Browsing by department "Bio- ja ympäristötieteellinen tiedekunta"

Proteostasis is used by cells to maintain proteome health and understanding the biological mechanisms underpinning proteostasis is important. Despite many studies on small molecule-mediated inhibition of Sec61-dependent protein translocation, a knowledge gap exists in the quality control pathway(s) of pharmacologically-displaced secretory polypeptides. Genetic screens can be used to discover proteostasis regulators of pharmacologically-displaced secretory polypeptides. Near-haploid human HAP1 cells with an inducible Tet-on GFP reporter (reporter HAP1 cells) can be used as a cellular tool to screen for human host factors pertinent to proteostasis of secretory polypeptides. The isolation of haploid-enriched reporter HAP1 cells ensures that the inability to efficiently recover bi-allelic gene trap mutants is avoided. The use of haploid-enriched cells is a prerequisite to gene trap mutagenesis screens. Here, I present data on the isolation of haploid-enriched reporter HAP1 cells that could be used as a cellular tool in gene trap mutagenesis screens. A workflow for the isolation of haploid-enriched reporter HAP1 cells was optimised using a diploid reporter HAP1 cell line as a control. Both DNA content analysis and karyotyping showed that the isolated HAP1 reporter cell lines were haploid. In the haploid-enriched HAP1 reporter cells, the GFP-reporter compartmentalised in the ER, and a Sec61-translocon inhibitor CT8 could inhibit the GFP-mediated fluorescence. The haploid reporter HAP1 cell lines produced in this study are suitable for future gene-trap mutagenesis experiments.

Introduction and aims. Multiple different neurobiological alterations have been hypothesized to underlie Major Depression Disorder (MDD), but no unifying theory exists to explain the mechanisms of the disorder. The aberrant brain dynamics in MDD can be seen in the alterations of long-range temporal correlations (LRTCs), which have been proposed to be an indication of criticality in healthy brain. Alterations in LRTCs have been suggested to reflect deficiencies in excitation-inhibition (E/I) balance, neuromodulation or connectivity patterns, which have also been proposed to be the underlying mechanisms of MDD. There has been controversy whether the pathology is related to attenuated or increased LRTCs, and the sources of altered brain dynamics have not yet been localized. The aim of this study was to find in which frequency bands and where in the brain the neuronal LRTCs are altered in MDD on source level. In addition to analyzing the correlations between neuronal LRTCs and depression severity in parcel level, we studied correlations in functional networks to get a better understanding of the system level alterations in MDD. We also studied whether behavioral LRTCs correlate with depression severity or with behavioral performance. Methods. We investigated the long-range temporal correlations in a cohort of 19 depressed subjects by using magnetoencephalography (MEG) for recording brain activity during resting state and response inhibition task and performed DFA analysis on the amplitude envelopes of cortical oscillations. The depression severity was measured with BDI-21 questionnaire. Results and conclusions. We found the LRTCs to be positively correlated with depression severity in the alpha frequency band (8–12Hz) predominantly in the limbic system that underlies emotional control. This result was supported by the parcel level analysis in which correlations between alpha band LRTCs and depression severity were observed in the orbitofrontal cortex and temporal pole, indicating that the hyper-activation of limbic system could explain the negative bias characteristic to depression. Positive correlations were also found in frontoparietal, ventral, and dorsal attentional networks that support cognitive control. Alpha band LRTCs correlated also with behavioral LRTCs during both resting state and task conditions. However, we observed more wide-spread correlations between alpha range LRTCs and depression severity than between neuronal LRTCs and behavioral LRTCs. Behavioral LRTCs correlated with depression severity, but not with behavioral performance. These results indicate that depression is characterized by vast alterations in the brain dynamics and imply that the wide range of different symptoms in MDD could be explained by alterations in the excitation/inhibition balance in the limbic system and cognitive networks.

Lymphedema is a progressive disease, resulting from abnormalities of the lymphatic system. It is characterized by swelling of one or more parts of the body, due to impaired lymph transport. Lymphedema has no definitive treatment and it can have serious effects on the patients' quality of life. However, the recent knowledge expansion of the molecular mechanisms regulating lymphangiogenesis provides new possibilities for the treatment of lymphedema. One of these mechanisms is the angiopoietin-TIE system. It has been shown recently that ANG2 is essential for the formation of lymphatic vasculature. Mice ANG2-deletion caused widespread lymphatic dysfunction, resulting in subcutaneous edema and chylous ascites. Lymphatic development is also regulated by TIE1 receptor, as its deletion in mice resulted in malformed jugular lymph sacs and severe edema. In this study, a model of autocrine TIE receptor activation has been established in endothelial cells to investigate the effect of the WT-ANG2 and four ANG2 mutants (T299M, N304K, C435S and R492Q) in TIE receptor phosphorylation and lymphedema. The role of these variants in direct cell adhesion has also been investigated in vitro using HeLa and lymphatic endothelial cells. The findings revealed that WT-ANG2 and soluble ANG2 variants induced both TIE2 and TIE1 activation in endothelial cells. We also found that N304K, C435S and R492Q mutants are secretion-deficients and retain the co-expressed WT-ANG2 inside the cells, causing a dominant negative effect in both TIE2 and TIE1 receptor activation which is likely associated with the lymphedema in the patients where the mutants were identified.

The human gut is inhabited by gut microbiota, a complex and diverse ecological community of trillions of microbes that affect both the normal human physiology and countless disease states and susceptibilities. Understanding the composition, functions and the causes and effects of changes in the microbiota is invaluable for understanding diseases that are connected to the microbiota and developing better treatments to the diseases. The gut microbiota varies between individuals and keeps changing over time. Behind the variability are e.g. the person’s age, genetics, diet, environment, and especially diseases and the use of antibiotics. When antibiotic use disrupts the gut microbiota, the changes can persist for years. Antibiotic resistance tends to increase after the use of antibiotics. Since antibiotic resistance in bacterial pathogens is considered a major health threat, the characterization of the human gut resistome (the antibiotic resistance genes (ARGs) found in the gut microbiota) is of great medical interest. Next-generation sequencing techniques have enabled studying also those microbe species that cannot be cultured at the moment. Metagenomics provides information on all genetic material collected from a given environment and enables searching for any sequences of interest within it, e.g. ARG sequences. The development of Parkinson’s disease (PD) is suspected to begin in the enteric nervous system and spread from there toward the central nervous system. The use of antibiotics could be linked to PD through their effects on gut microbiota, and since these effects are modified by the gut resistome, the aim of this study was to find gene sequences coding antibiotic resistance in human gut metagenomics data originating from stool samples of PD patients and healthy controls, and to find out potential differences in the occurrence of antibiotic resistance genes in the gut microbes of the two study groups. DeepARG was the chosen method for searching antibiotic resistance gene sequences in the metagenomics data. The statistical data analyses, including alpha diversity, multivariate analyses, and differential abundance analysis, were performed with the R statistical programming language in RStudio. DeepARG found 840 different ARGs in 192 samples. The ARGs belonged to 29 different ARG classes. The alpha diversity analysis showed a small estimated difference between PD and control groups indicating a possible slightly higher ARG diversity in the PD group. Multivariate analysis did not give any strong suggestions of definite biologically meaningful differences between the study groups. 16 ARGs were deemed differentially abundant in the study groups. BepE, cmeA, cmlv, dfrE, mefC, msrB, opcM, oprM and RbpA seemed to have increased abundance, and arnC, BN537_02049, dfrK, mgrA, murA, tet35 and tetT were suggested to have decreased abundance in PD patients compared to the healthy controls. These ARGs do not appear interconnected in any other way except for some sharing antibiotic types to which they offer resistance, and some having similar resistance mechanisms. In the light of an ongoing, unpublished epidemiological study of the connection between PD and the use of antibiotics it would seem that only three ARGs (msrB, mefC and dfrE) might be somehow relevant in PD development, but their effects, if any, are most likely minor. Eight ARG classes were shown to have differential abundance between PD patients and healthy controls. Bacitracin, fosfomycin and polymyxin classes showed decrease and chloramphenicol, fosmidomycin, puromycin, rifampin and sulfonamide classes showed increase in abundance in PD compared to controls. The change in the abundance of a certain ARG could reflect change in the abundance of the bacteria carrying that resistance gene. If so, the follow-up questions would be how much change in the abundance of bacteria is due to the use of certain antibiotics and how much is caused by environmental factors. It also remains to be studied whether specific antibiotics associated with the ARGs that in this study showed differential abundance in PD patients and healthy controls might have an actual role in PD development. The results of this thesis study are later to be combined with and further studied alongside information coming from ongoing studies on antibiotics use in general population and in PD patients. While this study did not concentrate its efforts into finding novel ARGs, the metagenomics dataset could also in the future be applied for that purpose.

The arylhydrocarbon receptor (AHR) is known for its xenobiotic role. In the last decades we have realized it has an important role even in normal physiology. Earlier studies have shown different circadian behavior in mice and rats when AHR is activated with the environmental toxoid TCDD. Also, AHR knock-out (AHRKO) mice have shown to adapt quicker to new lighting conditions. The aim of this study was to chart AHRs role on the circadian behavior in rats, by comparing daily eating and drinking habits under normal lighting condition for 7 days and for 7 days after a 12-hour light shift. Tissue samples to be used in continuing studies were taken after the 14 days long follow up. These studies will chart how the circadian timekeeping genes are expressed in the central (suprachiasmatic nucleus) and periphery (liver) cells in AHRKO rats after an adaptation to phase shift compared to wild type rats. This way the study will provide information that will help us understand the role of AHR in different species regarding behavior and in continuing studies gene expression. In our study no differences in drinking and eating activity could be seen between AHRKO and wild type rats. Both groups adapted to new lighting conditions equally fast.

The brainstem monoaminergic neuronal systems are involved in regulation of mood, reward system, memory processing etc. Any defects or damage in these cells lead to many neurological disorders. The brainstem inhibitory GABAergic and excitatory glutamatergic cells in turn control these neuromodulatory neurons. The glutamatergic neurons are found in the Laterodorsal tegmental nucleus (LDTg), Interpeduncular nucleus (IPN) as well as in the Ventral tegmental are (VTA). The LDTg in particular sends these glutamatergic projections to the VTA to regulate their Dopaminergic (DA) neurons. During embryonic development, the brainstem GABAergic and glutamatergic neurons, that regulate the monoaminergic systems, are produced in the ventral rhombomere 1. Their subtypes are known to express various transcription factors (TFs), such as Nkx6-1, Vsx2 and Skor1 marking the glutamatergic neuron precursors in the ventral rhombomere 1. In this thesis project, I studied the expression of another TF, Skor2 in the embryonic brainstem precursors. The basis of the experiment came from an embryonic brainstem single cell mRNA sequencing study performed earlier, where Skor2 expression was observed in the cluster of neurons containing Nkx6-1, Vsx2 and Skor1 expressing cells. Based on this, I hypothesized that Skor2 expression could be seen in glutamatergic precursors in the ventral rhombomere (rV2) domain as well as later in the LDTg nucleus derived from these precursors. To test this, I performed immunohistochemistry (IMHC) studies on a transgenic mouse line expressing Green Fluorescent Protein (GFP) from the Skor2 locus. In the second part of the thesis, I hypothesized that the Skor2 positive cells need this TF for their differentiation. To study this aspect, I performed similar IMHC studies on homozygous Skor2GFP/GFP mice, where Skor2 had been inactivated. My study showed that Skor2 positive cells expressed markers Nkx6-1 and Vsx2 and represented a specific subgroup of early embryonic post-mitotic precursors in the rV2 domain. Later in the brainstem, in contrast to my initial hypothesis, I did not observe Skor2 expression in the LDTg glutamatergic region. Instead, I observed Skor2 positive cells in a region more lateral to the Ventral and Dorsal tegmental nuclei of Gudden. In the homozygous Skor2 mutants, I observed no changes in cell fate during embryonic development. Based on my results, the TF Skor2 is expresses in the glutamatergic precursors and neurons in the rhombomere 1, but form a part of a new cluster of cells away from the LDTg. These neurons have not been studied in detail. However, the Ventral and Dorsal tegmental nuclei of Gudden have been shown to regulate memory and navigation. It is possible that the Skor2 expressing neurons also participate in these functions. Identification of specific molecular markers, such as Skor2, for these neurons now allows their focused functional studies. Skor2 and Skor1 are related TFs belonging to Ski family of transcriptional repressors and are seen to be expressed together. Further investigations into the roles and functional redundancy of these two TFs can be performed using mice carrying mutations in both of these genes.

Cornea is transparent layer of cells lying in front of lens. The corneal epithelium, a squamous epithelium, covers the ocular surface and ensures proper vision by preserving the integrity of the eye. Corneal epithelium is renewed continuously throughout life from a pool of stem cells (SC). There are still conflicting theories about the localization of stem cells required for the growth, renewal and maintenance of the corneal epithelium. Previous studies demonstrated that the limbus, located in the periphery of the cornea, serves as the stem cell niche (SCN) in adults. However, contrasting evidence from clonal analysis proposes that, in early postnatal life, renewal is fuelled by SCs located in the basal layer of the central cornea. There are alternate patterns of renewal in young and adult mouse cornea and that there is an important, transitional time frame called cornea maturation, when the adult patterns of gene expression, cell dynamics and tissue renewal are established. In the cornea, solid SC markers are still missing, yet studies on human limbal cells have suggested Bmi1 and C/EBPδ as limbal SC markers. There are, indeed, long-lived SCs in the central cornea and that the gene Bmi1 plays a role in these central corneal SCs. However, the physiological importance of these Bmi1+ cells remains obscure. The main aim of this project is to understand the fate and dynamics of these Bmi1+ cells and study the chronology of maturation of the cornea. In this study, I have also tried to correlate the growth of eye size with proliferation of corneal epithelial cells This study was conducted using few different kinds of transgenic mice (Mus musculus). To study the fate of Bmi1+ cells, two different mouse lines were crossed: Bmi1-CreER and ROSA26-LacZ. Mice carrying both alleles were used for lineage tracing experiments. Moreover, Hematoxylin-eosin staining was used to follow the eye morphology. Immunohistochemistry was performed to follow the chronology of maturation of the cornea, proliferation of corneal epithelial cells and the location of Bmi1+ cells in corneal epithelium. From this study, we can propose that cornea maturation is completed by the time of eyelid opening, which take place two weeks after birth. Krt19 is perfect for studying the chronology of the corneal epithelium, immunostaining of Krt19 separates the territory of limbus from central cornea enabling to distinguish limbus distinctly. Proliferating cells reside in basal layer of cornea. Bmi1+ cells found throughout the basal layer of the cornea that locally renews the corneal epithelium concluding Bmi1+ cells as the progenitor cells.

This master’s thesis addresses the role of intermediary organizations in sustainability transitions, specifically in the field of energy. The thesis discusses how intermediary organizations can diffuse and support the development of novel sustainable socio-technical niche-innovations through experiments. Theoretically, this thesis draws mostly upon the sustainability transition literature, particularly strategic niche management theory. Empirically, this thesis focuses on a case study of joint procurement of solar power plants that was implemented as a part of “New and innovative low-carbon business generates competitive advantage for companies and municipalities” (Välke) project in South-western Päijänne during 2016. Välke is a sub-project of Carbon-neutral municipalities (HINKU) and therefore the case study of this thesis links to other similar experiments in the HINKU network. The material of the thesis was collected through 9 semi-structured interviews with different stakeholders involved in the experiment and by using pre-existing secondary material. The material was analysed by qualitative content analysis using an analytical framework adopted from a previous study. The findings show that intermediaries support niche development by aggregating, circulating and applying lessons learned between and within joint procurement experiments. This was done by producing and disseminating documents, but more importantly through personal contact between intermediaries. The network of intermediaries showed hierarchical features as the coordinator of HINKU, the Finnish Environment Institute (SYKE), maintained the repositories of the learned lessons and also formed a link between municipal intermediaries involved with the experiments. Also, during the experiments the more experienced higher profile intermediaries – mostly SYKE – provided assistance to lower profile municipal intermediaries. This support included providing knowledge, but also raising the confidence of lower profile intermediaries. Following, the lower profile intermediaries provided similar assistance to the participants of the experiments. However, the intermediary roles were not stable as lower level intermediaries were adopting some of the roles of the higher profile intermediaries after gaining experience during the experimentation. In order to support the Finnish solar niche, the intermediaries went beyond mere aggregating, circulating and applying lessons between and within experiments. They were actively initiating new experiments in adjusted formsin new geographical locations and advocating the niche. This was partially linked to the strategic nature of the intermediaries, as they were established to catalyse activities that would lead to mitigation of greenhouse gas emissions. However, this active support for a particular socio-technical innovation contradicted the pursuit of intermediaries to be viewed as neutral and credible actors. Partially as a consequence – but also because of the phase of solar niche development in Finland and lack of resource and interest – SYKE chose to withdraw from future joint procurements of solar power plants for private actors. However, SYKE was planning to utilize the governance innovation of joint procurements in order to support other niche-innovations with sustainability gains. The findings show that intermediaries can accelerate energy transitions, at least on a regional scale. They emphasize the importance of cooperation and personal contact between intermediaries and the ability of intermediaries to utilize governance innovations. Also, the findings support stronger inclusion of intermediaries in governance frameworks to hasten energy transition and achieve wider sustainability goals. However, the thesis shows that particularly public intermediary organizations have to work under unclear mandates.

Reactive oxygen species (ROS) are one of the prominent groups of signal compounds that are produced in stress conditions such as excess light. Nuclear protein RADICAL-INDUCED CELL DEAT (RCD1) is sensitive to ROS and controls the expression of organelle components, e.g. mitochondrial alternative oxidases (AOX), thus balancing the redox-status of a plant cell. Plants have fast responses to fluctuating light conditions that happen even before gene expression: i.e. readjusting the capability to receive light energy between the two photosystems by state transitions and increasing the capacity to remove excess energy by non-photochemical quenching (NPQ). Various small auxiliary proteins function in these fast acclimation events. However, many of them are identified on gene level only. The goal of this master’s thesis is to describe the role of a hypothetical protein, PPD8 in Arabidopsis thaliana. We evaluate how PPD8 is associated with RCD1 and a chloroplast thiol-regulator enzyme NTRC. We created double (rcd1 ppd8) and triple mutant plant lines (rcd1 ppd8 ntrc) by crossing single knockout lines ppd8, rcd1 and ntrc. Photosynthetic performance, NPQ and sensitivity to ROS were observed in each line by using two different chlorophyll fluorescence measurement methods: pulse-amplitude-modulation (PAM) and novel OJIP imaging fluorometry. The leaves were exposed to methyl viologen (MV), which accelerates the chloroplastic ROS production in light, and also to hypoxic conditions in order to study how the effect of MV is altered in low concentrations of oxygen. Additionally, we examined the amount of photosynthetic proteins and stoichiometry of photosystems in ppd8, rcd1 and rcd1 ppd8 by immunological methods. Finally, PPD8 gene with attached hemagglutinin encoding tags was generated by cloning and reintroduced back to the ppd8 knockout lines. Plants lacking RCD1 are very tolerant against MV and ROS, but when rcd1 was crossed with ppd8 the resistance was suppressed. Both rcd1 ppd8 and ppd8 exhibited elevated chlorophyll fluorescence and NPQ values. The removal of PPD8 gene had an impact on the abundance and the stoichiometry of photosynthetic proteins reducing the plants’ performance. When RCD1, PPD8 and NTRC were simultaneously absent the plants had major defects: their NPQ and fluorescence values were drastically increased. Furthermore, several results hinted towards possible issues in the function of ATP synthase in ppd8 background plants. It is also known that NTRC regulates ATP synthase: taken together, the results suggest that PPD8 is necessary for a fully operative ATP synthase and photosynthetic machinery. By reintroducing PPD8 to knockout line ppd8, the phenotype could be reverted back to wild type -like, thus confirming the significance of the PPD8 gene product in plant.

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.