Browsing by department "Bio- ja ympäristötieteellinen tiedekunta"
Now showing items 1-20 of 231
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(2019)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.
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(2019)Elevernas intresse för naturvetenskaper har minskat och en del elever anser att naturvetenskapliga läroämnen är onödiga och att undervisningen i dem är gammaldags. Ungdomar spenderar dessutom mycket mindre tid i naturen än tidigare vilket har lett till att deras kunskap och engagemang för miljön minskat. I dagens värld, med avancerad teknik och komplexa miljöproblem, är det dock viktigt att ungdomarna utvecklar en vetenskaplig och ekologisk läskunnighet för att kunna göra välinformerade och hållbara val. Syftet med den här avhandlingen var att bidra till en förnyelse av undervisningen i biologi genom att skapa aktiverande uteundervisningsuppgifter om växter. Utomhuspedagogik fungerade som didaktisk grund till uppgifterna eftersom undervisningen utförs utomhus och inkluderar aktiverande, undersökande, upplevelsebaserade, samt helhetsskapande och ämnesöverskridande element. Den här typen av undervisning föreskrivs i den nya läroplanen och forskningen påvisar dessutom att den höjer elevernas intresse och lärande i biologi samt främjar deras miljöengagemang. Det antogs finnas ett behov av färdiga undervisningsuppgifter eftersom uteundervisning är tidskrävande att planera och lärarna rapporterat om tidsbrist på arbetet. Uppgifterna fokuserade på växter eftersom kännedomen om dem generellt är sämre än kännedomen om djur och eftersom det befintliga utbudet av växtrelaterade undervisningsuppgifter är bristfälligt. Kvalitativa intervjuer och en webbenkät genomfördes med biologilärare i årskurs sju och åtta i grundskolan med avsikt ta reda på skolornas praktiska förutsättningar för uteundervisning, lärarnas åsikter om uteundervisning, samt deras önskemål jämte behov av uteundervisningsuppgifter. Fyra lärare intervjuades och 18 lärare svarade på webbenkäten. Enkäten analyserades och intervjuerna transkriberades och analyserades. Resultaten påvisade att lärarna saknar tid för att konstruera egna uteundervisningsuppgifter och att lärarna ansåg att färdiga uppgifter skulle underlätta deras arbete. Nio undervisningsuppgifter och en växtartlista skapades för att svara på lärarnas behov och inkluderade de åtta mest önskade arbetssätten. Utomhuspedagogikens fördelar utnyttjades i uppgifterna till exempel genom att mångsidigt begagna sinnesupplevelser och reflektion samt rörelse, samarbete och diskussion. Uppgifterna beaktade de praktiska förutsättningarna för uteundervisningen i skolorna genom att förlägga majoriteten av uppgifterna till hösten och våren och genom att göra uppgifterna anpassningsbara till olika naturtyper. Uppgifterna kan användas för att förverkliga läroplanens mål för utveckling av mångsidig kompetens samt de flesta målen för undervisningen i biologi, och inkluderade dessutom ämnesövergripande och helhetsskapande undervisning. Undervisningsuppgifterna som skapades kan användas för att förnya undervisningen i biologi och för att underlätta lärarnas arbete. Uppgifterna har, tack vare utomhuspedagogikens och det aktiva lärandets unika särdrag, potential att höja elevens intresse och lärande i biologi, kapacitet att utveckla kvalitativ kunskap och högre former av tänkande och dessutom utsikter att skapa ett personligt förhållande mellan eleven och naturen vilket i förlängningen kan leda till att eleven utvecklar en miljömedvetenhet och hållbar livsstil. En utmaning som synliggjordes var att lärarna inte verkar inse uteundervisningens kvalitativa mervärde utan istället antar en mer kvantitativ kunskapssyn vilket varken är förenligt med samtida forskning eller den nya läroplanen. Några av uppgifterna kommer att publiceras som en del av en nätpublikation som produceras inom ramen för ett lärarfortbildningsprojekt finansierat av Utbildningsstyrelsen och kommer således att bli tillgängliga för lärarna.
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(2019)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.
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(2019)Uterine leiomyoma (also known as myoma) is the most common neoplasia in women during reproductive age and it represents a burden for public health care. Approximately 70% of Caucasian women develop myomas, although only 25% of cases are symptomatic. The genetic background of myomas varies significantly and the most common genetic causes are mutations in genes Mediator complex subunit 12 (MED12), Fumarate hydratase (FH) or YEATS Domain containing 4 (YEATS4) , rearrangements affecting the High Mobility Group AT-hook 2 (HMGA2), and deletions in COL4A5/6 locus. MED12 mutations represent the most common genetic alteration in myomas, being present in approximately 70% of cases. Genome organization comprises different levels of complexity, spanning from regulation of individual genes to changes in the architecture of large portions of chromosomes. Literature offers massive evidence of changes in genome organization among different cell types and between several tumor and related healthy cells, but information about these changes in myoma is lacking. The aim of this study is to determine the main features of genome organization in myomas belonging to the aforementioned five genetic subclasses, in order to identify which are the underlying common pathways that are dysregulated in the neoplasia. This is achieved by mapping regions of open chromatin in myomas and related my-ometrium samples with ATAC-seq. Sample’s clustering seems not to be individual-dependent, while tumors belonging to FH, YEATS4 and COL4A5/6 subclasses form distinct clusters, unlike MED12 and HMGA2 subclasses. Six open chromatin regions located within genes were identified in 19/25 tumors and not in myometrium. Seven myometrium-specific open chromatin regions were identified in 21/25 myometria and in less than 10 tumors. As expected, Gene Ontology enrichment analysis revealed that myomas belonging to FH subclass are characterized by deregulation of metabolic pathways. Many of the identified genes in the open chromatin regions have been linked to other tumors in previous studies. Tumor-specific open chromatin regions locate within oncogenes, while myometrium-specific ones are found in proximity of tumor suppressor genes, suggesting a biological role in myomagenesis for these genes. Further investigation on the identified genes (e.g. transcriptional regulation, gene expression and protein level) and addi-tional studies on chromatin architecture are needed to fully unravel the mechanism of myomagenesis.
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(2020)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.
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(2020)This study focused on the spectral sensitivity of two Norwegian lake populations of opossum shrimp (Mysis relicta), with a common implantation history and a temporal separation of about 50 years. Previous findings have indicated a difference in the absorption maximum (λmax) between sea and lake populations of Nordic mysids that have been separated about 9 000 years. Between the population groups, the spectral sensitivity correlates to the Wavelength of Maximal Transmission (WLMT) in the habitats. This may be considered a form of adaptive tuning. It is not known if the species-specific mechanism is based on chromophore shift or opsin modification or a combination of both, neither is the timescale of the adaption well understood. The intent was to determine λmax of both populations, what chromophore(s) they use and possible variations of the opsin gene. By comparison to spectrometric data of the habitats, the study aimed to broaden the insight into the mentioned unknowns. The light conditions of the lakes were determined by spectrometry down to depths of three and five meters. As predicted a positive, lake-bound correlation between the attenuation coefficient (k) and WMLT was found. Single-rhabdom microspectrophotometry (MSP) was used to determine λmax of the visual pigment in situ. Absorbance spectra were analysed by manual fitting to mathematical pigment templates and by script-based automation. Neither the chromophore nor differences in λmax could be determined, due to a small sample size that limited the statistical power of the results. The opsin genes from both populations were sequenced. No differences expected to have an effect on spectral sensitivity were found. Spectral tuning could not be demonstrated to have occurred in the populations due to the limited sample size. Nor did the results give support for any new findings on the mechanism or the time scale of spectral tuning among mysids. To answer the proposed questions of the study, additional sampling of both populations is needed.
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(2020)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.
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(2019)Despite recent advances in understanding, diagnosis and treatment of cancer, this complex and versatile disease remains one of the leading causes of death worldwide. New and rapid diagnostic methods are needed to detect cancers at their early stages of development, thus enabling earlier prognosis, better risk assessment and more efficient treatment of the disease. There has been an increasing interest in specific molecular biomarkers as the hallmark for cancer research, and the detection of these markers from liquid biopsies using advanced molecular diagnostics methods provides major advantages over the conventional imaging methods currently used in oncology. The aims of this thesis were to examine the applicability of a novel molecular method, SIBA® (Strand Invasion Based Amplification), for the detection of cancer biomarkers, and to develop an assay targeting androgen receptor splice variant 7 (AR-V7) mRNA. The AR-V7 is proposed as a treatment-response biomarker in patients with castration-resistant metastatic prostate cancer (mCRPC). The expression of this variant can indicate resistance to hormonal therapies used for the treatment of advanced prostate cancer. Prostate cancer is the most common cancer after lung cancer in men worldwide and can gradually develop into a highly advanced lethal form, mCRPC, that is not responsive to androgen deprivation therapies. Positive AR-V7 status is suggested to represent the phenotype of this advanced stage of prostate cancer, and its detection can assist in treatment selection for the mCRPC patients. SIBA is a novel isothermal method for the amplification and detection of nucleic acids. The technology offers significant advantages over the more conventional molecular detection method, polymerase chain reaction (PCR), since the amplification reaction occurs at constant temperature and does not require sophisticated laboratory equipment for the thermal cycling. Reverse transcription SIBA (RT-SIBA) enables reverse transcription of RNA to cDNA as well as the simultaneous amplification and detection of the cDNA in one-step reaction under isothermal conditions. The method displays both high analytical sensitivity and specificity to the target nucleic acids. The RT-SIBA technology has not formerly been applied for the detection of human DNA or RNA. The main finding of this thesis was, that the RT-SIBA technology can be applied for rapid detection of specific molecular cancer biomarkers such as the AR-V7 mRNA. In this study, two RT-SIBA assays targeting the full-length androgen receptor (AR-FL) mRNA and the AR splice variant 7 mRNA were developed and optimized. Performance of the assays were evaluated by testing RNA isolates from AR-V7 positive and negative prostate cancer cell lines in the presence of human whole blood and plasma in the reaction. The developed RT-SIBA assays provided high analytical sensitivity and specificity: low copies of the target mRNA were amplified within 20 minutes without the production of non-intended amplicons. The results suggest that the RT-SIBA technology can be utilized for easy and rapid detection of AR-V7 and AR-FL mRNA directly from liquid sample material without a need for time-consuming sample treatment. Further performance evaluation using real AR-V7 positive clinical samples from mCRPC patients is necessary for the reliable validation of the developed assays.
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(2018)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.
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(2019)Increased intestinal permeability and its role in autoimmune, metabolic and chronic intestinal diseases is under extensive research as the “leaky gut” is considered as a potential target for preventive and therapeutic strategies in wide range of diseases. Zonulin, an eukaryotic analogue of Vibrio cholerae derived Zonula occludens toxin, which induces tight junction disassembly, has recently become a popular serum-based biomarker of intestinal permeability in biomedical research, even though the link between serum zonulin levels and functional measures of intestinal permeability has never been validated properly in humans. In addition, surprisingly little is known about the location and regulation of zonulin expression in the humans despite the protein was discovered almost two decades ago. Zonulin, also known as pre-haptoglobin-2, is an uncleaved precursor form of haptoglobin that is abundantly expressed in the liver. Zonulin, in turn, based on studies on rats, rabbits and monkeys, is expressed in the small intestine and stimulated by exposure to bacteria and gliadin, but no other stimulators have been described so far. It is also unclear, if different bacteria can induce different responses in zonulin secretion as only the effect of gram-negative enterobacteria has been documented so far. The aim of this study was to evaluate the effect of selected intestinal bacteria and of two known upregulators of haptoglobin, interleukin-6 (IL-6) and bacterial lipopolysaccharide (LPS), on zonulin secretion in vitro. The impact of two gram-positive probiotic bacteria (Lactobacillus rhamnosus GG & Bifidobacterium bifidum) and of two commensal gram-negative bacterial strains (Escherichia coli DH5α & Escherichia coli RY13) were tested for zonulin secretion in HT-29 intestinal epithelial cells, in addition to IL-6. Two separate lineages of immortalized human hepatocytes were tested for zonulin secretion by stimulation of LPS and IL-6. In addition, different immunological methods were assessed for quantification of zonulin, as the potential cross-reactivity of our primary analysis method, a commercial zonulin ELISA kit from Immundiagnostik AG that is also the main method used in the published zonulin studies, became more evident at the beginning of this thesis project. The main findings of this study were that the widely used commercial zonulin ELISA from Immundiagnostik AG is not specific for zonulin, but instead cross-reacts at least with complement C3, in line with the results published by other group during this work. Our further experiments comparing the signals of the above-mentioned zonulin ELISA and complement C3 ELISA for serum samples showed that there was only weak correlation between the obtained signals, suggesting that the zonulin antibody does not directly bind to complement C3. By using dot blot, western blot and immunoprecipitation, we found that the cross-reaction only occurred in native conditions. Based on zonulin ELISA measurements of the cell culture media from the in vitro experiments, very low signal was obtained for both intestinal and hepatic cells. Among the tested bacteria, only exposure to Lactobacillus rhamnosus GG led to a significant increase in the release of target protein. In hepatic cells, LPS had no effect, while IL-6 led to a significant increase of zonulin ELISA signal in one of the tested hepatic lines. However, it is currently difficult to differentiate if the low detected “zonulin” levels in this study are due to low level of secretion, or rather due to the lack of a proper method to detect zonulin. Taken together, these observations suggest that the most commonly used zonulin ELISA and other related, commercially available antibody-based methods for zonulin detection should be utilized with caution, as these antibodies cross-react with other protein(s). Hence, the serum “zonulin” cannot be considered as a biomarker of intestinal permeability until the captured protein(s) are identified, and similarly the anticipated effects of intestinal bacteria on zonulin expression cannot be reliably investigated with the currently available antibodies.
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(2020)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.
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(2020)Tiivistelmä – Referat – Abstract Proteins differ from one another on the basis of their amino acid sequences, display a different spatial shape and structure, and have different functions. The linear order of amino acid residues are chained to one another by peptide bond. The ß-strands and α-helices can be considered as the key components present in the three-dimensional structure of a protein. There are several bioinformatic methods involved to predict structure and function of protein such as searching sequencing similarities, multiple sequence alignment, characterisation of domains, solvent accessibility, and modelling three-dimensional structures at atomic level. The main focus of this study was to build the three-dimensional structure models and then compare the homologues regions in different models. 36 reviewed capsid L1 and L2 protein sequences of human papilloma virus subtypes were selected based on 65% sequences similarity from Universal Protein database. We utilised several computational algorithms in this study for the analysis of protein sequences for the evolutionary relationship and modelled the three-dimensional structures of capsid L1 and L2 proteins of oncogenic human papilloma virus subtypes. For domains analysis in the protein sequences, we used Simple Modular Architecture Research Tool algorithms and predicted secondary structure of protein using Protein Prediction Protein 4.0 tool. I-TASSER Iterative threading assembly refinement algorithms were utilised for three-dimensional structure modelling of capsid L1 and L2 proteins. We found out a different evolutionary relationship and conserved residues in capsid L1 protein of human papilloma virus and L2 protein of human papilloma virus, and their different level of effect on the protein structure. We also predicted three-dimensional structure models for capsid L2 protein of human papilloma virus subtypes 41 and 13 which are folded completely differently from the rest L2 proteins. X-ray crystallography study is suggested for the determination of three-dimensional structure of L2 protein for understanding their contribution in viral assembly process.
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(2020)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.
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(2019)Tiivistelmä - Referat - Abstract Background. Platelets are known to contain ample amounts of brain derived neurotrophic factor. Previous spectrophotometric studies carried out in Pia Siljander’s lab have shown that BDNF is secreted from activated platelets packed in extracellular vesicles. For this project we wanted to 1) confirm that BDNF really is secreted in extracellular vesicles (EVs)2) find out how the choice of agonist affected the BDNF cargo of the platelet derived EVs, and 3) find out if the BDNF is packed into EVs of certain densities rather than others. Methods. The platelets were isolated from platelet concentrates by size exclusion chromatography. The isolated platelets were then activated by thrombin and collagen co-stimulation (TC) and by Ca2+ionophore, respectively. The platelet activation produced extracellular vesicles (PEVs) which were isolated by differential ultracentrifugation. The isolated PEVs were then analysed by flow cytometry, ELISA and Western blot for EV typical membrane surface proteins and for their BDNF content. As we were interested finding out whether BDNF is enriched in PEVs to certain populations, density gradient centrifugation was performed. These samples were also analysed by Western blot and by ELISA. The size distribution and concentration of PEVs in all samples was analysed by Nanoparticle tracking analysis. Results and conclusions. This study confirmed that platelets secrete PEVs as a response to agonists. PEVs with higher BDNF concentration were produced using TC co-stimulation as compared to PEVs derived from the Ca2+ionophore. The result implies that BDNF is actively packed into PEVs for instance as a thrombogenic response. Based on the density gradient results it seems that BDNF was packed into certain population of PEVs with a density between 1.112 g ml-1 and 1.132 g ml-1 corresponding to a particle diameter of less than 500 nm. The finding that BDNF is actively packed into TC co-stimulation derived PEVs of a certain population is interesting from a theragnostic point of view, since EVs are likely to be key players in the development of new cell-based therapies. Had there been more time, it would have been interesting to optimize both the density gradient protocol and the ELISA analysis. This optimization of methods would make the process more efficient, less prone to sample loss, not to mention that there would be less intra-assay variation.
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(2018)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.
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(2019)The consumption of omega (n-) 3 polyunsaturated fatty acids (PUFA) from fish has been associated with lower rates of cardiovascular diseases with one mechanism being lowering LDL cholesterol levels in blood. When incorporated into LDL particle n-3 PUFAs can modify the lipid composition and reduce atherogenicity of the particle, e.g. by influencing inflammatory processes. The effects of n-3 PUFA of plant origin are less studied. This study investigated the effects of Camelina sativa oil (CSO), a rich source of alpha-linolenic acid (ALA), on lipid species of human LDL including phosphatidylcholines (PC), lysophosphatidylcholines (LPC), sphingomyelin (SM), triacylglycerols (TAG) and cholesterol esters (CE). A total of 38 subjects with a history of impaired fasting glucose, were randomly divided into two groups; CSO (ALA 10 g/day) and the control group (limited fish and ALA intake) for 12 weeks. Blood samples were collected from the subjects at the beginning and at the end of the experiment after 12 weeks. LDL particles were isolated from blood and the lipids were analyzed by mass spectrometry. The CSO affected more the LDL core lipids (TAG and CE) than lipid species of the shell (PC, LPC, SM). CSO is high in ALA and linoleic acid (LA). Thus, the diet reduced mole fractions of lipid species containing saturated acyl chains while acyl chains in the core lipids with ALA, LA and EPA, that is formed in the body from ALA, were increased. Based on the results, having CSO in the diet changed the LDL particle lipid composition in a favorable direction for cardiovascular health.
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(2020)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.
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(2019)Cerebral dopamine neurotrophic factor (CDNF) belongs to the the family of neurotrophic factors that are evolutionary conserved, having a unique structure, with two domains: C-terminal domain and the N-terminal domain, and a cysteine bridge. It is known to be involved in the repair of the dopaminergic neurons when studied in the animal models of PD, which shows their different mode of action as compared to other neurotrophic factors, highlighting their therapeutic potential. Analysis of the crystal structure shows that CDNF and MANF consist of two domains: the saposin-like N-terminal domain with five α-helices stabilized by three disulphide bridges, and presumably unstructured C-terminal domain with a disulphide bridge. Characteristic feature of saposin-like proteins is their ability to interact with membranes or lipids. The lipid interaction may be crucial for the activity of CDNF and MANF proteins. In the first part of this project, the binding of CDNF was tested with several oxidized lipids, using two methods; Co-sedementation assay and lipid fluorescence assay;with two different types of probes. According to the results, CDNF seemed to show binding with POVPC. The second part of the project involved testing the binding and internalization of CDNF to mouse myoblast cells in the presence of oxidized lipid; POVPC. It was observed that CDNF seemed to show binding to the cell surface of the mouse myoblast cells (C2C12) and is also observed to be internalized to the cells as well. However, as these are the preliminary results, so we need to further test the binding between the protein and other lipids and devise more precise protocols for the testing the internalization to the cells.
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(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.
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(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.
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