Assembly of Micro Systems with the High Precision Robot Parvus

In recent years, the development of micro systems has been widely discussed in research articles concerning a decrease in size, an increase of complexity and the variety of materials used. In contrast, manufacturing and especially assembly processes of millimetre-sized products with high complexity did not play a significant role. Conventional precision robots that provide adequate accuracies for micro assembly are relatively large and expensive. These machines have to be operated in clean rooms, which results in high costs of maintenance. These days, the assembly technology of electronic production and conventional assembly robots is often no longer suitable for the assembly of hybrid micro systems. The increasing gap between millimetre-sized products and the production machines has lead to a high proportion of manual assembly in the manufacturing process of microproducts. Assembly costs that sometimes account for up to 80 % of the costs of micro systems retard the commercialisation and bulk production of these products. [1] Especially for small and medium-sized businesses, new concepts for flexible and lower-cost micro assembly have to be found

Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

“Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery

Biomolecular interactions

In der vorliegenden Arbeit wird der Weg vom Einzelmolekül über den Molekülverband bis hin zur lebenden Zelle exemplarisch an drei biologischen Systemen beschrieben: Im ersten Teil wird die Konformationsvariabilität des initial als starr betrachteten, größten Makromoleküls lebender Zellen, der DNA, auf atomarer Ebene unter Einsatz der beiden am besten dafür geeigneten Methoden, der Einkristall-Röntgenstrukturuntersuchung und der hochauflösenden NMR-Spektroskopie, analysiert. Ziel ist hierbei, die Bedeutung einzelner molekularer Interaktionen für die Stabilisierung einer spezifischen Konformation zu erfassen, wobei sich der Auflösungsbereich von der elektronischen Interaktion einzelner Atome und Moleküle über die Ausbildung spezifischer Wasserstoffbrückenbindungen bis zu der Koordination konformationsstabilisierender Kationen und dem Einfluß der Basenfolge des analysierten DNA-Fragmentes auf die Konformation erstreckt. Im zweiten Teil wird die Struktur eines hochmolekularen (190 kd), homodimeren Transmembranproteins, des humanen Transferrinrezeptors, im physiologischen Kontext einer Phospholipidmembran bestimmt und die Bindung seines etwa 80 kd großen Liganden Transferrin in vitro quantifiziert. Da die oben genannten physikalischen Verfahren zur Strukturaufklärung nicht für die Analyse membraninsertierter Proteine geeignet sind, kommen hier alternative Methoden zur Anwendung. Obwohl der Transferrinrezeptor zu den am besten untersuchten Membranproteinen zählt, differieren die publizierten Dissoziationskonstanten für die Bindung seines Liganden Transferrin um mehr als das Hundertfache. Vor diesem Hintergrund ist zum einen Ziel dieser Arbeit, ein Verfahren zur möglichst genauen Quantifizierung der Bindung unmarkierter makromolekularer Interaktionspartner zu entwickeln und zum anderen dieses Verfahren mit den zuvor verwendeten Methoden im Hinblick auf seine Genauigkeit zu vergleichen. Im dritten Teil soll schließlich die Brücke vom Einzelmolekül zur lebenden Zelle am Beispiel eines im Rahmen dieser Arbeit entdeckten neuen Zelladhäsionsmoleküls, des LI-Cadherins, geschlagen werden. Hierunter fallen zunächst die Isolierung, Klonierung und Sequenzanalyse des Proteins. Es folgen seine Expression in verschiedenen Zellsystemen und die Untersuchung seiner zelladhäsiven Eigenschaften, die sich mit herkömmlichen Methoden nicht an isolierten Molekülen, sondern nur im zellulären Kontext unter der gleichzeitigen Bindung vieler Tausender Einzelmoleküle nachweisen läßt. Um die Funktion des LI-Cadherins im lebenden Organismus aufklären zu können, wird im Rahmen dieser Arbeit nicht nur dessen Expressionsmuster in den einzelnen Geweben unter physiologischen Bedingungen, sondern auch bei pathologischen Veränderungen analysiert. Ausgehend von Sequenzmotiven, welche das LI-Cadherin von den klassischen Cadherinen unterscheiden, wird auf verschiedene biophysikalische Methoden zurückgegriffen, um die dadurch bedingten funktionellen Eigenschaften auf molekularer Ebene aufzuklären. Schließlich wird das zum LI-Cadherin strukturhomologe Ksp-Cadherin in die funktionellen Untersuchungen einbezogen und auf der Basis der genomischen Organisation von LI- und Ksp-Cadherin ein Modell für die phylogenetischen Entwicklung beider Proteine, welche die Familie der 7D-Cadherine bilden, abgeleitet.This thesis describes the path from a single molecule over molecular assemblies to living cells by analyzing three biological systems on different scales: In the first part, the largest macromolecule within the cell, DNA, which has initially been considered to be rigid, is being studied on atomic scale with respect to its conformational dynamic using the two most suitable methods, single crystal X-ray diffraction analysis and high resolution NMR spectroscopy. The specific goal of this project was to determine the molecular interactions that stabilize a particular conformation. The study covers a range reaching from the electronic interactions between single atoms over the function of particular hydrogen bonds and cation coordinations to the conformational impact of variations in the nucleotide sequence. In the second part, the structure of a large (190 kd), homodimeric transmembrane protein, the human transferrin receptor, is being analyzed in its native molecular environment, the phospholipid membrane. In addition, the binding of its 80 kd ligand transferrin is quantified using purified receptor and ligand. Since the above described biophysical methods are not suitable for solving the structure of membrane-inserted proteins, complementary methods were applied, in particular electron microscopy. Although the human transferrin receptor is one of the best studied transmembrane proteins, the published binding constants for its ligand transferrin vary by more than two orders of magnitude. In this respect it was also the goal to invent a simple and exact method for determining binding constants of unlabeled macromolecules and to compare its accuracy to that of other methods. In the third part, the gap is finally being filled between macromolecules and living cells by scrutinizing the structure and function of a novel cell-cell adhesion protein discovered in our laboratory, LI-cadherin. Described are first the purification, cloning and sequencing of LI-cadherin, followed by its expression in various cell systems and the analysis of its adhesive function, which has been studied in the cellular context where thousands of molecules interact simultaneously. In order to understand the function of LI-cadherin in the living organism, its tissue expression pattern was examined under physiological and pathological conditions. The functional impact of the observed differences in the primary structure of LI- and classical cadherins has in addition been probed with various biophysical methods. Finally, the structurally and functionally related Ksp-cadherin was included into the study. Based on a detailed analysis of the genomic organisation of both proteins, a phylogenetic model was derived that explains a common origin of LI- and Ksp-cadherin, which are thus grouped together in the 7D-cadherin protein family

Structural model of phospholipid-reconstituted human transferrin receptor derived by electron microscopy

AbstractBackground: The transferrin receptor (TfR) regulates the cellular uptake of serum iron. Although the TfR serves as a model system for endocytosis receptors, neither crystal structure analysis nor electron microscopy has yet revealed the molecular dimensions of the TfR. To derive the first molecular model, we analyzed purified, lipid-reconstituted human TfR by high-resolution electron microscopy.Results: A structural model of phospholipid-reconstituted TfR was derived from 72 cryo-electron microscopic images. The TfR dimer consists of a large extracellular globular domain (6.4 × 7.5 × 10.5 nm) separated from the membrane by a thin molecular stalk (2.9 nm). A comparative protein sequence analysis suggests that the stalk corresponds to amino acid residues 89–126. Under phospholipid-reconstitution conditions, the human TfR not only integrates into vesicles, but also forms rosette-like structures called proteoparticles. Scanning transmission electron microscopy revealed an overall diameter of 31.5 nm and a molecular mass of 1669 ± 26 kDa for the proteoparticles, corresponding to nine TfR dimers. The average mass of a single receptor dimer was determined as being 186 ± 4 kDa.Conclusions: Proteoparticles resemble TfR exosomes that are expelled by sheep reticulocytes upon maturation. The structure of proteoparticles in vitro is thus interpreted as being the result of the TfR's strong self-association potential, which might facilitate the endosomal sequestration of the TfR away from other membrane proteins and its subsequent return to the cell surface within tubular structures. The stalk is assumed to facilitate the tight packing of receptor molecules in coated pits and recycling tubuli

Influence of a Heterologous (ChAdOx1-nCoV-19/BNT162b2) or Homologous (BNT162b2/BNT162b2) Vaccination Regimen on the Antibody and T Cell Response to a Third Vaccination with BNT162b2

Emerging numbers of SARS-CoV-2 infections are currently combated with a third vaccination. Considering the different vaccination regimens used for the first two vaccine doses, we addressed whether the previous vaccination influences the immune response to the booster. Participants for this prospective study were recruited from among healthcare workers. N = 20 participants were previously vaccinated with two doses of BNT162b2, and n = 53 received a priming dose of ChAdOx1-nCoV-19 followed by a BNT162b2 dose. Participants were vaccinated with a third dose of BNT162b2 in December 2021. Antibody concentrations were determined after vaccination, and in a subset of n = 19 participants, T cell responses were evaluated. Anti-S concentrations and IFNγ production increased during the first 21 days. The choice of the first and second vaccineshad no influence on the final outcome of the booster vaccination. Before booster vaccination, antibody concentrations were lower for older participants but increased more strongly over time