Browsing by Title
Now showing items 141-160 of 1112
-
(2020)The human cerebral cortex is characteristically large and folded, which can be majorly attributed to the high number and variety of neural progenitors during embryonic development. Radial glial cells are essential neural progenitors during neurogenesis. In addition to giving rise to new cell types, they also provide scaffold for migrating newborn neurons. Radial glia are known to portray peculiar characteristics in their cell division process, including unique migratory behavior as well as specifically regulated cleavage furrow orientation. While these processes of radial glial division have been studied extensively, the underlying molecular mechanisms are still largely unknown. ABBA (actin-bundling protein with BAIAP2 homology) and NEDD9 (neural precursor cell expressed, developmentally downregulated 9) are proteins, which are both known to be expressed in certain radial glia progenitors during embryonic development, while they are mainly absent in neurons. ABBA has a defined role of regulating plasma membrane deformation and actin polymerization in radial glia, while NEDD9 expression levels are a known factor in the correct progression from mitosis to cytokinesis. An interaction between ABBA and NEDD9 has previously been identified in a yeast two-hybrid screen done for the embryonic mouse brain. The aim of this thesis was to validate the interaction between ABBA and NEDD9 biochemically. First, their interaction was evaluated by doing co-immunoprecipitation assays on the endogenous proteins from C6 cells. The second approach was to test, whether their interaction is directly mediated by the N-terminal SH3-domain of NEDD9 and the proline-rich C-terminal portion of ABBA. This was done by doing biochemical binding assays using purified proteins and domains of interest. While co-immunoprecipitation of the two proteins gave results indicating an interaction, I could show that there is no direct binding between NEDD9 SH3-domain and ABBA, suggesting that the interaction might require other domains or be indirect. Together, these results provide valuable information that will help characterize what roles of ABBA and NEDD9 play in cortical development and beyond.
-
(2020)APECED (Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal-Dystrophy) is a severe, multiorgan autoimmune disease caused by mutations in the AIRE (autoimmune regulator) gene. APECED is a rare disease, however in Finland the frequency is significantly high (1:25 000) and APECED belongs to the ‘Finnish Disease Heritage’. The most common mutation worldwide is the so-called Finn-major mutation R257X that results in a truncation of the AIRE protein, which disrupts the indispensable functions of AIRE. Immune reactions towards body’s own components are typically prevented with various central and peripheral immune tolerance mechanisms. AIRE is essential for the proper development of central and peripheral tolerance and the absence of functional AIRE leads to a loss of immune tolerance and various autoimmune manifestations. Recent studies have suggested that AIRE also has functions in stem cells and actively contributes to the regulation network of pluripotency. Currently, the development of induced pluripotent stem cell (iPSC) technology has opened opportunities for precision medicine and for defining the cure for genetic diseases, such as APECED. The ultimate objective of our research group is to examine whether APECED could be cured via autologous, gene-corrected cell transplants with the use of induced pluripotent stem cells (iPSCs). As a requirement for such later therapeutic use and iPSC differentiation, the APECED patient-derived iPS cells needed to be characterized in detail. To assess, whether AIRE R257X mutation, present in APECED patients’ iPSCs, would cause defects in their stemness properties, the expression of AIRE and classical stem cell markers were examined with qPCR and immunocytochemistry and compared to healthy control iPSCs. The iPSC cells were also treated with spontaneous differentiation -inducing dimethyl sulfoxide (DMSO) to study, whether AIRE R257X mutation would affect the spontaneous differentiation of iPS cells. To further investigate the stemness and early developmental phase properties of APECED patient derived iPSCs, self-aggregated embryoid bodies (EBs) were generated and cultured. Immunocytochemistry was used to examine whether APECED EBs differ in stemness, proliferation or apoptosis from healthy individual’s EBs. The comparative Ct method (ΔΔCt) i.e. fold change revealed that APECED iPSC clones expressed all the classical stem cell markers similarly to healthy control iPSCs. DMSO treatment reduced the expression of stem cell markers in both healthy and APECED-derived iPSCs. The immunostaining results of iPSCs were consistent with the qPCR analysis. The overall growth properties as well as the immunocytochemical assays of stemness, proliferation and apoptosis markers did not show any significant difference between the APECED patient and healthy control derived EBs. Together the results indicate that the R257X mutation of the APECED patients does not affect stem cell properties such as stem cell marker expression and colony or the EB formation of the iPSCs. The results are contrary to previous studies in mice demonstrating the interspecific difference between mouse and human and denoting the importance of human samples completing the studies with animal models. As the APECED patient derived iPSCs did not exhibit any defects in their stemness properties, the later iPS differentiation and therapeutic use could be accomplished without hindrance. However, future work is still needed, as the small sample size in this preliminary test might introduce some biases to the results and hindered a relevant statistical analysis. Nevertheless, this thesis project was the first time APECED patient-derived iPSCs were characterized and has provided new information about the effect of AIRE mutation in APECED patient derived iPSCs.
-
(2019)After birth, stem cells act as the source of reparative and regenerative potential in various tissues. Among different tissues and organs in human body, tooth is one of the organs which does not undergo continuous regeneration. Therefore, tooth regeneration must be studied in a different animal, which possesses continuously growing teeth. In mouse, the incisor undergoes continuous growth which is fueled by the interaction between epithelial and mesenchymal stem cell compartments located at its apical end. The inferior alveolar nerve, which supports mandibular dentition, and its surrounding blood vessels (combinedly known as neurovascular bundle or NVB) were previously shown to act as a source of the mesenchymal stem cells during incisor growth and regeneration. However, the regulation of the cells in the NVB is not well understood. The primary aim of my master’s thesis was to characterize the effect of the Hh pathway modification on cellular properties of the NVB and the MSCs within it. The Ptch2 KO mouse model used in this study demonstrated increase in the number of blood vessel in the NVB. Additionally, analysis of the structure of skin in the mouse model was the second aim of my project, which showed significant increase in the thickness of the dermis at the postnatal day 1. Collectively, the change in structure of skin and NVB showed that Ptch2 might regulates the cellular properties of tooth mesenchyme and dermis by modulating the structural components of the NVB of continuously growing mice incisor and skin, respectively.
-
(2018)Staphylococcus aureus is a commensal bacterium in humans and approximately 30% of healthy people carry it as part of their microbiome, in the nasal cavity and skin, without any harm. However, it is an opportunistic pathogen that causes severe infections in immunocompromised and hospitalized patients. Typical infections caused by S. aureus are wound and skin infections, pneumonia and urinary tract infections in people with a medical implanted device such as for example a catheter. S. aureus has gained resistance to virtually all antibiotics over the years of excessive antibiotic consumption, making treatment nearly impossible in some cases. MRSA, methicillin resistant S. aureus, is a worldwide problem in hospitals and the mortality rate is still rising. One of the most common MRSA lineages is USA300, a community-acquired MRSA, which is notorious not only for its antibiotic resistance but also for its ability to form prolific biofilms. Biofilm production combined with antibiotic resistance complicates treatment of S. aureus even further. A detailed understanding the molecular mechanisms of biofilm formation might bring us closer to a cure for infections caused by MRSA biofilms. The study comprised two parts. First, characterize the phenotype of the mutants under static and dynamic conditions, test the minimal inhibitory concentrations (MIC’s) for antibiotics and verify the gene knockout by real-time RT-PCR. Second, study gene function by transduction to the parental strain USA300-UAS391 EryS and a MRSA strain TCH1516 EryS to study the gene function in a different bacterial background. The methods used were cell culturing for static and dynamic biofilm as well as growth curve, fluorescence microscopy, antibiotic susceptibility testing and real-time RT-PCR. In total seven strains were selected for characterization. The chosen seven knockouts were ΔHAD (HAD-superfamily hydrolase, subfamily IA, variant 1), non-coding region, ΔausA (non-ribosomal peptide synthetase), ΔoppA (Oligopeptide ABC transporter substrate-binding protein), ΔclfB (clumping factor B), ΔampA (cytosol aminopeptidase), and ΔpgsA (CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase). General characterization showed a few changes in biofilm formation for the genes ΔoppA, ΔausA, ΔHAD and ΔpgsA. Especially ΔpgsA is interesting because of increased ciprofloxacin resistance. The real-time RT-PCR showed some altered gene expression patterns, but no connection to poor biofilm formation. With fluorescence microscopy the growth patterns of USA300 transposon mutant strain biofilms could be described. To verify the results of the characterization, further experimentation is needed, such as RNA sequencing and complementation. Also expanding the studies to other gene hits of the screening is recommended.
-
(2019)LCMV Lymphocytic choriomeningitis virus is a rodent-borne pathogen belongs to Arenaviridae family. Most of the studies have referred Mus musculus as the main reservoir of the LCMV. It has been detected in pet rodents, laboratory rodents, and wild mice. Humans be infected with LCMV through the ingestion or inhalation of sources contaminated with rodent feces, urine, or both. LCMV infection can be asymptomatic, present with mild symptoms, or it can cause aseptic meningoencephalitis (AME) and teratogenic effects in infants. However, clinical cases of LCMV infection have been rarely reported, and there is only fragmental knowledge on the presence and prevalence of LCMV infections around the world. Likewise, the genetic characteristics of the circulating LCMV strains and impact of LCMV on public health have remained poorly characterized. This study was performed in the Southern Iraq, due to the lack of comprehensive information about LCMV in this area. There were three main aims in this thesis. First, to assess the prevalence of LCMV among the healthy human population in the Nasiriyah region, southern Iraq. Second, to assess whether LCMV infections can be associated with neurological manifestations. Third, to characterize the genetic variation and evolutionary history of LCMV strains circulating in southern Iraq. Serum and CSF samples were collected from patients and healthy people in Nasiriyah governorate in the Southern Iraq. Serum samples were screened for LCMV using Immunofluorescence assay (IFA) to detect IgG and IgM antibodies. Real-time PCR was used to detect LCMV genome. In order to confirm the PCR positive samples, we sequenced these samples by Next-generation sequencing. The serological assay results showed 12.22% IgG prevalence of LCMV among healthy people and 7.36% IgG prevalence among patients with neurological symptoms. The IgM prevalence was 1.25% among the patients with acute infections. From symptomatic patients, we sequenced partial L-segments of two new LCMV strains. The phylogenetic tree constructed on the basis of all known LCMV strains suggested that these new LCMV strains from Iraq are genetically distant from the previously known LCMV strains and form a novel sub-cluster within LCMV species. This study is the first survey of LCMV in the Southern Iraq. LCMV appears to be a rather common infection in Iraq. I reported new strains of LCMV that are circulating in the study site and most likely is the causative agent of the central nervous system-associated clinical manifestations in these patients. For future work, I’m aiming the detection of other Arenaviruses spreading in the Southern Iraq.
-
(2020)Tiivistelmä – Referat – Abstract Plant lives and grows in variable environment and climate conditions. Everyday plants can be confronted with a variety of abiotic (temperature, light, salt, water availability) and biotic stress (pathogens, insects etc). These abiotic and biotic stress can halt plant growth and influence crop productivity. Plant has evolved signaling mechanism and different responses to adapt or respond with these unfavorable environmental conditions. Our group’s previous research identified a new mutant in the model plant Arabidopsis thaliana with a striking phenotype – when the plants ages it progressively becomes yellow and eventually the entire plant is white. The mutant was named “white” after its striking appearance. The phenotype is associated with increased accumulation of mRNA transcript for stress and senescence regulated genes. Mapping of the mutation identified a 4 bp deletion in a gene EGY1 that encodes a metalloprotease located in the chloroplast. To identify molecular mechanisms that regulate this unusual type of premature senescence, a suppressor mutants screen was performed in the white mutant, and three suppressors that restore normal appearance to the plant was identified. Mapping of one of these suppressors, identified a mutation in STAY GREEN1 (SGR1) as a likely candidate. SGR1 encodes the protein that catalyze the first step in chlorophyll breakdown, removal of Mg2+ from chlorophyll. The overall aim of my master thesis was to understand the molecular mechanisms behind the development of the age and chlorophyll related phenotypes in the white mutant and its two suppressors S1 and S2. Furthermore, with gene expression analysis, plant stress and senescence responses were studied in white, S1 and S2. By complementation method I proved that mutations in SGR1 gene caused the development of suppressor mutant phenotype and restoration of wild type allele of SGR1 gene restore white phenotype in suppressor mutant. Measurements of chlorophyll concentration provided further evidence that the mutation in SGR1 stabilizes the suppressor mutant phenotype, stops chlorophyll breakdown and keep the leaves green. Gene expression study using qPCR with marker genes provided insight of molecular changes within these phenotypes.
-
(2021)Enhancers are important regulatory elements of DNA, that are bound by transcription factors (TFs) to regulate gene expression. Enhancers control cell type specific gene expression and they can form structures called super-enhancers, that consist of multiple normal enhancers and are bound by high numbers and variety of transcription factors. These super-enhancers are important for defining cell identity and changes in the super-enhancer landscape have been linked to different cancers. In this project, characterization of super-enhancers and their transcription factors composition between primary and cancer cells were studied using genome-wide next-generation sequencing data from multiple assays, such as ChIP-seq, RNA-seq and ATAC-seq. The focus of the project was on the data processing and analysis to identify and characterize the super-enhancers. Analyses included GSEA, heatmap binding analysis, peak and super-enhancer calling and IGV analysis. This project used pancreatic HPDE cell line for primary cells and different cancers with endodermal origin as cancer cell lines. The goal of the thesis was to try show characteristic features of super-enhancers and their features in normal and cancer cells. Data analysis showed that distinct super-enhancers can be identified in cancer cells and defined super-enhancers had typical strong binding for specific transcription factor and histone modification such as histone 3 lysine 27 acetylation (H3K27ac) mark of active enhancers. Super-enhancer regions were located in highly accessible chromatin regions of the genome, and genes that were associated with HPDE super-enhancers could be shown to have association with cell identity. Peak and super-enhancer calling counts varied between cell lines for transcription factors, histone modifications and super-enhancers. Visualization of super-enhancers was successful and could show transcription factor binding and active enhancers that establish the super-enhancer structure. Comprehensive analyses allowed us to characterize typical features of super-enhancers and show differences in the numbers of super-enhancers between primary and cancer cell lines and cancer cell lines of different organ types. Analysis of the transcription factor binding showed unique peaks on some of the super-enhancers, and these peaks might have a role in inducing the super-enhancer structure.
-
(2021)Uterine leiomyomas are benign smooth muscle tumors arising in myometrium. They are very common, and the incidence in women is up to 70% by the age of 50. Usually, leiomyomas are asymptomatic, but some patients suffer from various symptoms, including abnormal uterine bleeding, pelvic pain, urinary frequency, and constipation. Uterine leiomyomas may also cause subfertility. Genetic alterations in the known driver genes MED12, HMGA2, FH, and COL4A5-6 account for about 90 % of all leiomyomas. These initiator mutations result in distinct molecular subtypes of leiomyomas. The majority of whole-genome sequencing (WGS) studies analyzing chromosomal rearrangements have been performed using fresh frozen tissues. One aim of this study was to examine the feasibility of detecting chromosomal rearrangements from WGS data of formalin-fixed paraffin embedded (FFPE) tissue samples. Previous results from 3’RNA-sequencing data revealed a subset of uterine leiomyoma samples that displayed similar gene expression patterns with HMGA2-positive leiomyomas but were previously classified as HMGA2-negative by immunohistochemistry. According to 3’RNA-sequencing, all these tumors overexpressed PLAG1, and some of them overexpressed HMGA2 or HMGA1. Thus, the second aim of this study was to identify driver mutations in these leiomyoma samples using WGS. In this study, WGS was performed for 16 leiomyoma and 4 normal myometrium FFPE samples. The following bioinformatic tools were used to detect somatic alterations at multiple levels: Delly for chromosomal rearrangements, CNVkit for copy-number alterations, and Mutect for point mutations and small insertions and deletions. Sanger sequencing was used to validate findings. The quality of WGS data obtained from FFPE samples was sufficient for detecting chromosomal rearrangements, although the number of calls were quite high. We identified recurrent chromosomal rearrangements affecting HMGA2, HMGA1, and PLAG1, mutually exclusively. One sample did not harbor any of these rearrangements, but a deletion in COL4A5-6 was found. Biallelic loss of DEPDC5 was seen in one sample with an HMGA2 rearrangement and in another sample with an HMGA1 rearrangement. HMGA2 and HMGA1 encode architectural chromatin proteins regulating several transcription factors. It is well-known that HMGA2 upregulates PLAG1 expression. The structure and functionality of HMGA2 and HMGA1 are very similar and conserved, so it might be that HMGA1 may also regulate PLAG1 expression. The results of this study suggest that HMGA2 and HMGA1 drive tumorigenesis by regulating PLAG1, and thus, PLAG1 rearrangements resulting in PLAG1 overexpression can also drive tumorigenesis. A few samples, previously classified as HMGA2-negative by immunohistochemistry, revealed to harbor HMGA2 rearrangements, suggesting that the proportion of HMGA2-positive leiomyomas might be underestimated in previous studies using immunohistochemistry. Only one study has previously reported biallelic inactivation of DEPDC5 in leiomyomas, and the results of this study support the idea that biallelic loss of DEPDC5 is a secondary driver event in uterine leiomyomas.
-
(2024)The sensitivity of our conscious visual system comes remarkably close to the sensitivity limits imposed by the quantal nature of light. This exquisite sensitivity is made possible by the rod bipolar pathway (RBP), the most sensitive neural circuit studied to date, which allows us to consciously perceive light stimuli producing, in total, fewer than a dozen single-photon absorptions in rod photoreceptors. One of the central features of the RBP is the pooling of signals arising in thousands of rod photoreceptors scattered over the surface of the retina (spatial integration) into individual retinal ganglion cells (RGC), which subsequently encode visual scene as a train of action potentials and transfer these signals to the brain. However, the ultimate limits of sensitivity and the retinal circuitry underlying non-conscious vision at the absolute threshold of visual sensitivity are poorly understood. Here, we utilized the pupillary light reflex (PLR) as a functional readout of the non-conscious visual system to simultaneously measure and compare the threshold sensitivities of the conscious and non-conscious visual systems across different spatial scales in dark-adapted human observers. For this purpose, we designed, built, and calibrated an apparatus capable of producing precisely calibrated stimuli across five orders of magnitude in intensity, and four orders of magnitude in size. We find that the PLR and conscious vision express stimulus size-dependent differences in their threshold sensitivities, where when utilizing stimuli covering the whole visual field the PLR matches the sensitivity of conscious vision, by responding to stimuli producing, fewer than three photon absorptions spread over a pool of ten thousand rod photoreceptors, but when utilizing small stimuli the threshold sensitivity of the PLR falls short by an order of magnitude as compared to conscious visual system. Additionally, we find that the PLR produces a constant response to a constant number of photons (complete spatial summation), for stimulus sizes of up to 570µm in diameter. Thus, the PLR is capable of complete spatial summation over a retinal area 9-fold larger than conscious vision. Our results are consistent with RBP input into both visual systems, with each visual system providing a readout to the brain through separate RGCs.
-
(2019)The TTN gene encodes a giant muscle protein called titin that regulates the function of muscle sarcomere and interacts with several other muscle proteins. Mutations in TTN are associated with a broad range of skeletal and cardiac muscle disorders termed titinopathies. Previous studies have shown the importance of unusual TTN splicing events in patients with TTN-related cardiomyopathies and muscular dystrophies. In this project, we characterized eight TTN splicing variants to further expound on the pathogenesis of titinopathies and to enhance the diagnostic accuracy for patients with TTN mutations. In addition, we also made a comparative analysis of five different RNA/cDNA sequencing techniques to extrapolate on which approach is most suitable to study splicing variants in TTN gene. Skeletal muscle samples of six patients were analyzed in this study who were previously detected with TTN variants in a compound heterozygous state from a targeted next-generation sequencing assay. Our results from traditional Sanger sequencing methods, second-generation (Illumina RNA-Sequencing) and third-generation sequencing (Single-molecule real-time sequencing) methods showed distinct splicing events in the form of partial or complete exon skipping, intron retention, and in few instances showed multiple splicing effects rendered by a single variant. Complying with the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, the splicing variants were classified as pathogenic, likely pathogenic or variant of uncertain significance primarily on the basis of our experimental data. To address which sequencing method is most promising for analyzing TTN splicing variants, Illumina RNA Sequencing is very efficient, though, the combination of Illumina RNA Sequencing with long-read sequencing could be ideal. Our results further demonstrate that a near full-length titin is vital for survival until birth, and further studies are needed to understand the pathophysiology mechanism of congenital titinopathies.
-
(2024)Intrinsically disordered proteins (IDPs) consist of charged, and polar amino acids, lacking bulky hydrophobic residues and they do not have a single well-defined 3D structure. They are found in all domains of life with higher abundance in eukaryotes, covering approximately 30% of the eukaryotic proteome. IDPs have key roles in many biological processes from cell signaling to phase-separation phenomena. Particularly, disordered protein regions serving as linkers, have been found in many multidomain proteins and they play a decisive role in the protein’s function. In the present thesis we aim to identify the correlation between sequence and rigidity disordered linkers, utilizing a synergistic method of Nuclear Magnetic Resonance (NMR) experiments and Molecular Dynamic (MD) simulations. For that purpose, glycine and proline rich disordered linkers which are widely utilized for constructing fusion proteins were used. Additionally, we aim to characterize the rigidity of the the disordered repetitive domain of the major ampullate type I dragline silk protein, using the same approach which served to connect the two terminal folded domains inside the protein. Dragline silk has been under thorough investigation due to its favorable mechanical properties and applications in material science. NMR spin relaxation times T1, T2 and hetNOE, are highly sensitive probes to motional timescales of IDPs, but they are difficult to interpret in terms of molecular dynamics. Here, we use the spin relaxation times to validate the MD simulations which in turn are set to interpret the linkers’ internal motions. Using the quality evaluation approach QEBSS, the best simulations were identified as the best description of the conformational ensemble, based on the comparison with the experimental spin relaxation times. Systematic differences in spin relaxation times correlate with systematic changes in the linkers rigidity, proving that spin relaxation times can be used to detect disordered linker rigidity. Prolines are shown to induce a comparatively expanded conformation ensemble with significantly slower dynamics whereas glycines offer flexibility. The ensemble of the repetitive domain of the silk protein showed conformations with intermediate rigidity. We also demonstrate that the synergy of NMR and MD simulations can be used for characterizing the rigidity-sequence interplay in short glycine and proline disordered linkers and silk protein systems. Being able to tune the properties of flexible and rigid linkers can be fundamental for understanding different biological systems and for protein engineering purposes. Bioengineering applications include designing and optimizing fusion protein linkers that in the long term be useful for drug design and developing protein-based biomaterials.
-
(2020)Fluctuating light conditions can cause light stress for plants. The photosynthetic apparatus can be damaged by the excess light. Light stress causes formation of reactive oxygen species in chloroplasts. Arabidopsis thaliana’s mutant radical induced cell death1 (rcd1) is tolerant to this stress. In my thesis I used a compound called methyl viologen which causes the formation of reactive oxygen species in chloroplasts. It has been used as a herbicide. By using this compound, we can make the light stress worse and see bigger differences between the rcd1 mutant and the wild type. We identified the causative gene of rcd1’s chloroplastic stress tolerance, clarified the dependence of growth light intensity for chloroplastic stress tolerance and explored possible structural differences at the cellular level between the wild type and rcd1. Finding the genes that prevent light stress would allow a light stress tolerant crop production which could make food production easier in hot and dry areas of the world. My thesis is a part of a screening study where rcd1 mutants were screened for lowered tolerance to light stress. The amount of stress of the leaves was defined by measuring the chlorophyll fluorescence. Two most promising lines which got damaged by methyl viologen were called #20 and #54. For these a backcrossing was made with the rcd1. Clear correlation was found from their offspring between the phenotype and the methyl viologen tolerance. The correlation was strongest in the line #20 so we focused on it. Small and yellowish pale individuals which resembled their parents were the most sensitive to methyl viologen. These individuals were selected for the sequencing. Candidate genes were in the chromosome 3. The most promising one was called AT3G29185 or BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE1 (BFA1). We ordered bfa1 mutant’s seeds. We found that bfa1 mutant was itself sensitive to methyl viologen proving our observation. We discovered that methyl viologen tolerance is growth light dependent. The individuals that grew under higher intensity of light were more tolerant to methyl viologen in both the wild type and rcd1 mutant. We didn’t find structural differences at the cellular level by confocal microscopy. Thus, they can’t explain the differences in the methyl viologen tolerance.
-
(2024)Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motoneurons (MN) within the central nervous system (CNS), leading to muscle atrophy and eventual paralysis. Cause of death is in most cases due to respiratory failures 3-5 years after diagnosis. ALS can occur idiopathically without any know causes or it can be associated with certain genetic mutations. One of these known factors is a point mutation in the superoxide dismutase 1 (SOD1) gene, particularly the G93A mutation is known to affect the functionality of SOD1. SOD1 is an enzyme that metabolizes reactive oxygen species (ROS) and the SOD1-G93A mutation limits this functionality and propagates endoplasmic reticulum (ER) stress signalling cascades. Mutated SOD1 cannot be broken down by the cell, and hence it is associated with activation of protein degradation (ERAD) system with a prolonged ER stress signalling, followed by apoptotic cellular response. Although SOD1-G93A mutation has been widely studied, the basic mechanisms of the disease are not fully understood. Mesencephalic astrocyte derived neurotrophic factor (MANF) is an evolutionary conserved protein with trophic properties. MANF has been researched as novel treatment in a range of neurodegenerative diseases, such a Parkinson’s. MANF has been shown to promote cell survival but has limitation as an administered drug treatment. In this study we used transgenic SOD1-G93A mouse model with male mice to study the effects of a novel MANF variant for ALS. Disease progression and histology were used to assess the treatment efficacy.
-
(2022)Chronic stress has been linked to the pathogenesis of various disorders, such as generalized anxiety disorder, depression, and post-traumatic stress disorder (PTSD). Stress-induced hyperexcitability of the basolateral amygdala (BLA) has implications in anxiety-like behavior. Promising evidence points to the direction of GluK1 subunit containing kainate receptors (KARs) having a role in the modulation of GABAergic transmission in the lateral amygdala (LA). The aim of the present study was to investigate whether dysfunction of KARs contribute to stress-induced amygdala hyperexcitability and anxiogenesis in mice. Chronic restraint stress (CRS) is an animal model simulating chronic psychological stress. An in situ hybridization experiment was performed to investigate how CRS affects expression levels of GluK1 in the different neuronal populations in the LA. These data show that CRS leads to downregulation of GluK1 expression in the parvalbumin-positive (PV+) interneurons specifically. Patch clamp recordings of spontaneous inhibitory postsynaptic currents showed that CRS did not affect synaptic GABAergic transmission to the principal neurons in the LA. Lastly, conditional knock-out (cKO) mice that have the Grik1 gene knocked out selectively in the PV-expressing interneurons showed no change in anxiety-like behavior after CRS while their wild-type counterparts demonstrated an increase in anxiety-like behavior observable in the elevated plus maze test. Thus, ablation of GluK1 in PV+ interneurons affects the stress-induced anxiogenesis. Due to low number of animals, it cannot be confirmed yet whether the deletion leads to stress resilience or a phenotype where even regular handling is an aversive experience comparable to physical restraint. GluK1 KAR modulation of PV+ interneuron excitability and its susceptibility to stress-related alterations is only a recently discovered phenomenon, and even though this study provides some insight into the underlying mechanism, further research is needed. Systematic characterization of the mechanism could provide a novel tool for understanding and treating stress-related pathological anxiety, possibly helping patients suffering from anxiety disorders resistant to current treatments available.
-
(2023)As the most common mental disorder, anxiety disorders present a major burden to healthcare worldwide and a challenging problem to overcome for the ones suffering from it. Recently, researchers have started to recognize that the relationship between sleep and anxiety disorders is bidirectional; disturbed sleep is a potential risk factor for the progression of anxiety and anxiety can lead to sleep disturbances. However, the neural mechanisms underlying anxiety and sleep problems are still poorly recognized. In this study, we used a chronic sleep fragmentation (SF) paradigm to investigate how disturbed sleep alters anxiety-like behavior in mice and what are the potential underlying neuronal mechanisms. This model was chosen because we wanted to focus on a common form of disturbed sleep in humans rather than total sleep deprivation. We measured anxiety-like behavior in the light-dark box and open field tests right after the 2-week SF period and again after a week of recovery. Additionally, we performed immunohistochemical analysis to study prolonged cell activity (transcription factor ∆FosB), parvalbumin (PV) interneurons and perineuronal net (PNN) structures in the medial prefrontal cortex (mPFC) of the mice. Changes in mPFC activity and related brain areas are associated to anxiety in humans and anxiety-like behavior in rodents alike. Similarly, changes in PV interneurons and PNNs, that regulates PV cell function, are associated to anxiety-like behavior. However, PV interneurons and PNNs have not been previously studied in a setting that combines sleep fragmentation and anxiety-like behavior. We found that chronic SF increases anxiety-like behavior in female mice and that this effect persists at least for a week. Conversely, we did not observe significant increase in anxiety-like behavior in male mice. Both female and male mice showed decrease in ∆FosB in the mPFC suggesting that SF treated mice had lower overall levels of cell activity. Similarly, we found that SF treated mice had decreased PV interneuron intensity in both sexes which could indicate changes in the cell activity. However, the pattern of changes in the IHC results was not identical in males and females. Based on the IHC results, we suggest that SF affects neuronal processes in both sexes but the disparity in them could explain the difference in the behavioral effect. This thesis shows that disturbed sleep can lead to increased anxiety-like behavior in rodent models and recognizes potential targets to study the mechanisms behind the phenomena.
-
(2021)This thesis is a case study that examines the reasons for the lack of citizen participation in the planning process of Kalasataman keskus, and, more generally, in the planning of megaprojects. The main observation of this thesis is that there are several reasons for this. Based on the interviews of main characters taking part in the planning of Kalasataman keskus and the planning documents of Kalasataman keskus (as well as the previous research on the topic) the reasons for lack of citizen participation were: the location of planning area with few inhabitants, the large size of the planning project, technical difficulty of the planning project, the weight on the commercial aspects of the planning, and the view of the planners (relying on experts in the planning). When these results were viewed together with the previous research, it was noted that, as the previous research suggests, the traditional practices of urban planning hinder citizen participation in planning. (For example, seeing that urban planning relies on the technical knowledge of experts.) Moreover, based on the findings of the thesis as well as the previous research, it is possible to see that when the tradition, which emphasizes expert knowledge, is paired with a planning project where the city has a commercial partner, the structures and procedures of planning tend to exclude citizens’ views from the planning process. Partly based on such findings, the thesis suggests that, if the intention is to strengthen citizen participation in, especially large, planning projects, the city should aim to strengthen, for example, local community organizations.
-
(2022)Circular economy (CE) is often offered as a solution to mitigate climate change and more efficient resource use. However, the socio-cultural side of transformation to CE is widely overlooked in the academic literature (Kirchherr et al., 2018) and in the context of CE, consumer-citizens are often framed as “consumers”, “users” and in terms of “acceptance” of new products and modes of provision (Hobson & Lynch, 2016). In fact, taking part in CE can be quite laborious and the notion of consumption work highlights the time, skills and access needed to participate in circular consumption (Hobson et al., 2021). Existing research on CE skills are scarce, outdated and focused on only one practice at a time. The research gap of citizens’ CE skills has been identified by several researchers (e.g. Hobson et al., 2021; Wieser, 2019) and this thesis aims to fulfil the gap by adopting a qualitative approach. The data on which this research is based on, consists of semi-structured interviews with 20 Finnish citizens who have been active in implementing zero waste lifestyles and responsible consumption principles that are relevant for CE. By exploring their everyday practices related to CE, I identify six skill categories that the active citizens utilise to take part in CE. Particularly (1) manual skills were identified by the interviewees as central to performing circular activities. They include skills such as sewing and technical skills that enable repair and repurposing materials. The interviewees possess (2) divergent thinking skills and abilities to think creatively, for example about the ways you can use a certain item. They are also skilful in questioning consumption related social norms. (3) Research and communication skills are central for active citizens as they are trying to figure out the most sustainable options and inspire others with humour and positivity to take part in the circular economy. The interviewees describe often utilising (4) organising and prioritising skills that revolve around time management. They need to make decisions and prioritise certain actions that preferably are quite influential in terms of their carbon footprint. Moreover, when buying products second hand they should start looking for the items early and with rental options, the need should be anticipated and planned. Another identified set of skills are (5) household skills. They include maintenance skills of household goods and clothes, cooking skills to avoid food waste by using creativity and planning as well as recycling skills on sorting different fractions. The respondents also described (6) skills brought by experience. Knowledge on different second hand marketplaces and the skills to recognise good quality on materials and items enable circular practices. One of the main contributions of this thesis is consolidation of various sets of citizen skills relevant for the CE into a single framework. The findings further illustrate that consumer-citizens are doing a multitude of CE activities that require consumption work and certain skills. The findings provide information on how citizens engage and coordinate CE practices on the household level by prioritising and planning, a topic on which research has been lacking (Hobson et al., 2021). The skills of “thinking outside the box” are also a new set of skills that emerged from the interviews and it has a clear connection to the “unlearning” of noncircular consumption practices (Wieser, 2019). The identified skills could be taught more through formal and informal education channels, but it should be considered, how infrastructure, companies and services can ease people’s participation in CE. Findings of the thesis offer insight on the domestic reality of CE and how it could be improved in the Finnish context.
-
(2019)Classical and rapid-acting antidepressant drugs have been shown to reinstate juvenile-like plasticity in the adult brain, allowing mature neuronal networks to rewire in an environmentally-driven/activity-dependent process. Indeed, antidepressant drugs gradually increase expression of brain-derived neurotrophic factor (BDNF) and can rapidly activate signaling of its high-affinity receptor TRKB. However, the exact mechanism of action underlying drug-induced restoration of juvenile-like plasticity remains poorly understood. In this study we first characterized acute effects of classical and rapid-acting antidepressant drugs on the interaction between TRKB and postsynaptic density (PSD) proteins PSD-93 and PSD-95 in vitro. PSD proteins constitute the core of synaptic complexes by anchoring receptors, ion channels, adhesion proteins and various signaling molecules, and are also involved in protein transport and cell surface localization. PSD proteins have in common their role as key regulators of synaptic structure and function, although PSD-93 and PSD-95 are associated with different functions during development and have opposing effects on the state of plasticity in individual synapses and neurons. Secondly, we investigated changes in mobility of TRKB in dendritic structures in response to treatment with antidepressant drugs in vitro. We found that antidepressant drugs decrease anchoring of TRKB with PSD-93 and PSD-95, and can rapidly increase TRKB turnover in dendritic spines. Our results contribute to the mechanistic model explaining drug-induced restoration of juvenile-like neuronal plasticity, and may provide a common basis for the effects of antidepressant drugs.
-
(2021)As the effects of climate change have become increasingly more visible in recent years, interest in climate adaptation has grown in both research and policy contexts. However, although Southeast Asia is one of the regions most vulnerable to climate change impacts, there has not yet been an effort to comprehensively track how Southeast Asian countries and communities are adapting to climate change. I apply a systematic review methodology developed for adaptation research to map adaptation responses identified in the Asian Development Bank’s (ADB) projects in Southeast Asia in 2016-2020. My results show that close to a fifth of the ADB’s adaptation projects in Southeast Asia is implemented in Cambodia, while Thailand and Timor-Leste are the least covered countries. In general, the characteristics of my examined projects are relatively similar to global adaptation trends. Flooding, drought, storms, and other heavy rainfall events are the most frequently addressed climate hazards by both the projects I examined as well as by UNFCCC climate fund projects and by adaptation responses documented in scientific papers. The sectors addressed and actors targeted by ADB projects were also typical to multilateral funding institutions, focusing on the agricultural and water sectors as well as national and local governments and farmers. Capacity building was the most frequent adaptation response category, indicating that adaptation implementation as delivered by the ADB is still in a relatively early phase in most Southeast Asian countries. In addition to results related to climate adaptation in Southeast Asia, I also demonstrate the applicability of a systematic review methodology for tracking climate change adaptation responses implemented by multilateral development banks, given sufficient information is made available on relevant projects.
-
(2017)Climate change causes climatic risks (hazard-exposure-vulnerability) that are experienced in agriculture as problems with increased precipitation, droughts, pest invasions and weather variability. Agriculture needs to adapt to these changing conditions to secure its continuation in future. It is the farmers, who in last hand take action for adaptation. Farmers are recognized as a stakeholder group in agricultural with plenty of skills to tackle varying weather conditions. In fact, farmers are already implementing adaptation measures, although it is not always driven by or aimed at climate change explicitly. Adaptation policies aimed at farm-scale are being planned and developed. The Intergovernmental Panel on Climate Change (IPCC) is the leading scientific source of adaptation policy recommendations. Currently it is recommending risk management approach for managing the known and unknown climatic risks that societies and sectors like agriculture are facing. In agriculture, for example, crop loss insurances are part of climate risk management. Farmers are making adaptation decisions at farm-scale based on their own beliefs and experiences, on information from variable sources, and guided by policies and legislation. The primary driver for taking adaptive action at farm, according to protection motivation theory (PMT), however, is the perception of risk – if the risk is assessed high enough and the adaptation is assessed possible. In this thesis, PMT and the theory of risk perception are used to explain farm-scale adaptation. Adaptation is examined as climate or weather variation driven adaptation measures implemented by farmers autonomously or guided by policies. A case study approach and stakeholder interviews were used because of the novelty of the study topic. By examining the case of ‘adaptation in Uusimaa agriculture’ through the perceptions of interviewed farmers and extension officers, an overview on farm-scale adaptation with its variety of influencing factors, and better understanding of risk perceptions as adaptation drivers is reached. Findings of the study show that farmers in Uusimaa are taking adaptation measures, but adaptation policies are not yet guiding adaptation at farm level and that farmers are divided by the ways they respond to climatic risks. Further studies on adaptation policies and agricultural adaptation should recognize the varying risk responses, the need for better adaptation policy guidance and farmers’ adaptation experiences and skills.
Now showing items 141-160 of 1112