Browsing by Title
Now showing items 204-223 of 1114
-
(2021)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.
-
(2021)As water flow encounters an object on the sea floor, its hydrodynamics change. Accelerated currents and vortices develop around the object with changing intensity as a function of distance from its proximity. This leads to erosion and aggradation of sediment, known as scour. Studies focusing on formation processes of scour often involve locating visible scour sites by sonar scanning the geomorphology of the seafloor. However, the effects of scour on macroinfauna and small-scale sediment characteristics are not visible in sonar images. In this Master’s thesis, scour at a shipwreck of a timber-built historic sailing ship, the Joskär shipwreck, was first identified by scanning the study area with side-scan sonar, and by measuring water depth contours around the shipwreck by scuba diving. Sediment samples were then taken inside the area assumed to be under the most pressure from scour. Samples from three separate distances on two transects drawn outwards from the hull of the shipwreck were collected and analysed for sediment grain size, organic content, and species assemblages of macroinfauna. In addition, macrofauna were analysed for individual lengths, number of individuals, diversity index, and functional groups. All samples were collected with a core tube sampler operated by a scuba diver. The methods used in this Master’s thesis widen the concept of scour past the sole physical processes observable with sonar to a more holistic level that considers the quality of biological, geological, and chemical characteristics of the benthic environment. The results of the present Master’s thesis show that the quality of the sediment near Joskär shipwreck varies within a relatively small scale. Organic content of the sediment was the most potent descriptor of scour at the study site, exhibiting a consistent decreasing trend as distance to the shipwreck increased on both sampled transects. Sediment grain size became finer as distance to the shipwreck increased. However, compared to grain size, based on visual observations of the sediment samples, shell debris content of the sediment could possibly act as a better measure of presence of scour. The variability of characteristics of macroinfaunal communities as a function of distance from Joskär shipwreck was not a viable tool to describe the presence of scour, as no consistent trends of the variables were observed. As no control site was included in the study design, the characteristics of the benthic environment inside the scour around Joskär shipwreck could not be compared to the seafloor unaffected by scour. Further research could reveal possible variation between these distinct habitats, and that way produce valuable indicators of scour. The hypothesis in the present thesis was that macroinfaunal assemblages and sediment characteristics would exhibit variation between the sampling sites as a function of distance from the shipwreck. The observed trends of sediment characteristics validated a part of the hypothesis, showcasing the utility of sediment characteristics in describing scour at Joskär shipwreck. However, a part of the hypothesis was rejected, as no consistent trends of macroinfaunal features were present.
-
(2019)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.
-
(2024)Educational technology is advancing rapidly, with VR (virtual reality) emerging as a promising branch of XR (extended reality) technology for educational purposes. Utilizing head-mounted displays (HMD), immersive VR experiences immerse users in a virtual environment, limiting their awareness of the physical world. VR proves valuable in education by complementing traditional teaching methods, offering experiences impossible in the physical realm. Studies indicate enhanced affective factors, understanding, motivation, and memorization among students. In biology education, VR serves as a visual aid, helping students grasp complex biological concepts difficult to visualize from a two-dimensional textbook. It also shows potential in supplementing hands-on activities like laboratory work and anatomical dissections, experiences outside classrooms, and sustainability education. However, challenges persist in VR's educational application, including uncertainty about learning outcomes, health concerns, high costs, and a general lack of expertise in VR design and pedagogical implementation. Educational VR design has thus far lacked a foundation in pedagogy and learning theories. This thesis aims to address this gap by reflecting on the development of a pedagogically meaningful VR experience within sustainability education. Collaborating with the Global Campus project of the University of Helsinki, the thesis introduces a VR experience integrated into the immersive virtual sustainability learning experience, Serendip. The design process involved literature research, user and expert interviews, and consideration of learning theories such as constructivist learning, experiential learning, flow theory, gamification, CTML, SDL, and CLT. Specific aspects of VR design, like immersion levels and prior knowledge of users, were also considered. The thesis's significance lies in pioneering pedagogy-based design for educational VR, particularly addressing complex, abstract, and multidisciplinary subjects. It emphasizes the need for collaboration among pedagogy, content, and VR animation experts in future educational VR design. This work serves as a potential template and inspiration for further research in the field, aiming to refine the integration of pedagogical principles into VR experiences for education.
-
(2023)Arctic ecosystems face drastic changes in community structure due to warming, shrubification, permafrost loss, and other environmental changes. Due to the spatial heterogeneity of these ecosystems, understanding such changes on a local scale requires high-resolution data. Earth observation using satellite imagery and aerial photography has become a staple in mapping large areas and general patterns. Advances in sensor technology, the proliferation of unmanned aerial vehicles (UAVs), and increases in processing capacity enable the use of higher spatial and spectral resolutions. As a result, more detailed ecological observations can be made using remote sensing methods. In this thesis, I assess how increased spectral resolution affects the remote-sensing based modelling of plant communities in low-growth oroarctic tundra heaths. Based on a large field observation dataset, I estimate biomass, leaf area index, species richness, Shannon's biodiversity index, and fuzzy community clusters. I then build random forest models of these with image data of varying spectral, spatial, and temporal specifications and topographical data. Finally, I create maps of the vegetation. Leaf area index and biomass are best estimated of the response variables, with R2 values of 0.64 and 0.59, respectively, with multispectral data proving the most important explanatory dataset. Biodiversity metrics are best estimated with R2 values of 0.40–0.50 with the most important explanatory variables being topographical and hyperspectral, and community cluster with R2 values of 0.27–0.53, with the importance of various explanatory variables depending on the cluster being estimated. These results can help choose a suitable high-resolution remote sensing approach for modelling plant communities in similar conditions.
-
(2024)The RNA splicing process is an important part of gene expression in which the introns are removed from the pre-mRNA so that the mature mRNA only contains protein coding exons. The splicing process is executed by the spliceosome in two subsequent transesterification reactions that occur partly co-transcriptionally. In the first step an intron lariat is formed between the exons. This is followed by splicing of the intron lariat and ligating the exons together. Genetic variants that affect the splicing of a particular gene are called splicing variants and they may disrupt the normal splicing process. Splicing variants can be both exonic or intronic and have effects on splice site recognition, activate cryptic splice sites or create new splice sites. These changes can lead to for example exon skipping or intron retention in the transcript. Diagnosing splicing variants is challenging because of the unknown functional effects of the variants. Splicing prediction tools can help predict the possible effects of variants. Different sequencing methods enable the detection of aberrant splicing transcripts and thus may help in variant interpretation. The aim of this master’s thesis was to develop a detection method suitable for the diagnostic laboratory for RNA splicing variants in congenital disorders. The methods that were tested included RNA and Sanger sequencing. First, the patient selection was performed using splicing predictions and previous research on the variants. Secondly, after receiving patient samples, RNA was extracted, and its integrity measured. The laboratory work was then divided into two parts, the other leading to the RNA sequencing and the other to Sanger sequencing. Before Sanger sequencing primer design, RT-PCR, PCR and analysis of the PCR fragment sizes was performed. RNA sequencing was preceded by RNA library preparation. The studied variants in this thesis were BRCA2 c.476-3C>A, MSH2 c.2005+3A>T and CYLD c.2350+5G>A. The PCR fragment analysis and Sanger sequencing was able to detect an aberrant splicing transcript with exon skipping on two patients caused by a variant in the CYLD gene. The RNA sequencing results confirmed the aberrant splicing transcript. In addition, fragment analysis showed evidence of a possible splicing isoform with skipping of two exons caused by a variant in the BRCA2 gene that was not expressed enough to show on the Sanger sequencing results. The RNA sequencing detected a splicing transcript with exon skipping in two BRCA2 patients. However, this was not the same transcript as interpreted from the fragment analysis results and no results in the RNA sequencing indicated a transcript with skipping of two exons.
-
(2024)Cereal viruses are of great economic importance as they reduce crop yield which poses a threat to food security. Early detection of these viruses in cereal plants can help in disease control and future management strategies. This study aimed to develop a detection method and study the presence of three cereal viruses (barley yellow dwarf virus – BYDV, cereal yellow dwarf virus – CYDV, and oat sterile dwarf virus – OSDV) in oat plants grown summer of 2023. These viruses were previously detected in barley plants by siRNA sequencing in the year 2021. Total RNA was isolated from oat leaf samples collected from test plots in the Viikki research farm in July and August 2023. The experimental setup had 60 plots in total where 56 of the plots had oat plants with different undergrown plants making up five biodiversity levels and 4 of the plots were bare fields. Virus infections were detected through real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for all samples and RNAseq (RNA sequencing) of 20 samples from July across different plant biodiversity levels in the test plots. BYDV and CYDV which are transmitted by aphids were detected in 88% and 3% of the plots, from the July collections through RT-qPCR. The result from the RT-qPCR was further validated through RNAseq of 20 samples from the July set. From the August samples, BYDV was detected in 29% of the plots while there was no record of CYDV. OSDV was not detected in any of the samples. Based on the RNAseq, BYDV-PAV, a BYDV isolate that is prevalent in Finland was found in 70% of the sequenced samples while BYDV-PAS and BYDV-GAV were recorded in 20% and 5%, respectively. The significant difference in the viral incidence in the samples could be attributed to the difference in timing of the sample collection. This study was successful in developing a reliable method for the detection of three cereal viruses from the oat samples. Also, three isolates of BYDV including BYDV-PAV, which has been reported to be prevalent in Finland were found. Further studies will be required using samples across various locations in Finland to validate the occurrence of these viruses especially CYDV, which has not been previously reported in Finland.
-
(2021)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.
-
(2022)In the past few years, there has been an increased consideration on the stem cell niche as a key factor to regulate stem cell maintenance and differentiation. Research on characterization of the stem cell microenvironment boosted after the determination of long-term three-dimensional (3D) tissue cultures, or so-called organoids. Organoids are derived from stem cells which self-organize in 3D multicellular structures upon embedding in an extracellular matrix mimic, such as Matrigel®. Their main advantage is these structures resemble the architectural distribution of the tissue of origin in vivo. Likewise, the cellular components of organoids vary depending on multiple variables as the tissue of origin and the growth factors they have access to. As a result of advances in this technique, some stem cell niches have been well characterized, as in the case of intestinal stem cells (ISCs), while others remain elusive as in case of the human gastric stem cells (hGSCs). Along with the remarkable development of 3D cultures, the interest of ECM proteins in stem cell regulation increased. Matrigel® is a rich matrix composed of several adhesive proteins such as laminins and collagens. Aside from providing structural support, the extracellular matrix (ECM) proteins forming this matrix contribute to cell adhesion and signalling. However, Matrigel® composition cannot be modified or even well-characterized due to its origin from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Additionally, it has been demonstrated that contains a high batch-to-batch variability. Other techniques to study the effects of individual ECM proteins have been used such as coating of tissue culture plates with ECM proteins. However, the biomechanical properties in this model are far from being physiological. Therefore, although preliminary results can be obtained using this technique, results extrapolation to an in vivo model can be questioned. To date, there is a lack of a reproducible, high-throughput and reliable technique to test the effect of ECM proteins on human gastric stem cells behavior. This Master’s thesis presents a novel transwell device containing a polyethylene glycol (PEG)-based hydrogel enriched with human ECM proteins to test their effect on human gastric stem cell regulation. Preliminary results showed that gastric organoid-derived epithelial cells (GODE) grown on hydrogels with ECM proteins that are localized at base of the gastric glands, such as Laminin-211, had a higher stem cell marker expression than the control grown on ECM proteins that are uniformly localized in vivo. Additionally, when GODE were grown on hydrogels containing ECM proteins that are localized at the surface of the native gastric epithelium, expression of surface gastric mucins markers was enhanced. These preliminary results highlight the utility of the optimized transwell device to further shed light on how the human gastric stem cells are regulated and what is the effect of the ECM proteins surrounding them.
-
(2022)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.
-
(2019)There is significant reduction in number of approved drugs for acute myeloid leukemia in recent years. Partially it may be due to the failure of discovery and validation approach to new drugs as well as the complexity of the disease. Ex vivo functional drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia (AML). In ideal condition, an effective drug should eradicate the immature AML blasts, but spare non-malignant hematopoietic cells. However, current strategies like conventional cell viability assay fail to measure cell population-specific drug responses. Hence, development of more advanced approaches is needed. Using multiparameter, high-content flow cytometry (FC), we simultaneously evaluated the ex vivo sensitivity of different cell populations in multiple (10) primary AML samples to 7 FDA/EMA-approved drugs and 8 drug combinations. Amongst the 7 tested drugs, venetoclax, cytarabine and dasatinib were very cytotoxic with venetoclax had the highest blast-specific toxicity, and combining cytarabine with JAK inhibitor ruxolitinib effectively targeted all leukemic blasts but spared non-malignant hematopoietic cells. Taken together, we show that the ex vivo efficacy of targeted agents for specific AML cell population can be assessed with a cell phenotype, FC-based approach. Furthermore, we put an effort to analyze the potential of this assay and biomarkers to predict the clinical outcome of individual patients and future perspectives.
-
(2019)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.
-
(2022)Pain is a subjective feeling often difficult to interpret or study and thus, pain of those unable to communicate their pain is difficult to recognize. According to the new definition of pain by IASP (Raja et al 2020), verbal description is only one of the many behaviours that can be used to express pain, and the inability to communicate pain does not negate the possibility of experiencing it. This addition to the definition points out that non-human animals, too, even if they cannot express it in words, are capable of both experiencing and communicating pain. Can we as humans interpret a state of pain in an animal in a trustworthy way – and in a manner that would be respectful and non-invasive to the animal? Infrared thermography (IRT) is a technology based on using infrared radiation instead of normal light to form images. These images can be used to quantify the surface temperature of an object with high resolution. The intensity of the radiation emitted by the object being imaged depends on the surface temperature and for this reason thermal imaging enables detecting and measuring changes of surface temperature. Pain and stress might manifest physiologically as activation of the autonomic nervous system, which in turn might result in changes in surface temperatures of the body. These changes might be detectable with a thermal camera. If we could establish a link between certain intricate temperature changes of the head area to certain type of activation of the sympathetic nervous system resulting from pain, thermal imaging could have the potential to detect this. In this study I investigated if there were detectable temperature changes in animal patients before and after a standard examination conducted to each patient admitted to the Wildlife Hospital of Helsinki Zoo, where my data was gathered. Another question was whether the patients that had pain differed in their temperature changes as compared to other patients. The question at the heart of my research was whether there would be a change in peripheral facial temperatures of patients before and after the examination. Another question was whether thermal patterns would be different for pain- and non-pain patients. I found that for some parameters, the temperature differences between pain- and non-pain patients were indeed different, for example the crown temperature of birds seemed to change with examination for patients without pain but not for patients with pain. A more prominent finding was that temperatures decrease across many parameters after an examination as compared to prior to it, across all or many patient groups. My research does not univocally show that thermal imaging could be used to detect pain; rather it affirms the thought that the measurement of changes in peripheral temperatures could be a potential window to non-invasively detect some changes of activation of the sympathetic nervous system in animals.
-
(2017)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.
-
(2024)Background: The infant gut microbiome undergoes major temporal changes in the first year of life, crucial for supporting normal development and long-term health. The immense diversity of fiber structures in breast milk and later in solid foods pose unique selection pressures on the gut microbiome maturation by providing novel substrates for the microbiota. However, the longitudinal impact of complementary food-derived fibers on the taxonomic and functional maturation of the gut microbiome during the gradual transition from breast milk to solid foods is not well understood. Objectives: My objective was to examine how breast milk, its fiber and complementary food fibers in the broader context of overall infant diet may affect the gut microbiome bacterial species composition and support age-appropriate gut bacterial maturation trajectories during first year of life. Methods: Longitudinal and cross-sectional development of 68 infant gut microbiomes and 33 metabolomes were examined with linear mixed models to determine the impact of infant nutrition on gut microbiome taxa and functional development. Nutrition assessments were based on detailed quantitative weighted 3-day food records (months 3,6,9,12) and the intakes of total dietary fiber with its food sources and fiber fractions relied on current internationally approved CODEX-compliant values. Questionnaires were utilized to monitor when various complementary foods were introduced, enabling more comprehensive nutritional analyses. Bacterial species identification was based on MetaPhlAn2 quantification of bacterial species from metagenomic data and metabolomic profiles were generated using four liquid chromatography-mass spectrometry (LC-MS) methods. Results: My examinations place the previously described sequential trajectories in infant gut microbiome maturation into detailed fiber-dependent nutritional context relying on metagenomic species identification. I discovered 176 complementary food derived fiber-bacterial species associations. The majority of the associations (147, 84%) were positive whereas breastfeeding and related variables tended to be inversely associated with the same species, showing strongest inverse correlations to later trajectory species indicative of slower maturation. Both bacterial species and metabolomic profiles displayed pronounced longitudinal shifts in response to solid food fibers. Each introduction of novel dietary source of fiber associated to diversification of the microbiome revealing fiber-species specific temporal patterns. Conclusions: The longitudinal analyses highlight that sufficient fiber intake from appropriate sources during the weaning period likely function to build capacity for the species permanence in the more diverse and stable mature gut microbiome composition and function reached in later childhood.
-
(2019)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.
-
(2020)The Miocene epoch (c. 23-5 million years ago) was a noteworthy geological time period in which significant changes took place both in the climate regimes as well as in vegetation characteristics, bringing about novel adaptations in many herbivorous lineages. These adaptations constituted morphological, dietary, and ecological factors in a relatively short period of evolutionary time. Among these herbivores were the proboscideans, the living and extinct elephants, which were among the most dominant and largest herbivores at the time. Despite that proboscideans were diverse and large group of hundreds of species, yet the understanding of dietary and ecological patterns of majority of Miocene sympatric species is still limited. The aim of this study was to analyse the molar surfaces of Miocene proboscideans (e.g. Deinotherium and Gomphotherium) from Eurasia to provide a reconstruction of the feeding preferences of the study species based on observed dental wear. The dental wear indicates the abrasiveness of the diet, thus allowing broad categorization to either browser (<10% grass in the diet), mixed-feeder (10-90% grass in the diet) or grazer (>90% grass in the diet). Secondly, this study aimed for providing estimation of the environmental characteristic and vegetation patterns of the study localities by comparing to the previous studies and to hypsodonty value (proxy of general openness and aridity of the environment). Proboscidean dietary signals from the key localities of Maragheh (Iran) and Pannonian basin (Austria) were compared with the paleobotanical studies. Thus, the general estimation of spatial and temporal variation of the environment characteristics in the study localities were based on these parameters. The materials of fossilized molars were analysed by mesowear angle method, in which the measured angles show the diet abrasiveness due the nutritional targets’ differences. The results allowed the reconstruction of the feeding preferences which suggested that majority of Miocene proboscideans were browsers and browse-dominated mixed feeders or pure mixed-feeders. Instead, Choerolophodon pentelici was found grass-dominant mixed-feeder. The wide spectrum of feeding preferences allowed diet flexibility according available vegetation and also these sympatric species to co-exist by niche partitioning. Thus, demonstrating clearly the connection between diets and environments thought the diet. As a conclusion, in the diet of the paleocommunities of proboscideans had, on average, more grass-dominant components in open and dry environment likely due the presence of grass-dominant vegetation. Instead, in the wet conditions the closed-canopy forest environments enhanced browsing. Further, the results indicated shift in feeding preferences of proboscideans prior to Miocene climate and environment changes. These results are in line with the findings of the previous studies of modern elephants’ diet- environment relationships. The further studies would provide insight to the relative amounts of the grass in the diet of Miocene proboscideans.
-
(2022)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.
-
(2024)Accumulating evidence indicates that the plasticity-inducing effects of conventional antidepressant drugs like fluoxetine are mediated by their direct binding to TrkB. TrkB is the receptor of the brain-derived neurotrophic factor (BDNF), a neurotrophic factor of critical importance for neuron survival and synaptic plasticity. In addition, it has recently been reported that LSD and psilocybin, two psychedelic compounds with therapeutic potential, also bind to TrkB with higher affinity than antidepressants. It has been proposed that the differences in binding affinity between conventional antidepressants and psychedelics may help explain the much faster and longer-lasting antidepressant effects of psychedelics. Psychedelics and classical antidepressants bind to the transmembrane domain of TrkB dimers, where they act as positive allosteric modulators by potentiating the action of endogenous BDNF. The transmembrane binding sites of LSD and fluoxetine, despite being partially overlapping, are distinct and induce different conformational changes when bound to TrkB dimers. However, it is still unknown whether there are differences in the TrkB dimerization dynamics and neurotrophic signalling pathways induced by psychedelics when compared to conventional antidepressants. In this study, we investigated whether psychedelics and classical antidepressants promote TrkB dimerization and neurotrophic signalling in a differential manner. The effects of psychedelics on the TrkB dimerization dynamics and neurotrophic signalling associated with plasticity were studied treating N2a cells and primary cortical neuronal cultures with LSD or fluoxetine. Dimerization of the TrkB receptor in the presence of experimental compounds is assayed by protein-fragment complementation assay (PCA). Results show a significant dimerization in cells treated with LSD, whereas non-significant response in the ones treated with fluoxetine. The phosphorylation state of the neuronal TrkB receptor in three different tyrosines (Y515, Y706, and Y816) was checked as a marker of its activation by Western blot. Primary cortical cultures were treated with classical antidepressant fluoxetine (10uM) or psychedelic LSD (100nM) for 1 hour, when their effects on TrkB phosphorylation were compared. This experiment showed a significant increase of phosphorylation in TrkB Y816 after LSD treatment in cortical neuronal cultures, while fluoxetine treatment showed no significant effect. This indicates that LSD is able to activate the BDNF-TrkB signalling pathway associated with PLCg1 recruitment and induction of plasticity at an early time point and with a much lower concentration than fluoxetine, which would support LSD’s much more potent antidepressant and plasticity-inducing effects when compared to fluoxetine’s. Together, these results suggest that psychedelics that bind to TrkB, like LSD, are more potent than classical antidepressants in inducing TrkB-BDNF signalling. Overall, this study provides further evidence that TrkB is a critical mediator of psychedelics’ actions on neurotrophic signalling preceding their plasticity-enhancing and antidepressant effects and sheds more light on the common and differential mechanisms used by psychedelics and conventional antidepressants to produce their therapeutic effects.
-
(2024)Degeneration of dopaminergic neurons in the substantia nigra is a primary pathological feature of Parkinson’s disease (PD). Current treatment strategies available are not disease-modifying but rather symptom-alleviating due to limitations in understanding the molecular event(s) that trigger the degeneration of dopaminergic neurons. While several animal and cellular models exist to study the disease progression and pathophysiology, their translational relevance to humans is debatable. In this thesis, our goal was to replicate a published protocol designed to differentiate SH-SY5Y human neuroblastoma cell lines into dopaminergic neurons. Our study shows successful differentiation and characterization of SH-SY5Y cells into dopaminergic neurons, but also the limitations in reproducibility. This finding highlights the challenges of technical variability, but also the reliability of existing protocols by emphasizing the need for more consistent reproducible methods as it could be significant for improving this cellular model.
Now showing items 204-223 of 1114