeneity of telomere lengths and transcriptomes in different cell lines. Telomere related proteins, specifically those involved in shelterin complex, are expressed highly in cancer cell lines and LPS-stimulated monocytes compared to the non-stimulated monocytes. In our study, SLC38A2, PURB, UBR3, SSR1, NCAPH2, AIMP2, PHF21A genes were highly correlated with telomere in mutual way and can therefore be considered as new biomarkers/novel candidates for telomere-related studies. The importance of these genes has been reported in aging/mortality. Concurrent with our findings, a recent report also suggested that NCAPH2 plays role in regulating telomere stability and maintenance through its interaction with TERF. [65] We found new genes in correlation with telomere regulation, and our findings are therefore of high importance in research of cancer, neurodegenerative diseases and aging. Further studies are, however, required as our data is limited by small number of samples and inability to properly validate our technique.