Entwicklung eines mRNA basierten Transkript Therapieansatzes zur Behandlung der Erkrankung Epidermolysis bullosa

Die Hauterkrankung Epidermolysis bullosa (EB) zeichnet sich durch eine Blasenbildung der Haut und Schleimhäute aus, die mit immensem Leid für die betroffenen Patienten verbunden ist. Seit Jahrzehnten werden intensiv neue Behandlungsoptionen gesucht, wobei sich bisherige Strategien auf gentherapeutische und zellbasierte Ansätze konzentrieren. Bis heute umfassen diese Untersuchungen den Einsatz von retroviralen Vektoren, die intradermale Injektion von Fibroblasten bzw. mesenchymalen Stromazellen, die intradermale Applikation von rekombinant hergestellten Proteinen, sowie die Durchführung einer Knochenmarksstammzelltransplantation. Im Rahmen dieser Dissertation sollten die bisherigen therapeutischen Möglichkeiten dahingehend erweitert werden, dass mit Hilfe von synthetisch hergestellten mRNAs eine fehlende Genexpression in Hautzellen wiederhergestellt wird. Dabei ist es gelungen, für eine Kontroll mRNA (codierend für das Fluoreszenzprotein DsRed) und zwei verschiedene Adhäsionsmoleküle der Haut (Desmoglein-3 und Kollagen 7) entsprechende mRNAs in vitro zu synthetisieren und aufzureinigen. Nach Etablierung eines Protokolls zur erfolgreichen mRNA Transfektion von humanen und murinen Keratinozyten und Fibroblasten war es möglich die Dsg3 codierende mRNA in Dsg3 knockout Keratinozyten bzw. die Col7 codierende mRNA in Col7a1 hypomorphen Fibroblasten in vitro zu expri-mieren. Der Nachweis der Expression konnte sowohl auf mRNA-Ebene mittels RT-PCR erbracht werden, als auch auf Protein-Ebene mittels immunhistochemischer Färbungen. In dieser Arbeit konnte nachgewiesen werden, dass Keratinozyten und Fibroblasten in Zellkultur mit mRNA transfiziert werden können und eine fehlende Genexpression dadurch kompensiert werden kann. Somit stellen diese Experimente den Ausgangspunkt für weiterführende in vivo Untersuchungen dar, mit dem Ziel, in den entsprechenden Mausmodellen eine Genkorrektur herbeizuführen. Sollte dies gelingen, stünde eine vielversprechende Therapieoption für Patienten mit EB zur Verfügung

The Three Dimensions of Functional T-Cell Tolerance: From Research to Practice

In this issue, Paek et al. describe two phenomena. First, they show that intermediate concentrations of a “transgenic” autoantigen may cause a lichen planus–like autoimmune disease. Second, and more importantly, they show that high doses of peptide antigen suppress the expression of the T-cell receptor and coreceptors, particularly CD8, and that this suppression improves this T-cell-mediated, destructive inflammatory skin disease that is similar to erosive lichen planus

Controlling the crystal structure of precisely spaced polyethylene-like polyphosphoesters

Understanding polymer crystallization is important for polyethylene-like materials. A small fraction of monomers with functional groups within the polyethylene chain can act as crystallization “defects”. Such defects can be used to control the crystallization behavior in bulk and to generate functional anisotropic polymer crystals if crystallized from a dilute solution. Due to their geometry, phosphate groups cannot be incorporated in the polyethylene lamellae and thus control chain folding and crystal morphology. Herein, the synthesis and crystallization behavior for three different long-chain polyphosphates with a precise spacing of 20, 30, and 40 CH2-groups between each phosphate group are reported. Monomers were prepared by esterification of ethyl dichlorophosphate with respective tailor-made unsaturated alcohols. Acyclic diene metathesis (ADMET) polymerization and subsequent hydrogenation were used to receive polyethylene-like polyphosphoesters with molecular weights up 23 100 g mol−1. Polymer crystallization was studied from the melt and dilute solution. Samples were characterized by differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). A change in crystal structure from pseudo-hexagonal to orthorhombic was observed from the “C20” to the “C40” polymer. Melting points and lamellar thicknesses increased with the length of the aliphatic spacer from 51 °C (“C20”) to 62 °C (“C30”) and 91 °C (“C40”). Values for the long periods in bulk (3.1 nm for C20, 4.8 nm for C30, and 7.2 nm for C40) obtained by SAXS and TEM are in qualitative agreement. The thickness of the crystalline part obtained by AFM and TEM increased from about 1.0 nm (C20) to 2.0 nm (C30) to 2.9 nm (C40). Our systematic library of long-chain polyphosphates will allow designing anisotropic polymer colloids by crystallization from solution as functional and versatile colloid platform

Measurements of metastable helium in Earth’s atmosphere by resonance lidar

Monitoring and predicting space weather activity is increasingly important given society’s growing reliance on space-based infrastructure but is hampered by a lack of observational data. Airglow at 1083 nm from metastable helium He(23S) in the thermosphere has long been a target for remote-sensing instruments seeking to fill that gap; however, passive measurements of He(23S) fluorescence are limited by low brightness, and interpretation of these observations is complicated by the > 500 km depth of the He(23S) layer. Here, we demonstrate a lidar instrument that is able to stimulate and detect He(23S) fluorescence, and we present measured profiles of He(23S) density. These measurements provide crucial validation to space weather models, support predictions of peak number density ( ~ 1 cm−3) and the dependence of density on altitude, solar zenith angle, and season, and extend by a factor of 4 the maximum probed altitude range by an atmospheric profiling lidar. These measurements open the door for the development of more sophisticated lidars: by applying well-established spectroscopic lidar techniques, one can measure the Doppler shift and broadening of the He(23S) line, thereby retrieving profiles of neutral wind speed and temperature, opening a window for studying space weather phenomena

Single-crystal Ih Ice Surfaces Unveil Connection between Macroscopic and Molecular Structure

Physics and chemistry of ice surfaces are not only of fundamental interest but also have important impacts on biological and environmental processes. As ice surfaces—particularly the two prism faces—come under greater scrutiny, it is increasingly important to connect the macroscopic faces with the molecular-level structure. The microscopic structure of the ubiquitous ice Ih crystal is well-known. It consists of stacked layers of chair-form hexagonal rings referred to as molecular hexagons. Crystallographic unit cells can be assembled into a regular right hexagonal prism. The bases are labeled crystallographic hexagons. The two hexagons are rotated 30° with respect to each other. The linkage between the familiar macroscopic shape of hexagonal snowflakes and either hexagon is not obvious per se. This report presents experimental data directly connecting the macroscopic shape of ice crystals and the microscopic hexagons. Large ice single crystals were used to fabricate samples with the basal, primary prism, or secondary prism faces exposed at the surface. In each case, the same sample was used to capture both a macroscopic etch pit image and an electron backscatter diffraction (EBSD) orientation density function (ODF) plot. Direct comparison of the etch pit image and the ODF plot compellingly connects the macroscopic etch pit hexagonal profile to the crystallographic hexagon. The most stable face at the ice–water interface is the smallest area face at the ice–vapor interface. A model based on the molecular structure of the prism faces accounts for this switch

Projetos de Aprendizagem Mediados por Ambientes Virtuais no Ensino de EngenhariaEl trica

As experiências relacionadas à metodologia de projetos de aprendizagem têm sua origem nas formulações de John Dewey no início do século XX. A concepção de projeto de aprendizagem defendida nesta pesquisa alia às formulações de Dewey as propostas da professora Lea Fagundes. Esta metodologia tem o aluno como responsável pela sua aprendizagem e reflete os conceitos construtivistas de Piaget. A integração e a construção de conhecimento via projetos é facilitada com o uso dos ambientes virtuais para apoio à aprendizagem que são softwares que permitem interações síncronas ou assíncronas além de possibilitar o registro de todo o caminho percorrido pelo estudante e de todas as atividades de uma disciplina. A partir de dados coletados, no período de 2003 a 2007, em atividades escolares de disciplinas do terceiro período letivo do curso de Engenharia Elétrica da Universidade Federal do Espírito Santo pretende-se analisar (a) em que medida os ambientes virtuais de aprendizagem e a apropriação das tecnologias de informação e comunicação acrescentam na formação do estudante de engenharia, em relação às práticas pedagógicas presenciais e (b) em que medida os projetos de aprendizagem contribuem para o enfrentamento dos problemas que temos, hoje, no ensino de engenharia elétric

Molecular scale structure and dynamics at an ionic liquid/electrode interface

The structural arrangement and dynamics of ions near the IL/electrode interface during charging and discharging was studied by a combination of time resolved X-ray reflectivity and impedance spectroscopy.</p

NKG2D Signaling Leads to NK Cell Mediated Lysis of Childhood AML

Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression

Haploidentical hematopoietic stem cell transplantation as individual treatment option in pediatric patients with very high-risk sarcomas

Background Prognosis of children with primary disseminated or metastatic relapsed sarcomas remains dismal despite intensification of conventional therapies including high-dose chemotherapy. Since haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is effective in the treatment of hematological malignancies by mediating a graft versus leukemia effect, we evaluated this approach in pediatric sarcomas as well. Methods Patients with bone Ewing sarcoma or soft tissue sarcoma who received haplo-HSCT as part of clinical trials using CD3+ or TCRα/β+ and CD19+ depletion respectively were evaluated regarding feasibility of treatment and survival. Results We identified 15 patients with primary disseminated disease and 14 with metastatic relapse who were transplanted from a haploidentical donor to improve prognosis. Three-year event-free survival (EFS) was 18,1% and predominantly determined by disease relapse. Survival depended on response to pre-transplant therapy (3y-EFS of patients in complete or very good partial response: 36,4%). However, no patient with metastatic relapse could be rescued. Conclusion Haplo-HSCT for consolidation after conventional therapy seems to be of interest for some, but not for the majority of patients with high-risk pediatric sarcomas. Evaluation of its future use as basis for subsequent humoral or cellular immunotherapies is necessary

CRISPR medicine for blood disorders: Progress and challenges in delivery

Blood disorders are a group of diseases including hematological neoplasms, clotting disorders and orphan immune deficiency diseases that affects human health. Current improvements in genome editing based therapeutics demonstrated preclinical and clinical proof to treat different blood disorders. Genome editing components such as Cas nucleases, guide RNAs and base editors are supplied in the form of either a plasmid, an mRNA, or a ribonucleoprotein complex. The most common delivery vehicles for such components include viral vectors (e.g., AAVs and RV), non-viral vectors (e.g., LNPs and polymers) and physical delivery methods (e.g., electroporation and microinjection). Each of the delivery vehicles specified above has its own advantages and disadvantages and the development of a safe transferring method for ex vivo and in vivo application of genome editing components is still a big challenge. Moreover, the delivery of genome editing payload to the target blood cells possess key challenges to provide a possible cure for patients with inherited monogenic blood diseases and hematological neoplastic tumors. Here, we critically review and summarize the progress and challenges related to the delivery of genome editing elements to relevant blood cells in an ex vivo or in vivo setting. In addition, we have attempted to provide a future clinical perspective of genome editing to treat blood disorders with possible clinical grade improvements in delivery methods