Browsing by discipline "genetik"

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.

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.

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.

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.

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.

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.

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.

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.

Intellectual disability (ID) is a clinically diverse and genetically heterogeneous disorder characterized by central nervous system defects of varying severity resulting in substantial impairment of intellectual and adaptive functioning as expressed in conceptual (IQ<70), social and practical adaptive skills diagnosed before 18 years of age. The condition is referred to as non-syndromic when ID is the only clinical feature and syndromic when ID is accompanied by specific other features, for example, Down syndrome. Intellectual disability is one of the largest unsolved problems of health care with a prevalence of 2-3% in the population. There is a 30-40% excess of male versus female patients in ID which refers to over-representation of X chromosomal defects causing ID. In this study, exome sequencing of the X chromosome was applied in order to identify genes and their mutations in two Finnish families with intellectual disability of unknown cause. The mutations were identified using Agilent Sure select array that covers almost 93% of the coding region of the chromosome. Exome sequencing resulted in 11 variations in total. Segregation of these variants was studied using PCR, ExoSAP-IT purification protocol and BigDye® Terminator v3.1 Cycle Sequencing Kit. Eventually, two novel mutations were identified: one for each family. Both mutations reside in genes that have previously been shown to cause X-linked intellectual disability. Both of the mutations were absent in over 120 control DNA samples. In one family with three affected males, a novel splice mutation was identified in discs large homolog 3 (DLG3), which encodes synapse-associated protein 102 (SAP102). The mutation is located at the splice site in intron 1 (500+1 G>C) and its effect on protein function needs to be analyzed at the RNA-level using cDNA-sequencing. The clinical phenotype of the three affected brothers is mild to moderate intellectual disability. In the other family with three severely affected male patients, a novel mutation in exon 12 was identified on glutamate receptor, ionotropic, AMPA 3 (GRIA3) resulting in amino acid glycine (GGG) changing to arginine (CGG) at codon 630 (G630R). GRIA3 belongs to AMPA receptors implicated in the regulation of several biological processes. Our findings elucidate the power of exome sequencing in the diagnosis of rare, genetically heterogeneous disorders like intellectual disability. The results obtained will help in assessing the prognosis of the disease, in estimating the risk of the disorder to other family members, and in facilitating the development of future therapies for these devastating disorders. The results also further confirm the role of DLG3 and GRIA3 in human cognitive development.

Isolating mechanisms of the species usually prevent interspecific hybridisation. At times, these mechanisms might break down temporarily and lead to the birth of interspecific hybrids. Introgression is a term related to a set of consecutive backcrossings in which the hybrids reproduce with one of their parental species. It is characterised as a long process associated with alleles which are transferred from a population of one of the parental species to a population of the other parental species. Introgression is adaptive if phenotypic variation is increased in the recipient population by the genetic variants of the donor population and maintained by natural selection. The Baltic grey seal (Halichoerus grypus) and the Baltic ringed seal (Pusa hispida botnica) have interbred when they were kept in captivity in a shared pond. According to the findings from a previous study, interbreeding could have happened in the wild as well. The purpose of this study is to examine the proportion of introgression between the Baltic grey and ringed seals. The genomewide introgression is analysed using Patterson’s D-statistic, F4-ratio test and specific introgression intervals defined from the seals of analysed data. Introgression is assumed to have contributed intraspecific morphological variation detected in phocine teeth. Therefore, it is also examined whether the genes involved in tooth development express signs of introgression in the grey and ringed seals and whether the introgression intervals include potential variants. The results of Patterson D-statistic and F4-ratio test show both hybridisation and introgression between the Baltic grey and ringed seals. Based on the introgression intervals, a longer period has passed since the species interbred. Similar proportions of introgressed DNA as those defined from the genomes of the ringed seals have been detected in brown bears, bovines and modern humans. Furthermore, several genes affecting the shape of a developing mammalian tooth show signs of introgression in the seals. The individuals also carry variants in their introgression intervals. Introgression and the variants can account for the intraspecific morphological variation in the phocine dentitions. Potential introgressed genome intervals in the regulatory sequences of the tooth genes might also affect phocine tooth shape, which should be examined more in the future.