Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.Autoimmuunisairaudet, kuten diabetes, ovat huomattava kansanterveydellinen ongelma. Yleensä autoimmuunisairaus syntyy usean perinnöllisen ja ympäristöstä johtuvan tekijän summana. APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) on peittyvästi periytyvä harvinainen autoimmuunisairaus. Siinä yksi virheellinen geeni riittää taudin puhkeamiseen. Potilailla esiintyy useita eri elimiin kohdistuvia autoimmuunisairauksia, jotka ovat seurausta kudostuhosta mm. hormonirauhasissa. APECED-taudissa virheellinen geeni, AIRE (autoimmune regulator), koodaa kateenkorvassa toimivaa geenien ilmentymisen säätelijäproteiinia. AIREn säätelemät kohdegeenit ohjaavat kehittyviä puolustussoluja, T-soluja, erottamaan kehon omat rakenteet vieraista. AIRE on näin ollen osoittautunut hyvin tärkeäksi ns. immunologisen toleranssin synnylle. AIREn toiminnan mekanismeista tiedetään kuitenkin vielä varsin vähän. Tässä väitöskirjatutkimuksessa on pyritty valaisemaan AIRE-proteiinin toimintaa tutkimalla erilaisissa solumalleissa AIREn vuorovaikutuksia itsensä ja muiden molekyylien kanssa. Väitöskirjassa kuvattiin mahdollinen mekanismi APECEDin poikkeukselliselle, yhdessä perheessä havaitulle, vallitsevalle periytymiselle. Kyseisen perheen jäsenillä esiintyvä viallinen AIRE-proteiinin muoto kykenee sitomaan tervettä AIRE-proteiinia ja estämään sen toiminnan. Tutkimuksessa kuvattiin lisäksi kaksi uutta proteiiniperhettä, joiden kanssa AIRE on vuorovaikutuksessa. Importiini alfa -proteiini säätelee AIREn tumakuljetusta sitoutuen sen yksiosaiseen klassiseen tumakuljetussignaaliin. AIREn vuorovaikutus E3 SUMO ligaaseina toimivien PIAS-proteiinien kanssa viittaa AIREn ja sumoylaation yhteyteen. Sumoylaatio on yksi keskeisiä tuman toiminnallisen järjestyksen säätelijöitä. Tutkimuksessa osoitettiin, että AIREa itseään ei sumoyloida, mutta AIRE tehostaa SUMO1- ja PIAS1-proteiinien sijoittumista pistemäisiin tumarakenteisiin. Kahdella mallipromoottorilla saadut tutkimustulokset tukevat aiempaa havaintoa, jonka mukaan AIRE pääosin aktivoi kudosspesifisiä geenejä ja inhiboi useissa kudoksissa toimivia ns. ylläpitogeenejä. Lisäksi AIRE- ja PIAS1-proteiinit yhdessä säätelevät insuliinipromoottorin, tunnetun AIREn säätelykohteen, aktiivisuutta osoittaen, että vuorovaikutus on biologisesti merkityksellinen. Uusien AIREn kanssa vuorovaikutuksessa olevien proteiinien tunnistaminen lisää tietoamme siitä, mitkä säätelyreitit osallistuvat immunologisen toleranssin syntyyn ja ylläpitoon. Tämä on tärkeää, jotta ymmärtäisimme paremmin autoimmuunisairauksien syntymekanismeja

Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation

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Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.Peer reviewe

Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease

Toward Corneal Limbus In Vitro Model : Regulation of hPSC-LSC Phenotype by Matrix Stiffness and Topography During Cell Differentiation Process

A functional limbal epithelial stem cells (LSC) niche is a vital element in the regular renewal of the corneal epithelium by LSCs and maintenance of good vision. However, little is known about its unique structure and mechanical properties on LSC regulation, creating a significant gap in development of LSC-based therapies. Herein, the effect of mechanical and architectural elements of the niche on human pluripotent derived LSCs (hPSC-LSC) phenotype and growth is investigated in vitro. Specifically, three formulations of polyacrylamide gels with different controlled stiffnesses are used for culture and characterization of hPSC-LSCs from different stages of differentiation. In addition, limbal mimicking topography in polydimethylsiloxane is utilized for culturing hPSC-LSCs at early time point of differentiation. For comparison, the expression of selected key proteins of the corneal cells is analyzed in their native environment through whole mount staining of human donor corneas. The results suggest that mechanical response and substrate preference of the cells is highly dependent on their developmental stage. In addition, data indicate that cells may carry possible mechanical memory from previous culture matrix, both highlighting the importance of mechanical design of a functional in vitro limbus model.Peer reviewe

Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium

In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.Public Library of Science open acces

Kunniaan liittyvä väkivalta : Opas naisen hoitotyöhön

Tämä opinnäytetyö oli kehittämistyö, jossa tarkoituksena oli tuottaa kunniaväkivaltaa käsittelevä hoitotyö opas kätilöille ja kätilöopiskelijoille. Opas on tarpeellinen, sillä naisten hoitotyössä työskentelevien on tärkeää ymmärtää kunniaväkivallan problematiikkaa ja saada toimintamalleja kunniaväkivaltaa kokeneen naisen kohtaamiseen. Opas on tarkoitettu ensisijaisesti Metropolia Ammattikorkeakoulun terveysalan loppuvaiheen kätilöopiskelijoille ja täydennyskoulutuksen opiskelijoille. Materiaalia voidaan hyödyntää myös naisen hoitotyössä, esimerkiksi neuvoloissa ja äitiyspoliklinikoilla. Tässä kehittämistyössä tarkastelemme kunniaan liittyvää väkivaltaa naisen hoitotyön ja kätilötyön näkökulmista. Kehittämistyön aineisto on kerätty alan uusimmista tutkimuksista ja tiedeartikkeleista sekä asiaa tukevasta kirjallisuudesta. Opas antaa tietoa kunniaan liittyvästä väkivallasta ilmiönä, sen esiintyvyydestä Suomessa ja lain asettamista velvoitteista. Materiaalin tavoitteena on myös auttaa tunnistamaan kunniaan liittyvän väkivallan riskisignaaleja sekä tarjota keinoja kunniaväkivaltaa kokeneen naisen auttamiseksi. On tarkoitus, että oppaan avulla saa nopeasti päivitettyä tietonsa kunniaan liittyvästä väkivallasta ja että se antaa työkaluja yllättäviinkin tilanteisiin, joihin terveydenhuollon ammattilainen joutuu eri-ikäisiä naisia hoitaessaan.The purpose of this thesis was to develop a guide booklet about honour related violence for midwives and those who study midwifery. This guide is useful because it is important that nursing staff that take care of women understand the problems of honour related vio-lence and get models how to face woman who is a victim of violence. The guide is de-signed primarily for the health care students of midwifery who will graduate soon and up-dating training students in Metropolia University of Applied Sciences. It can be used also, for instance, in maternity and child welfare clinics. In this thesis honour related violence is studied from the perspectives of midwifery and taking care of women. The references of the thesis were collected from the newest researches, research journals and professional literature. The guide offers knowledge about honour related violence as a phenomenon, the occurrence in Finland and the obligations which are set in law. It aims also to help to recognize the signals of honour related violence and offers tools which have been created to help the victims. The purpose is that the learning material offers the reader a quick update on the phenomenon and gives tools to unexpected situations which health care professional faces when taking care of different-aged women

Directed Differentiation of Human Pluripotent Stem Cells towards Corneal Endothelial-Like Cells under Defined Conditions

The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method.publishedVersionPeer reviewe