Ein Mehr-Zustands-Mehr-Episoden-Modell in diskreter Zeit zur Analyse klinischer Studien unter Beruecksichtigung unbeobachteter Heterogenitaet

Die in der Theorie der Verweildaueranalyse ueberwiegenden zeitstetigen Ansaetze gehen davon aus, dass keine Bindungen existieren. Diese Voraussetzung ist in der Praxis jedoch problematisch, so dass zeitdiskrete Ansaetze der Datensituation besser angepasst sind. Im medizinischen Kontext steht zumeist die Ueberlebenszeit von Patienten im Mittelpunkt. Neben diesem Ereignis koennen jedoch andere sich gegenseitig ausschliessende Ereignisse/Zustaende (competing risks) in verschiedenen Episoden, wie etwa der Gesundheitszustand im Krankheitsverlauf, von Interesse sein. In der vorliegenden Arbeit wird ein zeitdiskreter parametrischer Ansatz zur Analyse von competing risks fuer Mehr-Episoden-Modellen vorgestellt. Angelehnt an die Theorie der generalisierten linearen Modelle wird ein Multinomiales-Logit-Modell zur Modellierung der Hazardrate verwendet. Fuer die neben den beobachteten Einflussgroessen bestehende unbeobachtete Heterogenitaet wird eine Normalverteilungsannahme getroffen. Die Maximum-Likelihood-Schaetzung wird mittels des Newton-Raphson-Verfahrens durchgefuehrt, die noetige Integralapproximation erfolgt ueber die Gauss-Hermite-Quadraturtechnik. Mit der vorgestellten Methode werden Daten von 476 Patienten einer Hirntumorstudie ausgewertet

Binding of His-tagged fluorophores to lipid bilayers and giant vesicles

His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the biofunctionalization of membranes and vesicles. To measure the coverage by the membrane-bound molecules, it is useful to study molecules that are fluorescent as well. Here, we use two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescin isothiocyanate (FITC), both of which are tagged with a chain of six histidines that bind to achor lipids within the bilayers. This His-tag is much smaller in size than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. Several protocols for the preparation of GUVs have been developed. We apply and compare three well-established protocols based on polyvinyl alcohol (PVA) hydrogel swelling, electroformation on platinum wires, and electroformation on indium tin oxide (ITO) glass. For the same nanomolar concentration in the exterior solution, the coverage by His-tagged FITC is much lower than the one by His-tagged GFP. However, for both GFP and FITC, we find that the binding of the His-tagged molecules to the anchor lipids depends strongly on the preparation method. The highest binding affinitiy is obtained for electroformation on platinum wires. PVA gel swelling gives rise to a somewhat smaller binding affinity whereas electroformation on ITO glass leads to essentially no binding. Furthermore, the binding affinitiy is also observed to depend on the pH of the aqueous solution, with a relatively weak and strong pH-dependence for His-tagged GFP and His-tagged FITC, respectively

Integrin α_IIbβ₃ activation and clustering in minimal synthetic cells

Platelet adhesion and activation are mediated by integrin αIIbβ3 clustering, which is crucial for the hemostatic function of platelets. In an activated state, integrins provide the connection between the extracellular matrix and the actin cytoskeleton through a variety of cytoplasmic proteins, such as talin. Here, droplet-based microfluidics is applied to generate cell-sized giant unilamellar vesicles (GUVs) with a defined molecular composition to quantify the adhesion of integrin αIIbβ3-containing protocells in relation to the number of integrin–talin head domain (THD) complexes. Furthermore, it is shown that THD induces integrin clustering in protocells adhering to fibrinogen. The formation of this molecular link, which has, so far, only been observed in vivo, is an essential step in synthetic cell design to recapitulate integrin-mediated bidirectional signaling across the membrane. These results pave the way for further quantitative investigations of protein–protein interactions between integrins and associated proteins and their assembly within such defined, but complex, synthetic cells. An essential future step to mimic the complex interaction between cells and their environment will be to combine synthetic approaches with peptide chemistry to guide the molecular mechanisms involved in integrin binding and activation

Biomarkers in melanoma

Biomarkers are tumour- or host-related factors that correlate with tumour biological behaviour and patient prognosis. High-throughput analytical techniques--DNA and RNA microarrays--have identified numerous possible biomarkers, but their relevance to melanoma progression, clinical outcome and the selection of optimal treatment strategies still needs to be established. The review discusses a possible molecular basis for predictive tissue biomarkers such as melanoma thickness, ulceration and mitotic activity, and provides a list of promising new biomarkers identified from tissue microarrays that needs confirmation by independent, prospectively collected clinical data sets. In addition, common predictive serum biomarkers--lactate dehydrogenase, S100B and melanoma-inhibiting activity--as well as selected investigational serum biomarkers such as TA90IC and YKL-40 are also reviewed. A more accurate, therapeutically predictive classification of human melanomas and selection of patient populations that would profit from therapeutic interventions are among the major challenges expected to be addressed in the futur

Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion

The interaction of specifi c surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand´s spacer chemistry and length reveal signifi cant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Biomarkers in melanoma

Biomarkers are tumour- or host-related factors that correlate with tumour biological behaviour and patient prognosis. High-throughput analytical techniques—DNA and RNA microarrays—have identified numerous possible biomarkers, but their relevance to melanoma progression, clinical outcome and the selection of optimal treatment strategies still needs to be established. The review discusses a possible molecular basis for predictive tissue biomarkers such as melanoma thickness, ulceration and mitotic activity, and provides a list of promising new biomarkers identified from tissue microarrays that needs confirmation by independent, prospectively collected clinical data sets. In addition, common predictive serum biomarkers—lactate dehydrogenase, S100B and melanoma-inhibiting activity—as well as selected investigational serum biomarkers such as TA90IC and YKL-40 are also reviewed. A more accurate, therapeutically predictive classification of human melanomas and selection of patient populations that would profit from therapeutic interventions are among the major challenges expected to be addressed in the future

Sampling Local Fungal Diversity in an Undergraduate Laboratory using DNA Barcoding

Traditional methods for fungal species identification require diagnostic morphological characters and are often limited by the availability of fresh fruiting bodies and local identification resources. DNA barcoding offers an additional method of species identification and is rapidly developing as a critical tool in fungal taxonomy. As an exercise in an undergraduate biology course, we identified 9 specimens collected from the Hendrix College campus in Conway, Arkansas, USA to the genus or species level using morphology. We report that DNA barcoding targeting the internal transcribed spacer (ITS) region supported several of our taxonomic determinations and we were able to contribute 5 ITS sequences to GenBank that were supported by vouchered collection information. We suggest that small-scale barcoding projects are possible and that they have value for documenting fungal diversity

Palladium nanoparticles by electrospinning from poly(acrylonitrile-co-acrylic acid)-PdCl2 solutions. Relations between preparation conditions, particle size, and catalytic activity

Catalytic palladium (Pd) nanoparticles on electrospun copolymers of acrylonitrile and acrylic acid (PAN-AA) mats were produced via reduction of PdCl2 with hydrazine. Fiber mats were electrospun from homogeneous solutions of PAN-AA and PdCl2 in dimethylformamide (DMF). Pd cations were reduced to Pd metals when fiber mats were treated in an aqueous hydrazine solution at room temperature. Pd atoms nucleate and form small crystallites whose sizes were estimated from the peak broadening of X-ray diffraction peaks. Two to four crystallites adhere together and form agglomerates. Agglomerate sizes and fiber diameters were determined by scanning and transmission electron microscopy. Spherical Pd nanoparticles were dispersed homogeneously on the electrospun nanofibers. The effects of copolymer composition and amount of PdCl2 on particle size were investigated. Pd particle size mainly depends on the amount of acrylic acid functional groups and PdCl2 concentration in the spinning solution. Increasing acrylic acid concentration on polymer chains leads to larger Pd nanoparticles. In addition, Pd particle size becomes larger with increasing PdCl2 concentration in the spinning solution. Hence, it is possible to tune the number density and the size of metal nanoparticles. The catalytic activity of the Pd nanoparticles in electrospun mats was determined by selective hydrogenation of dehydrolinalool (3,7-dimethyloct-6- ene-1-yne-3-ol, DHL) in toluene at 90 °C. Electrospun fibers with Pd particles have 4.5 times higher catalytic activity than the current Pd/Al2O3 catalyst

Moving lessons: teaching sociology through embodied learning in the HE classroom

This chapter outlines an approach to classroom teaching that makes use of physical movement alongside more traditional lecturing methods when delivering lessons on abstract theoretical material. It develops the notion of embodied learning as a 'physical metaphor', outlining some examples of this practice that we have used in our recent work with a class of first year undergraduates. We argue that conceptualising students as embodied subjects, whose capacity to learn extends through and beyond their physical selves, educators are able to enhance classroom delivery by diversifying teaching activities and creating opportunities for enjoyable and memorable learning experiences. We advocate the reflexive, contextually-sensitive and level- appropriate use of this method, arguing that despite some limitations it can animate students' understanding of academic ideas in uniquely personalised ways

Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the “first come – first serve” principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells