Groß-Hardt, Rita

Lade...
Vorschaubild
Preferred name
Groß-Hardt, Rita
Official Name
Groß-Hardt, Rita
ORCID

Einstellungen

Gerade angezeigt 1 - 3 von 3
  • Lade...
    Vorschaubild
    Some of the metrics are blocked by your 
    consent settings
    Item-typ:Veröffentlichung,
    A novel platform for the synthesis of inorganic Janus nanoparticles with tailored cell interactions
    (2022-03-30)
    Kadam, Reshma Sampat
    ;
    Groß-Hardt, Rita
    ;
    Rezwan, Kurosch
    ;
    Rezwan, Kurosch
    In this thesis, we describe the preparation of Janus particles with face-separated compartments. These are anisotropic spherical colloidal particles of the inorganic origin. These compartmentalized particles have unique properties, such as different chemistries designed to specifically target bio applications. Biofunctionalized Janus particles with tailored surface chemistry in general have been gathering interest. The dual nature of the surface chemistry of Janus particles can be exploited to immobilize drugs, cell surface targets, and/or other functional molecules on both sides of the particle surface. There have been several reports and studies based on the preparation of Janus materials including several shapes and materials along with their widespread applications since a few years. The use of micro-sized particles with unique “Janus” character has been widely exploited over the years. However, there are certain limitations pertaining to the use of micro-sized particles when bio applications are concerned. In addition, there is a limited amount of research performed with nano-sized particles exhibiting the “Janus” character and their bio applications. In this thesis, we adapted the current state of the art to synthesize nano-sized Janus particles in bulk-quantities and used these particles to demonstrate bio applications including dual protein functionalization, agglomerate-free bacterial separation from mixtures and attachment to the cell surfaces of eukaryotic cells with minimal uptake. First, we established a model system for the scalable preparation of nanoscale Janus particles with dual protein functionalization with the proteins ferritin and streptavidin. We used 80 nm silica NPs (SiNPs) modified with azidosilane to prepare Pickering emulsions with molten wax as the droplet phase. The azide-functionalized SiNPs on the Pickering emulsion droplets were further subjected to face-selective silanization with biotin-polyethylene glycol (PEG) ethoxy silane. Afterwards, we grafted ferritin on the azide-functionalized side via a click-reaction and the biotin groups were conjugated with streptavidin which was labeled with ultra-small gold NPs. In order to elucidate the advantages and limits of our approach, we performed a detailed characterization of the particles at every process step. The results showed that this method represented a scalable platform for the versatile preparation of protein- nanoscale Janus NPs that can potentially be used with a wide variety of proteins. We further took advantage of the established method where protein-protein functionalization at the nanoscale was demonstrated, to prepare Janus SiNPs for bio application on a prokaryote-based system. We presented a scalable method for designing magnetic Janus NPs which are capable of performing bacterial capture while preventing agglomeration between bacterial cells. To this end, we prepared silica-coated magnetite (Fe3O4) Janus NPs functionalized with a bacteria-specific antibody on one side and PEG chains on the other, using the established wax-in-water emulsion strategy. These magnetic Janus NPs specifically interacted with one type of bacteria from a mixture of bacteria via specific antigen-antibody interactions. Contrary to bacterial capture with isotropically functionalized particles, the bacterial suspensions remained free from cell-NP-cell agglomerates owing to the passivation coating with PEG chains attached to the half of the magnetic NPs pointing away from the bacterial surface after capture. Selective magnetic capture of Escherichia coli (E.coli) cells was achieved from a mixture with Staphylococcus simulans (S.simulans) without compromising bacterial viability and with an efficiency over 80%. This approach is a promising method for rapid and agglomeration-free separation of live bacteria for identification, enrichment and cell counting of bacteria from biological samples. Furthermore, after the successful preparation of Janus NPs for the selective capture of bacteria, we prepared Janus NPs that are designed for the attachment to eukaryotic cell surface molecules with minimal cell uptake. To this end, we synthesized rhodamine-doped SiNPs functionalized with 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) on one hemisphere of the NP surface and high-molecular-weight long-chain PEG on the other one using the wax-Pickering emulsion technique. NP localization was studied with mouse fibroblasts in vitro. In these studies, the Janus NPs were attached to the cell surface and, in contrast to isotropic control particles, only negligible uptake into the cells was observed, even after 24 h of incubation. The study revealed that the prolonged attachment of the Janus NPs is most likely the result of an incomplete macropinocytosis process, and it seems to be independent from caveolae- and receptor-mediated endocytosis. Consequently, by design, these Janus NPs have the potential to firmly anchor onto cell surfaces for extended periods of time which might be utilized in various biotechnological and biomedical applications like cell surface tagging, magnetic manipulation of the cell membrane or non-invasive drug and gene delivery.
    Dissertation
      289  220
  • Lade...
    Vorschaubild
    Some of the metrics are blocked by your 
    consent settings
    Item-typ:Veröffentlichung,
    Analysis of ectomycorrhiza induced gene expression of selected poplar genes expressed in poplar fine roots
    (2020-11-30)
    Schnakenberg, Jana
    ;
    Groß-Hardt, Rita
    ;
    Nehls, Uwe
    ;
    Nehls, Uwe
    Ectomycorrhiza is the symbiotic interacting between fungal hyphae and plant roots. The fungus provides the plant with nutrients mobilized from soil and the plant delivers carbohydrates from photosynthesis in exchange. Former studies showed an up-regulation of genes coding for the transcription factor dehydration responsive element binding factor 1 (DREB1) and for one facilitator of the gene family sugar will be eventually exported transporter (SWEET1) in the model organism poplar. While SWEET1 was shown to be involved as glucose transporter in diverse associations like pathogenic interactions, rhizobia nodules and arbuscular mycorrhiza, no link between DREB1 and such biotic interactions is reported. The Arabidopsis DREB1 homolog was shown to be involved in the regulation process of abscisic acid signaling and glucose related pathways. Aim of the thesis was the in vivo analysis of promoter fragments of the mentioned genes. For analysis promoter reporter constructs should be expressed in transgenic composite poplar. To distinguish between transgenic and non-transgenic roots, firstly a second, visual marker under control of a constitutive promoter should be integrated into the plant transformation vector. This step was necessary, since no classical selection procedure can be performed during composite plant generation leading to transgenic and non-transgenic roots emerging from the transformed shoot. A nuclear targeted Td-Tomato under control of the nopaline synthase (NOS) promoter turned out to give clear signals in poplar roots and was chosen as visual selection marker. Furthermore different binary vectors were tested for their transformation efficiency in composite poplar. The pCAMBIA-based vector pCXUN showed compared to pBi121 and the p-Green-based vector pPLV significantly increased transformation efficiency. To monitor expression of the promoter of interest, nuclear targeted super yellow fluorescence protein (sYFP) and double green fluorescence protein (dGFP) were analyzed. DGFP showed higher signal intensity in poplar roots and distinct localization to the nucleus in comparison to sYFP and was therefore used as marker gene in the final binary vector. Furthermore a transient expression system for poplar leaves was established, to analyze the activity of ectomycorrhiza induced promoters for their root specificity. Different techniques were tested and the infiltration of the fragile poplar leaves turned out to be most successful. The transient expression in poplar turned out to be not as robust as the infiltration of model organism N. benthamiana, but it enables a fast, transient expression in poplar. The newly composed vector pCXUN04NOS was used to analyze promoter fragments of DREB1 (3.2 kb) and SWEET1 (3.4 kb) for ectomycorrhiza dependent expression localization in composite poplar and expression in leaf tissue. To compare expression in mycorrhized and non-mycorrhized roots, composite plants were mycorrhized using the ectomycorrhiza fungi Pisolithus microcarpus and Amanita muscaria. The generated results stand in contrasted to previous expression data, since no ectomycorrhiza induced expression could be detected. Furthermore both fragments showed expression in leaf tissue of N. benthamiana and P. tremula x alba. These results indicate that the investigated promoter fragments did not contain all cis-elements important for ectomycorrhiza specific expression.
    Dissertation
      605  427
  • Lade...
    Vorschaubild
    Some of the metrics are blocked by your 
    consent settings
    Item-typ:Veröffentlichung,
    Analysis of selected symbiosis repressed poplar genes upon ectomycorrhiza formation
    (2021-10-05)
    Immoor, Anneke
    ;
    Groß-Hardt, Rita
    ;
    Nehls, Uwe
    ;
    Nehls, Uwe
    Damit eine Symbiose zwischen Ektomykorrhizen und Pflanzen stattfinden kann, muss die Struktur und Funktionalität von Feinwurzeln angepasst werden. Nur so kann der Stoffaustausch von Zucker gegen Nährstoffe und weitere Metabolite stattfinden. Die grundlegenden Prozesse dieser Anpassungen sind dabei in großen Teilen noch unerforscht. Auf dieser Grundlage wurden in vorangegangenen Studien durch genomweite Genexpressionsanalysen drei Pappel Gene identifiziert, deren Transkription in Mykorrhizen 20 – 100 -fach niedriger war. Es handelte sich dabei um ein Kalzium Bindeprotein (Potri.2G2183), einen Aminosäure Transporter (Potri.2G0797) und ein Enzym aus dem Fettstoffwechsel (Potri.9G1040). Um die Proteine zu charakterisieren und den Mechanismus der Regulierung herauszufinden, wurden die Promotoren in dieser Arbeit auf mögliche cis-Elemente untersucht. Dafür wurden Promotor-Reporter Konstrukte mit Fluoreszenzproteinen hergestellt und dann mittels Agrobakterien zur Behandlung von Komposit Pappeln verwendet. Als Grundlage für diese Analyse konnte im Rahmen dieser Arbeit durch einen Wechsel von pPLV zum pCXUN basierten Vektorrückrat die Transformationseffizienz der Pappeln um 69.8% erhöht werden. Weiterhin wurde herausgefunden, dass weder die Orientierung der Fluoreszenz Kassetten auf der T – DNA, noch deren Größe einen messbaren Einfluss auf die Pflanzentransformationseffizienz haben. Dabei konnten Promoter Bereiche von Potri.2G2183 und Potri.2G0797 erfolgreich mittels PCR amplifiziert und trunkiert werden. Zur weiteren Charakterisierung der Promotoren, wurden diese und ihre Trunkierungen in eine eGFP-NLS Expressionskassette kloniert. Um potenzielle cis – Elemente durch Auswertung visueller Daten zu finden, wurde eine konstitutiv exprimierende tdTomato-NLS Expressionskassette in Tandem mit der Promoter Kassette geschaltet (Grün: Rot Verhältnis). Diese Verhältnisse können gegebenenfalls Aufschluss über Änderungen in der Genexpression durch die Mykorrhizierungen geben. Als Alternative zu diesen so genannten Doppelmarker Konstrukten aus eGFP und tdTomato wurde das Timer Protein DsRED-E5 etabliert. Weil das grüne Signal im Verhältnis zum roten Signal im Falle des Timer Proteins sehr schwach war, wurde die Bildaufnahme durch Variierung des der Kameraeinstellungen optimiert. In anschließenden Studien muss diese Methode nun auf Anwendbarkeit in Mykorrhizen getestet werden. Insgesamt muss die visuelle Quantifizierung noch weiter optimiert werden. Auch subzälluläre Lokalisierung der Gene basierend auf transienter Expression in Tabakblättern wurde durchgeführt. Da hier keine eindeutigen Signale detektiert werden konnten, muss das System weiter angepasst und optimiert werden.
    Dissertation
      326  227