Innovative Form- und Fügetechniken für biologisches Gewebe zur Fertigung nahtloser Transkatheter-Aortenklappenprothesen

Die Transkatheter-Aortenklappenimplantation ist ein minimalinvasives Verfahren, bei dem eine Aortenklappenprothese im zusammengefalteten Zustand innerhalb der nativen Klappe platziert und freigesetzt wird. Das Implantat besteht aus einzelnen, manuell vernähten, kollagenhaltigen Gewebekomponenten, die in einen Stent integriert sind. Durch den aufwändigen und fehleranfälligen Nähprozess wird dabei eine komplexe, dreidimensionale Gewebegeometrie erzeugt, die für die Funktionalität der Prothese unerlässlich ist. Gleichzeitig bilden die chirurgischen Knoten mechanische Schwachstellen, die potentiell zum Versagen des Implantats führen. Diese Dissertation befasst sich mit der Entwicklung innovativer Formgebungs- und Fügeverfahren für biologisches Gewebe zur Reduktion der Anzahl der Nähte bei Transkatheter-Aortenklappenprothesen. Hierzu wurden zwei miteinander kombinierbare Lösungsansätze verfolgt, die beide auf der chemischen Vernetzung des Gewebes mit Glutaraldehyd, einem reaktiven Quervernetzer, beruhen. Da die Vernetzung ein obligatorischer Prozessschritt bei der Fertigung des Implantats ist, um das Gewebe vor enzymatischer Degradation zu schützen, wird die Nahtreduktion somit ohne zusätzlichen Werkstoff im Endprodukt realisiert. Zunächst wird ein Prozess etabliert, mit dem unter Verwendung 3D-gedruckter Formkörper eine dauerhafte und präzise Formgebung des Gewebes erzielbar ist. Hierbei wird der Effekt ausgenutzt, dass der Vernetzungsprozess eine Konservierung des aktuell im Gewebe vorherrschenden Dehnungszustandes bewirkt. Der zweite Ansatz basiert auf der Ausbildung interfibrillärer Quervernetzungen zwischen überlappenden kollagenhaltigen Fügepartnern, durch die sich eine stabile, stoffschlüssige Verbindung erzeugen lässt. Im Anschluss an die Konzeptionierung und vollständige Gewebecharakterisierung werden die erarbeiteten Methoden zuletzt in den Herstellungsprozess von Transkatheter-Aortenklappenprothesen integriert. Diesbezüglich werden vier neuartige Prozessketten vorgestellt, mit denen sich Nähte bei der Fertigung des Implantats weitestgehend vermeiden lassen. Unter anderem wird als Endergebnis dieser Arbeit erstmals ein professionell gefertigter, voll funktionstüchtiger Prototyp mit einteiliger Gewebekomponente präsentiert. In Anbetracht der entwickelten Konzepte erscheint sogar eine vollständig nahtlose Prothese zukünftig möglich, sodass die vorliegende Arbeit die Basis zur Entwicklung einer neuen Generation 3D-geformter kardiovaskulärer Implantate bildet

Recovering the Imperfect: Cell Segmentation in the Presence of Dynamically Localized Proteins

Deploying off-the-shelf segmentation networks on biomedical data has become common practice, yet if structures of interest in an image sequence are visible only temporarily, existing frame-by-frame methods fail. In this paper, we provide a solution to segmentation of imperfect data through time based on temporal propagation and uncertainty estimation. We integrate uncertainty estimation into Mask R-CNN network and propagate motion-corrected segmentation masks from frames with low uncertainty to those frames with high uncertainty to handle temporary loss of signal for segmentation. We demonstrate the value of this approach over frame-by-frame segmentation and regular temporal propagation on data from human embryonic kidney (HEK293T) cells transiently transfected with a fluorescent protein that moves in and out of the nucleus over time. The method presented here will empower microscopic experiments aimed at understanding molecular and cellular function.Comment: Accepted at MICCAI Workshop on Medical Image Learning with Less Labels and Imperfect Data, 202

Origen de la casiterita detrítica en los aluviones recientes de Tirados de la Vega-Golpejas (Salamanca)

9 páginas, 6 figuras, 5 tablas.[ES] En el sector Tirados de la Vega-Golpejas han sido detectadas dos anomalías de casiterita en los aluviones del Regato de los Lentiscos y en otros existentes al sur de Tirados de la Vega. El estudio granométrico y morfométrico de éstas casiteritas. contrastado con el de la casiterita procedente del granito de Golpejas. permite asegurar que la anomalía del Regato de los Lentiscos se debe a la dispersión de la casiterita de aquel granito. mientras que la de Tirados de la Vega procede de filones estanníferos. La aplicación del análisis factoral corrobora este hecho y permite diferenciar las casiteritas que proceden de rocas graníticas de aquellas que vienen de filones estanníferos. Según esto se puede indicar que la capacidad de migración de la casiterita desde el yacimiento estannífero de Golpejas es de unos 3 Kms.[EN] Two cassiterite anomalies have been detected in the Regato de los Lentisco and Tirados de la Vega alluviums, in the Golpejas area (Salamanca). The granometric and morphometric parameters of these cassiterites have been compared with those shown by the cassiterite crystal ocurrying in the Golpejas granite. The comparison between the two groups of data allow us to conclude that the anomaly observed in the Regato de los Lentistos has been originated by the disperssion of the cassiterite from the primary deposit of Golpejas. On the contrary, the anomaly ocurring in the alloviums to the south of Tirados de la Vega shows a different origin, and is coming from stanniferous veins. The factor anylisis corroborates the differentiation mentionated above, and indicates a migration capacite of about 3 kms. from the stannifeous deposits of Golpejas for the alluvial cassiterite.Peer reviewe

In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Background: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide - particularly ATP-Mg - and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF-hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties. Results: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca2+-binding to the EF-hand motifs in the N-terminal domain of the carrier. Conclusion: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo

Standard for Synthesis of Customized Peptides by Non-Ribosomal Peptide Synthetases

The purpose of this RFC is to introduce a standardized framework for the engineering of customizable non-ribosomal peptide synthetases (NRPS) and their application for in vivo and in vitro synthesis of short non-ribosomal peptides (NRPs) of user-defined sequence and structure

HiCT: High Throughput Protocols For CPE Cloning And Transformation

The purpose of this RFC is to provide instructions for a rapid and cost efficient cloning and transformation method which allows for the manufacturing of multi-fragment plasmid constructs in a parallelized manner: High Throughput Circular Extension Cloning and Transformation (HiCT). Description of construct libraries generated by the HiCT method can be found at http://2013.igem.org/Team:Heidelberg/Indigoidine. This RFC also points out further optimization strategies with regard to construct stability, reduction of transformation background and the generation of competent cells

miTuner - a kit for microRNA based gene expression tuning in mammalian cells

The purpose of this RFC is to introduce a modular expression tuning kit for use in mammalian cells. The kit enables the regulation of the gene expression of any gene of interest (GOI) based on synthetic microRNAs, endogenous microRNAs or a combination of both

miMeasure – a standard for miRNA binding site characterization in mammalian cells

This RFC proposes a standard for the quantitative characterization of miRNA binding sites (miRNA-BS) in mammalian cells. The miMeasure standard introduces a ready-to-use standard measurement plasmid (pSMB_miMeasure, BBa_K337049) enabling rapid experimental characterization of any miRNA-BS of choice. We recommend a new standard unit, RKDU (relative knock-down unit) to describe the knock-down efficiency of a miRNA-BS in a specific cell type. pSMB_miMeasure allows for an easy and fast measurement of RKDU while providing effective normalization against variance stemming from differences in transfection efficiency and from other sources

Optogenetic control of gene expression in plants in the presence of ambient white light

Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR–Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology