Detektion von Nukleinsäuren durch postsynthetisch modifizierte Fluoreszenzsonden auf Basis photostabiler Cyaninfarbstoffe

Die Arbeit beschreibt die Synthese von 25 neuen Cyaninfarbstoffen, wobei eine große Photostabilität angestrebt wurde. Durch chemische Variation wurde die Halbwertszeit stark erhöht und zugleich eine gesteigerte Fluoreszenz erhalten. Ausgewählte Fluorophore wurden anschließend postsynthetisch an DNA gebunden und zu Energietransferpaaren kombiniert. Geeignete Paare dienten zudem in Nukleinsäuresonden der Detektion von miRNA, Verfolgung von Strangaustauschprozessen und Duplexinvasion in DNA

Consequences of climate change on resilience of the food production system in Marchfeld, Vienna Region

Das Konzept der Resilienz gewinnt an Akzeptanz und wird zunehmend in sozial-ökologischen Studien angewendet um die Anpassungsfähigkeit, die Pufferfähigkeit und die Lernfähigkeit der im System befindlichen Akteure unter sich ändernden Rahmenbedingungen zu analysieren. Im Rahmen des interdisziplinären Forschungsprojektes „Klimawandel und regionale Nachhaltigkeit“ zwischen der Universität Wien, Department für Ernährungswissenschaften und der Wirtschaftsuniversität Wien, Institut für Regional- und Umweltwirtschaft wurde die Resilienz des landwirtschaftlichen Kleinproduktionsgebietes Marchfeld in Hinblick auf den Klimawandel untersucht. Dabei wurde das integrative Nachhaltigkeitsmodell EASEY verwendet, welches ehemals für die Nachhaltigkeitsleistung börsennotierter Unternehmen entwickelt wurde. Mittels umfassender Literaturrecherche zur Lebensmittelwirtschaft im Marchfeld und auf der Basis von regelmäßig durchgeführten Gruppendiskussionen innerhalb der Forschungsgruppe wurde das EASEY Modell grundlegend an die Bedürfnisse regionaler Untersuchungen angepasst. In dieser Diplomarbeit wurde näher auf die drei Dimensionen „Prozesse“, „Anspruchsgruppen“ und „Ziele“ des EASEY Modells eingegangen und die Bezugsquellen konkretisiert. In weiterer Folge wurde ein neuer Satz von wirtschaftlichen, sozialen und ökologischen Indikatoren entwickelt, der auf die bereits vorhandenen Indikatoren des EASEY Modells zurückgreift, diese jedoch passend zur Bedarfslage durch Indikatoren aus der lokalen Datenrecherche sowie mit verhaltensbezogenen Indikatoren aus der Literatur ergänzt. Anschließend konnte die Situation der Marchfelder Lebensmittelwirtschaft anhand des Datensatzes beleuchtet und mit passenden Daten für Österreich verglichen werden. Die Ergebnisse weisen deutlich darauf hin, dass die Lebensmittelproduktion im Marchfeld profitabel, innovativ gestaltet und den wirtschaftlichen, gesellschaftlichen und ökologischen Herausforderungen gewachsen ist. Daher und aufgrund der bereits durchgeführten Anpassungsmaßnahmen (nicht zuletzt der beinahe flächendeckend vorhandenen Bewässerungssysteme) kann die Region hinsichtlich des Klimawandels derzeit als weitgehend resilient bezeichnet werden.The concept of resilience is increasingly being used to assess sustainability of social-ecological systems in case of disturbances or threats with the focus on adaptability, buffer capacity and self-organisation. In the context of an interdisciplinary research project “Climate Change and Regional Sustainability” between the University of Vienna, Department of Nutritional Sciences and the Vienna University of Economics and Business Administration, Institute for Regional- and Environmental Economics and Management, resilience of food production area Marchfeld with respect to climate change was analyzed by means of the integrative model EASEY, which was formerly developed to visualize sustainability performance of companies quoted on the stock exchanges. Via comprehensive literature research on the food system Marchfeld and frequently conducted group discussions within the project staff, the EASEY model was basically adjusted for regional assessments. Within this thesis, the single dimensions of EASEY, namely process-, stakeholder- and goal dimension were put in concrete terms. Further, a new set of economic, social and ecological indicators was developed, based on already existing indicators of EASEY, behaviour-based indicators from literature- which allow less precise metrics but comprehensive analysis – and indicators, derived from data research. By means of these indicators, situation in Marchfeld was compared with condition in Austria and then resilience of the food production area Marchfeld evaluated. Results clearly indicate that agricultural food production in Marchfeld is characterised by innovative, adaptive and profitable land-use management and can widely be termed resilient in case of climate change due to the possibility of irrigation and attributes mentioned before

A new hope? Possibilities of therapeutic IgA antibodies in the treatment of inflammatory lung diseases

Inflammatory lung diseases represent a persistent burden for patients and the global healthcare system. The combination of high morbidity, (partially) high mortality and limited innovations in the last decades, have resulted in a great demand for new therapeutics. Are therapeutic IgA antibodies possibly a new hope in the treatment of inflammatory lung diseases? Current research increasingly unravels the elementary functions of IgA as protector against infections and as modulator of overwhelming inflammation. With a focus on IgA, this review describes the pathological alterations in mucosal immunity and how they contribute to chronic inflammation in the most common inflammatory lung diseases. The current knowledge of IgA functions in the circulation, and particularly in the respiratory mucosa, are summarized. The interplay between neutrophils and IgA seems to be key in control of inflammation. In addition, the hurdles and benefits of therapeutic IgA antibodies, as well as the currently known clinically used IgA preparations are described. The data highlighted here, together with upcoming research strategies aiming at circumventing the current pitfalls in IgA research may pave the way for this promising antibody class in the application of inflammatory lung diseases

Development of a Wavelength-Shifting Fluorescent Module for the Adenosine Aptamer Using Photostable Cyanine Dyes

DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide–alkyne cycloaddition by two structurally different approaches: 1) as nucleotide modifications at the 2′-position of uridines and 2) as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications

Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids

The so-called acpcPNA system bears a peptide backbone consisting of 4′-substituted proline units with (2′R,4′R) configuration in an alternating combination with (2S)-amino-cyclopentane-(1S)-carboxylic acids. acpcPNA forms exceptionally stable hybrids with complementary DNA. We demonstrate herein (i) strand displacements by single-stranded DNA from acpcPNA-DNA hybrids, and by acpcPNA strands from DNA duplexes, and (ii) strand invasions by acpcPNA into double-stranded DNA. These processes were studied in vitro using synthetic oligonucleotides and by means of our concept of wavelength-shifting fluorescent nucleic acid probes, including fluorescence lifetime measurements that allow quantifying energy transfer efficiencies. The strand displacements of preannealed 14mer acpcPNA-7mer DNA hybrids consecutively by 10mer and 14mer DNA strands occur with rather slow kinetics but yield high fluorescence color ratios (blue:yellow or blue:red), fluorescence intensity enhancements, and energy transfer efficiencies. Furthermore, 14mer acpcPNA strands are able to invade into 30mer double-stranded DNA, remarkably with quantitative efficiency in all studied cases. These processes can also be quantified by means of fluorescence. This remarkable behavior corroborates the extraordinary versatile properties of acpcPNA. In contrast to conventional PNA systems which require 3 or more equivalents PNA, only 1.5 equivalents acpcPNA are sufficient to get efficient double duplex invasion. Invasions also take place even in the presence of 250 mM NaCl which represents an ionic strength nearly twice as high as the physiological ion concentration. These remarkable results corroborate the extraordinary properties of acpcPNA, and thus acpcPNA represents an eligible tool for biological analytics and antigene applications. © The Royal Society of Chemistry 2015

The immunomodulatory properties of the IgM- and IgA-enriched immunoglobulin preparation trimodulin

Severe infection diseases, like sepsis or sCAP, are well-known problems for public health. With the outbreak of SARS-CoV-2 and COVID-19, another infection disease with a dramatic impact on health and the economy comes into focus. Severe cases of sepsis, sCAP and COVID-19 lead to a dysregulated immune response associated with overwhelming inflammation. Modulation of the hyperinflammatory immune system is, therefore, a major goal of therapy. A promising option is the adjunctive therapy with trimodulin, an IgM- and IgA-enriched immunoglobulin preparation in clinical testing (comprising ~21 % IgM, 23 % IgA, and 56 % IgG). Available functional studies identify several modes of action, which were mainly attributed to the IgG or IgM component. In contrast, the role of IgA in these preparations was not unraveled yet. This thesis aim is to investigate the immunomodulatory properties of trimodulin in the context of severe infection diseases. A specific focus was set on the identification of functional roles for the additional IgA component. First, a cellular model system based on neutrophil-like HL-60 cells was established and comprehensively characterized. The induction of inflammation by bacterial stimuli (LPS and S.aureus) or by SARS-CoV-2-like particles was investigated and the underlying mechanism (FcR dependency, signaling pathways) identified. Next, anti-inflammatory effects of trimodulin on resting cells, as well as on inflammatory activated cells were analyzed. The following five synergistic mechanisms for immunomodulation by trimodulin were shown: (1) The direct binding of pro-inflammatory cytokines. (2) Targeting of ITAMi signaling by monomeric IgG and IgA species. (3) Activation of inhibitory ITIM signaling and increased FcγRIIB expression. (4) Modulation of cell phenotype by decreased FcγRIIA and FcαRI expression. (5) Displacement of immune complex and reduced phagocytosis. By comparing immunomodulatory effects to classical IVIg, trimodulin was shown to mediate stronger immunomodulation by better binding of cytokines, enhanced phenotype modulation, and activation of ITAMi signaling. Furthermore, the IgA component of trimodulin interacts with the FcαRI receptor and mediates FcαRI dependent phagocytosis of S.aureus. In conclusion, several synergistic modes of action are induced by trimodulin mediating powerful immunomodulation. Beneficial effects in comparison to IVIg can be attributed to the additional IgA component, by targeting FcαRI and inhibitory signaling. Differences in IgG subclass distribution and synergistic effects between monomeric and multimeric IgG, IgA, and IgM in trimodulin could be further reasons for improved immunomodulation by trimodulin. The in vitro models of this work depicting sepsis, sCAP and COVID-19 demonstrated potent immunomodulation of trimodulin. Nevertheless, these models are restricted by testing only one cell type and a limited number of cellular effector outcomes. Further work is necessary to extend the data set to further cell types and modes of action. Only with comprehensive knowledge about the modes of action in vitro, clinical data for sepsis, sCAP or COVID-19 patients can be supported and explained

Immunomodulation: Immunoglobulin Preparations Suppress Hyperinflammation in a COVID-19 Model via FcγRIIA and FcαRI

The rapid spread of SARS-CoV-2 has induced a global pandemic. Severe forms of COVID-19 are characterized by dysregulated immune response and “cytokine storm”. The role of IgG and IgM antibodies in COVID-19 pathology is reasonably well studied, whereas IgA is neglected. To improve clinical outcome of patients, immune modulatory drugs appear to be beneficial. Such drugs include intravenous immunoglobulin preparations, which were successfully tested in severe COVID-19 patients. Here we established a versatile in vitro model to study inflammatory as well as anti-inflammatory processes by therapeutic human immunoglobulins. We dissect the inflammatory activation on neutrophil-like HL60 cells, using an immune complex consisting of latex beads coated with spike protein of SARS-CoV-2 and opsonized with specific immunoglobulins from convalescent plasma. Our data clarifies the role of Fc-receptor-dependent phagocytosis via IgA-FcαRI and IgG-FcγR for COVID-19 disease followed by cytokine release. We show that COVID-19 associated inflammation could be reduced by addition of human immunoglobulin preparations (IVIG and trimodulin), while trimodulin elicits stronger immune modulation by more powerful ITAMi signaling. Besides IgG, the IgA component of trimodulin in particular, is of functional relevance for immune modulation in this assay setup, highlighting the need to study IgA mediated immune response