Browsing by discipline "Genetics"
Now showing items 1-20 of 48
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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)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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(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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(2017)Puberty is a process of physiological changes, through which an immature individual becomes sexually mature. In humans, timing of puberty is highly variable within and between sexes and populations. Timing of puberty represents a complex trait, which is controlled both genetically and environmentally. Precocious pubertal timing is associated with development of metabolic diseases later in life, such as obesity and diabetes, and other disorders as ovarian and testicular cancer. Despite the estimated high heritability (50-80%) of pubertal timing, its genetic background is still poorly understood. Recently, the genome-wide association studies (GWASs) revealed many novel pubertal timing associated loci. Nevertheless, molecular mechanisms behind these associations remain elusive. This thesis focused on gene vestigial-like family member 3 (VGLL3), which is associated with pubertal timing in humans and maturation in Atlantic salmon (Salmo salar). Since the main physical structures, such as the hypothalamus and the pituitary gland, needed in reaching puberty are evolutionary conserved and start to develop in vertebrates during embryogenesis, the aim was to study the expression pat-terns and role of vgll3 in zebrafish (Danio rerio) during this period. In order to localize expression patterns of the vgll3 gene in zebrafish embryos, a whole-mount in situ RNA hybridization (ISH) was performed. mRNA overexpression and morpholino oligonucleotide (MO) knockdown techniques were used to alter the vgll3 gene expression levels in 0-5 dpf zebrafish. The combined injections of both mRNA and MO were performed to validate MO specificity. The ISH experiment showed the expression patterns in 0-1 dpf embryos. The expression was ubiquitous up to 6 hours post fertilization becoming more localized to specific regions in the head and trunk of the embryos during the later stages. Altering vgll3 expression with high concentrations of synthetic mRNA or MO lead to phenotypical abnormalities such as shortened and curved body axis, pericardial and yolk sack edemas, deformed heads and eyes. However, it remained unclear if these malformations appear only due to the alteration of vgll3 expression levels. The results suggest that vgll3 may play an important role in the embryonic development. However, the study does not show that vgll3 has impacts on the pubertal timing in vertebrates by affecting the development of the structures required for sexual maturation.
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(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.
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(2020)Tiivistelmä – Referat – Abstract Telomeres are cap shaped structures at the very end part of each chromosome that protect DNA from degradation or unwanted chromosome-chromosome attachments. Telomere lengths show considerable heterogeneity in different cells of the same cell population. Reasons for heterogeneitiy and mechanisms inside cells causing them are not fully understood. In this study, we explored the correlation between telomere length and different gene expressions. First, using FACS technique we sorted each single cell into each well of 96-well plate. Second, we used SYBR green based qPCR for telomere length measurement. Third, we used Illumina-seq for sequencing extracted mRNAs. [6] We found a set of genes that were in strong correlation with telomere length, giving opportunity to explore the biological pathways. We compared pathways between different samples and found strong connections between genes involved in viral cycles and immune system with extracted genes that were in high correlation with telomere. We found heterogeneity of telomere lengths and transcriptomes in different cell lines. Telomere related proteins, specifically those involved in shelterin complex, are expressed highly in cancer cell lines and LPS-stimulated monocytes compared to the non-stimulated monocytes. In our study, SLC38A2, PURB, UBR3, SSR1, NCAPH2, AIMP2, PHF21A genes were highly correlated with telomere in mutual way and can therefore be considered as new biomarkers/novel candidates for telomere-related studies. The importance of these genes has been reported in aging/mortality. Concurrent with our findings, a recent report also suggested that NCAPH2 plays role in regulating telomere stability and maintenance through its interaction with TERF. [65] We found new genes in correlation with telomere regulation, and our findings are therefore of high importance in research of cancer, neurodegenerative diseases and aging. Further studies are, however, required as our data is limited by small number of samples and inability to properly validate our technique.
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(2020)The axolotl (Ambystoma mexicanum) has an astounding ability to regenerate entire lost body parts throughout its life. Significant progress has been made in recent years to understand the mechanisms of axolotl regeneration, but how the animal maintains its capacity for successful regeneration remains obscure. In mammals, the ability to repair damaged tissue drastically declines with age, in part due to the accumulation of senescent cells. However, in axolotls, the number of senescent cells does not increase upon aging. Low levels of chronic senescence in axolotls have been proposed to support their ability to regenerate even at an old age. Axolotls can efficiently clear senescent cells, but whether they can prevent the induction of senescence is not known. This thesis provides the first indication of a secreted anti-senescence activity from axolotl cells. Data presented here show that conditioned medium from cultured axolotl cells reduces senescence and increases proliferation in mouse embryonic fibroblast, a widely used model for spontaneous senescence. Remarkably, conditioned media from other tested cell types, namely cervical cancer cells and young mouse embryonic fibroblasts, did not considerably affect senescence, despite extensively increasing proliferation. Taken together, secreted factors from cultured axolotl cells seem to reduce senescence directly, and not by merely promoting proliferation. This observation forms a basis for future endeavors to determine whether preventing senescence facilitates regeneration in vivo.
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(2016)Background: Epithelial ovarian cancer is the most common type of ovarian cancer and is the most lethal gynecologic cancer due to its late diagnosis. Compared to ovarian cancer, endometrial carcinoma, as the most common gynecologic malignancy, is referred to as the “curable cancer”, as it can be detected early. As aberrant promoter methylation patterns are a common change in human cancer, detection of promoter methylation status may help in early diagnosis. In this study, we used a custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay as a rapid and easy method, to simultaneously detect the methylation status of multiple genes in ovarian and endometrial cancer samples. Aims: To design and test an MS-MLPA assay for analyzing promoter methylation of four genes associated with ovarian and endometrial cancers. The selected genes were HNF1 homeobox B (HNF1β), Ten-eleven translocation 1(TET1), L1 cell adhesion molecule (L1CAM), and AT-rich interactive domain 1A (ARID1A). These genes are known to have expression changes by DNA methylation. Methods: The promoter DNA methylation patterns of these four genes were analyzed in 15 cancer cell lines and 5 normal cell lines and DNAs using bisulfite sequencing. Six synthetic probe pairs were designed and optimized by applying them to cancer and normal cell lines and normal DNAs and comparing the results with those of bisulfite sequencing. Finally, the MS-MLPA assay was performed on patient specimens according to the MRC-HOLLAND MS-MLPA general protocol and methylation frequencies were calculated from MS-MLPA data. Results and conclusion: The MS-MLPA assay gave accurate methylation results with the 170 samples assayed. The HNF1B, L1CAM, and TET1 Genes were observed methylated in tumor samples whereas they were not methylated in the normal samples or showed very little methylation, suggested to be favorable diagnostic markers. MS-MLPA robustly and sensitively detects the promoter DNA methylation status.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1990)Ns. restriktioentsyymin keksimisen myötä on kehitetty uusi menetelmä tutkia yksilön perimää geenitasolla. Perimäns isältämä DNA pilkotaan lyhyemmiksi pätkiksi restriktioentsyymin tunnistamista paikoista. Nämä pätkät kokoerotellaan elektroforeesilla. Radioaktiivisella koettimella saadaan näkyviin ne perimän sisältämät pätkät, joissa on kyseisen koettimen tunnistama ns. minisatellittialue. Näin saatu juovasto on yksilöspesifinen, ja sitä nimitetään yksilön DNA-sormenjäljeksi (DNA-fingerprint). Koirilla menetelmää voidaan käyttää yksilön tunnistamiseen, isyystutkimuksiin ja rodun sisäsiittoisuuden arvioimiseen. Isyystutkimuksessa jälkeläisen jokaisen juovan on löydyttävä jommalta kummalta vanhemmalta. Rodun sisäsiittoisuutta tutkittaessa arvioidaan rodun eri yksilöiden juovastojen samankaltaisuutta. Tutkimuksessa asetettiin pohdittavaksi, voiko voimakas sisäsiivoisuus alentaa menetelmän tuloksellisuutta varsinkin isyysmäärityksiä tehtäessä. Aineistona käytettiin 3 bedlingtoninterrieriä ja verrokkiryhmänä 8 sekarotuista. DNA eristettiin 2 ml verinäytteestä (EDTA). Aineiston perusteella laskettiin todennäköisyydet, että isyystapaus jäisi ratkaisematta käytettäessä kahta koetinta. Bedlingtoneilla tämä prosentti oli 36.0% (eli 36 isyystapausta sadasta jäisi ratkaisematta) ja sekarotuisilla 6.4%. Saadut prosentit ovat muihin vastaaviin tutkimuksiin verrattuna melko suuria. Tulosten perusteella havaitaan sisäsiittoisuuden vähentävän menetelmän tuloksellisuutta merkittävästi. Menetelmän käyttö isyysmäärityksiin ei ole mielekästä rutiininomaisena menetelmänä johtuen menetelmän kalleudesta ja hitaudesta. Perinteisillä menetelmillä ratkeamattomissa kiistatapauksissa voidaan DNA-sormenjälkianalyysillä kuitenkin saada hyödyllistä lisätietoa. Myös eri koirarotujen sisäsiittoisuuden arvioimisessa voidaan menetelmää soveltaa tuloksellisesti.
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(2020)Upregulation of specific helpful proteins represents a possible method for preventing or treating human diseases. Endogenous upregulation (knockup) is the increase of a gene's expression only in cells in which it is already expressed, thus avoiding physiologically abnormal spatiotemporal patterning. A gene's three prime untranslated region (3′UTR) affects protein expression through stability regulation of RNA already transcribed, which suggests 3′UTR modification as a viable route for endogenous upregulation. Mammalian model organisms can be generated in order to test the effects of different 3′UTR modifications, but at great cost of time, effort, and money. If able to predict in advance with an in vitro assay whether an in vivo modification would cause a desirable or undesirable change, these costs could be substantially reduced. In this thesis project, an in vitro assay was used to compare the protein expression influence of twenty neurodegeneration-relevant mouse genes' 3′UTRs to that of a flip-excision cassette (flex-cassette) previously used for in vivo conditional knockup. The assay used was the Promega Dual-Luciferase Reporter Assay, in which plasmids expressing Renilla and Firefly luciferase as reporter and internal control are co-transfected into in vitro cells, then each luciferase's expression measured with its respective substrate and a luminometer. Transfections were carried out in three-well replicates and on multiple days. The aims of the project were the evaluation of the assay's ability to predict in vivo results, the suggestion of 3′UTRs which could be upregulated in vivo by the conditional knockup flex-cassette, and the identification of any trends in 3′UTR-based protein expression influence according to gene function. A number of gene 3′UTRs were identified which were either candidates for flex-cassette upregulation or candidates for use in the flex-cassette to upregulate other genes. However, the flex-cassette's in vitro results were only partially consistent with its previous in vivo results. Specifically, the lox sites in the flex-cassette was observed to lower expression level to a degree not observed in vivo. Additionally, in the course of the project a number of possible workflow improvements were identified, for which suggestions have been made in the text. As such, this in vitro approach requires further study in order to determine suitability for prediction of in vivo 3′UTR behaviour.
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(2020)There is a naturally reproducing Atlantic salmon population in the River Teno in northern Norway and Finland. The Teno population has a strong population structure and up to 28 subpopulations have been recognized. Estimation of effective population size is important in conservation of the subpopulations. Effective population size tells about genetic variation of a population and is among the most important concepts in conservation genetics. In this study, current and past effective population sizes of 28 subpopulations were estimated from high density SNP-data for 1137 individuals in total. The estimation was done with the linkage disequilibrium method and the effects of using different assumptions were studied. Current estimated effective population sizes in subpopulations were generally low and ranged from around nine to 272 individuals. Only four populations had a current effective population size bigger than 50 individuals. Past effective population sizes showed a clear declining trend from the most distant generations in all populations. The choice between physical and linkage map as well as female, male or average linkage map had an effect to estimates. Also, different sample size corrections resulted in different estimates. Furthermore, effective population size was estimated with temporal method in three populations. It was detected that the estimates from temporal and linkage disequilibrium method were different from each other. The results of this study suggest that Teno Atlantic salmon subpopulations have declined over the past 150 generations and are in risk of losing genetic variation due to current low effective population size. This should be taken into account in conservation plans.
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(2019)SH3 domains are relatively short and most common of modular protein binding domain in eukaryotes. They are present in proteins that play critical role in various cell signaling and regulatory pathway. Human genome encoded 296 types of SH3 domains have been successfully displayed in phagemid using classical PelB signal sequence and used for finding novel binding partners. However, given its shorter length and tendency to fold rapidly it is useful to understand if signal sequence that directs SH3 translocation through Co translational pathway is much more efficient in displaying these domains than the one that translocate protein post translationally. For the study, PelB signal sequence of phagemid displayed human SH3 library was replaced with DsbA signal sequence using round the horn PCR method (Site directed mutagenesis) and verified with agarose gel electrophoresis. Subsequently, infective phages were prepared. The infective titer of newly generated DsbAss based library was found to be higher than that of PelBss based library. Both libraries normalized at 1 x1012cfu/ml were panned against known protein targets MC159(Molluscum contagiosum 159), NCF2(Neutrophil cytosolic factor 2) and NS1(Nonstructural protein 1). Enrichment with DsbAss library was moderately higher for each antigen. However sequencing results showed that results for proteins panned with PelBss library were congruent with previous finding whereas DsbAss library selected some potential weak binders and nonspecific ones along with strong binders. Panning results of DsbAss with NCF2 was striking as all clones selected were NCF1 SH3 domains. Although further functional study was not performed. Based on the study, we concluded that both libraries have its own advantage. PelBss based library can be used for finding strong binders while DsbAss based library can be used for studying weaker interaction and functional role of NCF2-NCF1 SH3 domain interaction is still an open question.
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(2019)Nemaline myopathy (NM) is a rare congenital disorder, the most common of congenital myopathies. It affects primarily the skeletal muscles and it is recognised by nemaline bodies in muscle tissue samples and muscle weakness. Mutation of eleven genes are known to lead to NM and the most frequent disease-causing variants are either recessive NEB variants or dominant ACTA1 variants. Variants in NEB are thought to be well tolerated and only 7% of them are hypothesized to be pathogenic. Over 200 pathogenic NEB-variants have been identified in Helsinki and the majority occurred in patients as a combination of two different variants. The missense variants were speculated to have a modifying effect on pathogenicity by affecting nebulin-actin or nebulin-tropomyosin interactions. Nebulin is a gigantic protein coded by NEB and is one of the largest proteins in vertebrates. It is located in the thin filament of the skeletal muscle sarcomere. Enclosed by terminal regions, nebulin has an extensive repetitive modular region that covers over 90% of the protein. The repetitive zone comprises of 26 modules called super repeats (SR). SRs consist of seven simple repeats. There are seven conserved SDXXYK actin-binding sites at each super repeat, one per simple repeat, and one conserved WLKGIGW tropomyosin-binding site. Due to its enormous size and highly repetitive sequence, nebulin is one of the least studied proteins in vivo, in vitro or in silico. In the NM patient database used for this study, there are 70 families with verified pathogenic mutations and in 30 of them, there were additional missense variants in NEB. These missense variants can be pathogenic modifying factors or have no impact on the phenotype. Seven missense variants were selected to study the effect of these mutations on actin-binding capacity compared to wild-type nebulin using the SR panel constructed previously by Laitila and Lehtonen. Also, due to the differences in actin-binding capacity of SRs compared to each other, one of the aims was to determine whether corresponding mutations in different SRs would have a similar or different effect on actin-binding capacity. For this aim, one missense mutation in the strongly actin-binding SR 1, and one in the weakly actin-binding SR 7 were selected from the NM database, and corresponding variants were created. Also, an in-frame deletion in SR7 found in the ExAC database and the corresponding mutation in SR1 were constructed for this study. The actin-binding strength was determined using actin co-sedimentation assay and actin affinity assay. The results for co-sedimentation assay indicate that missense variants can have an effect on nebulin-actin interactions and, therefore, can be a possible cause for NM. The corresponding mutations had no correlation in their effect on actin-binding strength, just the opposite. S1-m-2 decreased actin-binding strength of SR1 and S7-m-2 had no effect on SR7. Likewise, S7-m-1 and S7-del-1 decreased actin-binding strength of SR7 and corresponding mutations had no effect on SR1. The selected missense mutations found in NM patients in SRs 2 and 4 decreased actin-binding strength, if located at the actin-binding sites and in SR 10 increased the actin-binding strength, if located at the actin-binding site. The change in actin binding strength was defined as significant if the P-value was below 0.005. The more accurate affinity assay was performed as a trial only for S16 and S16-m-1, a variant at a tropomyosin-binding site close to an actin-binding site. It indicated a difference in actin-binding affinity missed by the actin co-sedimentation assay. The results are preliminary, but show big promise and should be optimized and implemented in the future missense mutation affinity studies. In an attempt to understand if the effect missense mutations have on nebulin-actin interaction is based on the change in nebulin structure, the 3D-structure of each produced fusion protein was predicted in silico. Considering that the variants were produced as GST-fusion proteins, the position and effect of GST in them is also a point of interest. In order to predict the structure of these large proteins, a combined approach was implemented using I-TASSER (Iterative Threading ASSEmbly Refinement) software. The software uses ab initio modeling, threading methods and atomic-level structure refinement to build an accurate 3D-model of a protein from sequence. According to the predicted 3D models of the fusion proteins, the GST-part of the proteins folds into a globular structure and acts as a core around which the nebulin fragments fold. The GST does not bind to actin and is positioned on the inside, which indicates minimal effect on nebulin-actin interaction, but may be a reason for an alternative nebulin fragment folding. The accuracy of the default set of programs in software does not give the definitive answer of the possible effect missense mutations can have on structural changes. However, I-TASSER approach for 3D-modeling is promising with further software optimization and can possibly serve as an effective bioinformatic tool in the future.
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(2016)Epidermolysis bullosa (EB) is a group of hereditary skin disorders caused by mutations in the genes that code for adhesion molecules in keratinocytes. The symptoms of the disease include blisters and erosions in the skin as well as abnormalities in the mucosal membranes, nails and tooth enamel. Depending on the causative mutation the severity of the disease ranges from mild to lethal. This master s thesis was carried out in professor Hannes Lohi s canine genetics research group. It is a case study aiming to identify the genetic cause of EB present in Central Asian shepherd dogs. Furthermore, the mode of inheritance, frequency of the mutation in the breed and the effect of the mutation on the tissue were also examined. Primary research material included blood and tissue samples from a family of Central Asian shepherd dogs. Additional samples were obtained from other Central Asian shepherd dogs as well as from dogs of closely related breeds. All dogs included in this study are owned by private persons, and participation in this study was voluntary. The research methods used in this study included both wet laboratory experiments and bioinformatic in silico procedures. The genome of one affected dog was sequenced in order to identify the gene causing EB, and data from the sequencing was filtered with multiple programs according to recessive model. The model was decided after analyzing the pedigree of the affected dogs. After finding the likely causative gene the mutation was validated in a larger cohort with Sanger sequencing. Protein expression in the tissue of affected dogs was also studied using immunofluorescence staining. As a result of this study, a new mutation causing recessive dystrophic EB was identified. The mutation is specific to Central Asian shepherd dogs. Affected dogs have a homozygous mutation in the COL7A1 gene, which codes for collagen VII α1 protein. The mutation causes a premature stop codon in the mRNA sequence, which results in abnormal protein production and separation of skin layers. The frequency of the mutation allele in the sample is approximately 18 % and more than a fourth of the dogs in the sample are carriers. Based on the pedigree analysis, the mutation is relatively new and it is only found in a small population. The disease can be prevented from becoming more common in the breed with the help of a gene test, and the test will be available in the commercial MyDogDNA gene test panel.
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(2019)In this master’s theses, Finnish biology teachers’ needs for material for continuing education and educational material for upper secondary school in epigenetics were studied. Two sets of educational materials, continuing educational material for teachers and educational material for upper secondary school teaching was produced accordant with the results. Epigenetics is used to describe stable alterations in gene expression which do not consider mutations in DNA sequence. DNA methylation, histone modifications and chromatin remodelling are the main epigenetic mechanisms to affect gene expression. Epigenetic modification patterns can alter de novo, or they can be originated by some environmental factor. Epigenetic regulation was introduced as a new subject matter in National Core Curriculum for General Upper Secondary Schools 2015 in Finland. Epigenetics is a relatively new branch in biology, and as a result many teachers have not studied the subject matter at the university. Continuing educational material is needed to update their knowledge. Biology teachers’ needs for material in epigenetics were studied with a survey which was distributed to the Biology and Geography Teachers’ Union’s e-mail list subscribers. Data was analysed qualitatively and quantitatively. In order to produce continuing educational material for teachers, literature regarding teacher competence, adult education and Finnish biology teachers’ subject expertise was examined. The concept of constructivist learning, and conceptual change were applied in the production of educational material for upper secondary school teaching. Relevant scientific literature in epigenetics was gathered and used to produce both sets of materials. In the study survey, the teachers reported specific individual educational needs which were acknowledged in the production of both sets of materials, alongside literature listed earlier. The survey showed that one of 33 biology teachers had studied epigenetics at the university, 20 of 33 teachers independently and 13 of 33 hadn’t studied epigenetics at all. The extent of the teachers’ studies in epigenetics was most often elementary and the main motivation to study epigenetics independently was a desire to handle the basics. The most common resource teachers used to study epigenetics was non-scientific magazines. Among the teachers who had not studied epigenetics at all, lack of time was the most common reason mentioned. However, 14 teachers described epigenetics as an important subject matter. Three teachers reported that they lack the expertise in teaching epigenetics and three felt that textbooks don’t offer support in teaching epigenetics. Online material and expert lectures were the most common continuing education material forms requested. Regarding the content of the continuing educational material for teachers, the most common requests were that the material should include the basics of epigenetics and practical examples. The form of educational material for upper secondary school teaching was most commonly requested to be online text or educational video. Regarding the content of the educational material for upper secondary school teaching, the most common requests among teachers were a summary of the theory of epigenetics and practical examples. The continuing educational material for teachers produced in this thesis consists of an introduction part and four parts about different subjects in epigenetics. The titles of the parts are: 1) What is epigenetics? 2) Molecular mechanisms of epigenetic gene regulation 3) Epigenetic gene regulation and 4) Epigenetic inheritance. The material was designed in a way that texts 3) and 4) are possible to comprehend without studying text 2) about molecular mechanisms of epigenetic regulation. The educational material for upper secondary school teaching consists of seven parts. At the beginning there is an introduction to teachers which is followed by six separate parts for students. Each part has a “Rehearse before reading”-box which introduces students to the subject and encourages rehearsal of biological concepts and vocabulary which are necessary for comprehending each part. After each part there are questions which test students’ learning as well as encourage to apply freshly acquired knowledge about epigenetics to other biological contexts. Titles for different parts are: 1) What is epigenetics? 2) Epigenetics and nutrition 3) Epigenetics and exercise 4) Epigenetics and mental health 5) Epigenetics and tortoiseshell cats and 6) Epigenetic inheritance. The material has been designed in such a way that the parts can be taught and learned separately. References are provided at the end of both material sets. Both materials produced in this thesis meet the teachers’ requests revealed in the survey. The form of the continuing educational material for teachers is online material, which was one of the most common requests among the teachers who answered the survey. The contents of the material correspond to the teachers requests as well, since most requested contents were the basics of epigenetics and practical examples. The educational material for upper secondary school teaching was requested as online material with a summary of the theory of epigenetics and practical examples, and all these requests were met. Both materials were produced considering relevant theories on pedagogy and adult education. Results of this study cannot be applied nationally in Finland since the sample size was small. Therefore, national relevance of the materials cannot be predicted. Predictions about the impact of the materials on teachers’ and students’ understanding about epigenetics cannot be made either, since learning is an active process which requires effort from the learner. However, a strong case can be made for the produced material, because materials include relevant information and their pedagogic choices can be justified by the literature. This thesis uncovered many questions for future research. For example, the efficacy of the materials could be studied in a practical classroom situation. Other possible questions for research or study designs could be about biology teachers’ expertise and continuing education in Finland.
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(2017)Follicle-stimulating hormone (FSH) regulates mammalian reproduction. The hormonal function of FSH is exerted through its receptor, FSHR. Hormone binding leads to signal transduction by the synthesis of the second messenger (intracellular cAMP) and the activation of other downstream signaling pathways. FSH receptors are mainly found in ovaries and testes where FSH stimulates e.g. follicular growth and spermatogenesis, respectively. It has been previously shown that FSHR mutations are linked to infertility through abnormalities in the receptor function. For example, inactivating FSHR mutation (Ala189Val) leads to arrest of follicular development in females and reduced sperm counts in males. FSH action seems to be especially critical for folliculogenesis and essential for female fertility. The development of gonads and dysfunctions affecting reproduction are the focus of our research group. Studies of recent years have demonstrated extragonadal FSHR expression, including endothelial cells of female reproductive tract and developing placenta. The physiological relevance and function of this extragonadal FSHR is still not well known, especially during embryogenesis. In addition, the expression or function of the mutated FSHR has not been studied in any cells endogenously expressing the receptor. As a consequence, the purpose of this thesis was to study FSHR expression and function in human pluripotent stem cells (hPSCs) as a model for early human development. The study was conducted in a human embryonic stem cell line (hESC H9, 46, XX) and two human induced pluripotent stem cell lines (hiPSC HEL127.6 and HEL128.5; 46, XX). iPSCs were obtained from female patients carrying the A189V mutation in the FSHR gene. Cells were differentiated for 8/12 days using two in vitro cell culturing protocols for distinct differentiation attempts. The first protocol (protocol D) was designed for embryonal cell differentiation, the other (trophoblast protocol) for extraembryonal differentiation. Cells at different stages of differentiation were studied with qRT-PCR and immunocytochemistry. At d8, cells were stimulated with FSH for qPCR studies and for cAMP assay. Control cells (H9) differentiated by protocol D endogenously expressed functional FSHR. These cells responded dose-dependently to FSH stimulation by substantially increasing cAMP production, downregulating FSHR mRNA expression and altering inhibin gene expression. Patient-derived iPSCs carrying the mutation expressed FSHR as well but the receptors were non-functional as expected. Both H9 and patient-derived cells that differentiated into trophoblast-like cells with the other protocol, also expressed FSHR at low level but did not respond to FSH stimulation. Preliminary results with protocol D indicate that the cells resemble extraembryonic cell types. The study has revealed novel information and insight about extragonadal FSHR expression and function during differentiation. However, the exact identity and the biological role of these cells is yet to be confirmed.
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(2020)Tiivistelmä – Referat – Abstract Ewing sarcoma is a rare bone and soft tissues cancer that occurs mainly among children and young adults. It is an aggressive cancer. Treatment of Ewing Sarcoma Family of Tumours (ESFT) primarily includes surgery, radiation and chemotherapy. The treatment protocol depends on the presence of tumour metastases at the time of diagnosis. In the treatment of local tumours, the 5-year patient survival rate has increased from 50% to 70%. However, patients that have tumour metastases at the time of the diagnosis or have a recurrent disease, the five-year survival rate is only 25%. As the current treatment options have reached their limits, it is important to develop more advanced therapies. DNA methylation is an epigenetic event that affects gene expression. By comparing the methylation level of the DNA in gene promoter regions in ESFT cancer cells to the methylation level of DNA in gene promoter regions in normal cells it could be possible to discover genes and signalling pathways that are important in the development of ESFT and that could be potential drug target molecules. The aim of this study is to find out the genome-wide gene promoter DNA methylation status in Ewing sarcoma cell line samples and Ewing sarcoma patient tumour samples compared to a normal reference sample. Another aim is to find gene promoter regions that are differentially methylated in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample. Materials and Methods The Ewing Sarcoma cell line samples (12) were obtained from the Laboratory of Oncologi Research, Instituti Ortopedici Rizzoli Laboratory, Bologna, Italy. The Ewing sarcoma patient tumour samples were pre-isolated DNA samples already in Finland. The normal reference sample was a commercial mesenchymal cell line sample. From the Ewing Sarcoma cell line samples and the normal reference sample, DNA isolation was done by using phenol-chloroform method. DNA methylation profiling of the samples was performed by combining MeDIP (methylated DNA immunoprecipitation) protocol with 2-set promoter microarray hybridization protocol provided by Agilent Tecnologies company. DNA methylation data that was received from the microarrays was normalized and pre-processed with the Feature Extraction software provided also by the Agilent Technologies company. Visualization of the DNA methylation data was performed by using Chipster analysis software provided by CSC. To measure the level of DNA methylation at the gene promoter regions, a log2ratio value was calculated for every gene promoter region in all the sample types. To find gene promoter regions that were differently methylated, a log2 fold change value was calculated from the log2ratio values between the Ewing Sarcoma cell line cancer samples and the normal reference sample and between the Ewing sarcoma patient tumor samples and the normal reference sample for each gene promoter region. The log2 fold change value was also calculated between the Ewing Sarcoma cell line cancer samples and the Ewing Sarcoma patient tumor samples. After this a t-test was performed to determine the statistical significance of the log2 fold change values. Detection of genome-wide DNA methylation levels at the gene promoter regions in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample was performed by averaging log2 fold change values. The same calculation method was used to detect the differences in the genome-wide DNA methylation levels at the gene promoter regions between the Ewing sarcoma cell lines and the Ewing Sarcoma patient tumour samples. Results Differences in the DNA methylation levels at the gene promoter regions were detected between the Ewing Sarcoma cell line samples, patient tumour samples, and the normal reference sample. Genome-wide measurement of the DNA methylation levels at the gene promoter areas showed that the Ewing sarcoma cell lines had more DNA methylation at the gene promoter regions than the patient tumour samples and the normal reference sample. The patient tumour samples showed less DNA methylation at the gene promoter regions compared to the Ewing sarcoma cell lines and the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the reference sample were found 16. In the patient tumour samples, also 16 differently methylated gene promoter regions were found compared to the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the patient tumour samples were 56.
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(2015)Hampaat kehittyvät epiteelin ja mesenkyymin vuoropuheluna, vetovastuun ollessa ensin epiteelillä ja sitten mesenkyymillä. Ajatellaan, että ensin muodostuu ylä- ja alaleukaa reunustava epiteelijuoste eli hammasjuoste. Hammasjuoste paksuuntuu paikallisesti tulevilla etu- ja poskihampaan alueilla hammasplakodeiksi. Plakodit kasvavat muodostaen mesenkyymiin tunkeutuvat hammassilmut. Mesenkyymi tiivistyy silmun ympärille ja muodostaa vuorovaikutuksessa epiteelin kanssa kaikki hampaan kudokset epiteeliperäistä kiillettä lukuun ottamatta. Hampaan muodostumiseen vaikuttavat solubiologiset mekanismit ovat verraten huonosti tunnetut, etenkin hammasjuosteen ja plakodien osalta. Epiteelin ja mesenkyymin vastavuoroisesta signaloinnista taas on kertynyt runsaasti tutkimustietoa. Hyvin tunnettuja ovat esimerkiksi epiteelin primäärinen kiillekyhmy - ei-jakautuvista soluista koostuva signaalikeskus, joka ohjaa epiteelin kasvua silmuvaiheesta lakkivaiheeseen - sekä sekundääriset kiillekyhmyt, jotka määräävät hampaan nystermien paikat. Lisäksi jo 1990-luvun lopulla on saatu viitteitä varhaisemman, plakodivaiheen signaalikeskuksen olemassaolosta, mutta keskus on edelleen huonosti tunnettu eikä käsite ole vakiintunut kirjallisuudessa. Tämä Pro gradu-työ antaa lisätukea edellä mainitun varhaisen signaalikeskuksen olemassaololle. Työssä pyritään myös saamaan lisää tietoa varhaisimpien hampaan kehitysvaiheiden syntytavasta. Työssä hyödynnetään FUCCI-solusykli-indikaattorihiirtä ei-jakautuvien ja jakautuvien solujen erottamiseksi ja K17-GFP-siirtogeenisiä hiiriä hammasepiteelin erottamiseksi. Työssä tutkitaan hammasalueiden kehittymistä kuvantamalla alkiosta irrotettujen siirtogeenisten leukojen kehitystä hetki hetkeltä (ex vivo-aikapistekuvantaminen). Lisäksi tutkitaan hampaiden kehitystä kestävöityjen alaleukojen eri ikävaiheiden sarjoista sekä yhdistetään FUCCI-mallin antama tieto solujen jakautumisesta ja jakautumattomuudesta tunnettujen hammasalueen geenien ilmentymiseen whole mount in situ-hybridisaatiolla. Työssä etsitään myös parasta vasta-ainetta hammasepiteelin erottamiseen näytteistä, joilla fluoresoivaa hammasepiteelimarkkeria ei ole. Tässä työssä osoitetaan, että varhainen epiteelin signaalikeskus alkaa muodostua jo hammasjuostevaiheessa. Tällöin hammasalueelle ilmestyy yhtenäinen ei-jakautuvien solujen populaatio, joka myöhemmin rajoittuu kunkin plakodin alueelle. Nämä ei-jakautuvat solut ovat hammasepiteelin osajoukko ja ne ekspressoivat tunnettujen signaalireittien jäseniä Shh ja Dkk4, joiden ilmentyminen on liitetty varhaiseen signaalikeskukseen. Ei-jakautuvan solujoukon muoto muuttuu edelleen hampaanalun saavuttaessa silmuvaiheen. Vaikuttaa siltä, että varhaisen signaalikeskuksen solut päätyvät lopulta kiille-elimen varteen, joka aikanaan häviää. Ei-jakautuvien solujen alueen muodonmuutoksen mekanismi ei tässä työssä selvinnyt, vaikka sitä yritettiin ex vivo-kuvantamisella selvittää. Muussa leuassa solunjakautuminen on runsasta kaikissa tutkituissa kehitysvaiheissa ja ex vivo-kuvantaminen paljasti, että silmuvaiheessa ei-jakautuvien solujen populaation vieressä havaitaan erityisen voimakas solunjakautumisen aalto. On mahdollista että ei-jakautuvien solujen muodostama varhainen epiteelin signaalikeskus ohjaa tätä solunjakautumista ja siten silmun kasvua mesenkyymin sisään. Jatkossa tulee tarkemmin selvittää varhaisen signaalikeskuksen roolia hampaan kehityksessä. Kun tarkastellaan aiempia tutkimuksia tässä Pro gradu-työssä paljastuneiden seikkojen valossa, on syytä epäillä, että silmun kasvun lisäksi valmiiden hampaiden lukumäärä, sijainti ja koko ovat ainakin osittain riippuvaisia tämän varhaisen signaalikeskuksen säätelystä.
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(2018)Plants control the exchange of gases through the stomatal pores. Stomata are formed by guard cells and the closure of stomata are regulated via a complex signaling network in response to various biotic and abiotic stimuli, such as pathogens, elevated levels of CO2 and darkness. The leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) is part of the network regulating stomatal closure. GHR1 is an inaktive pseudokinase that can activate SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1), an anion channel that is crucial for stomatal closure, via interacting proteins. The exact role of GHR1 is still partly unknown, however, it has been suggested that GHR1 could function as a scaffold or as an allosteric regulator of additional components required for stomatal closure. The aim of this study was to identify novel interactors of GHR1. First stable plant lines expressing fusion proteins GHR1-YFP, GHR1W799*-YFP and plain YFP as a negative control were generated and from these lines fusion protein expression levels and the subcellular localization were studied. Next the plant lines were used for purifying GHR1 interacting proteins with the use of co-immunoprecipitation and identification of the proteins with mass spectrometry. The unlikely GHR1 interactor candidates were then filtered from the mass spectrometry data. The subcellular localization and the protein expression of the interacting proteins were studied with the use of internet databases. Literature of the GHR1 interacting proteins were studied in order to make possible connections with GHR1 and stomatal closure. In this study 38 GHR1 interactors were identified. Literature search revealed that many of the identified interactors had a known role in stomatal movements. These included proteins such as PLASMA MEMBRANE INTRINSIC PROTEIN2-1 (PIP2-1) and BETA CARBONIC ANHYDRASE 4 (BCA4), that are known to have a role in stomatal closure. Future work includes confirming the interactions with independent methods and studying the molecular mechanisms related to stomatal movements. The GHR1 interactome identified here for the first time reveals novel parts of the network regulating stomatal movements and thus increases our understanding of molecular mechanisms behind stomatal functions.
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