Cellular and Molecular Characterisation of Novel Lipopeptides with Anit-Myeloma Activities

The pleiotropic cytokine interleukin-6 (IL-6) is one of the major growth factors for multiple myeloma cells. It was previously shown that IL-6 induces the activation of Src family kinases Hck, Lyn and Fyn and that Hck is associated with the IL-6-receptor beta chain (gp130) via an acidic domain in gp130. In the first part of this thesis the acidic domain is narrowed down from 41 to 18 amino acids by a peptide-based functional screening assay. A derivative of the acidic domain, an 18mer lipopeptide, peptide 18AD, is characterised on the cellular and molecular level. IL-6-dependent growth of human and murine myeloma cells is inhibited with an IC50 of 25-30 µM by the addition of peptide 18AD to the growth medium. These cells remain unaffected by treatment with a control peptide with scrambled sequence (18sc). Furthermore, growth of IL-6-independent myeloma cells is not inhibited by peptide 18AD. In IL-6-dependent cells, peptide 18AD causes the same degree of apoptosis induction as IL-6 deprivation. On the molecular level it is shown by peptide competition assays that the association of Hck and gp130 is inhibited by peptide 18AD in a concentration dependent way. Peptide 18AD blocked the IL-6-induced activity of the Src family kinases Hck, Lyn and Fyn. Results from different factor-dependent cell lines demonstrate a common mechanism of the molecular peptide effects on Src family kinase activity, but the involved pathways downstream of the kinases appear to differ among species and cell lines tested. Treatment of human IL-6-dependent myeloma cells with peptide 18AD reduced the activating tyrosine phosphorylation of the signal transducer and activator of transcription 3 (STAT3), while STAT3 activation remains unaffected in murine cells. The Co-immunoprecipitations from different overexpressed receptor-deletionmutants and Hck confirms that the association is mainly due to the interaction between the kinase and a 9 amino acids spanning region within the acidic domain which carries the highest accumulation of acidic residues. Apparently, a sequence of 8-9 amino acids within gp130 with the prevalence of acidic side chains resembles a potential pseudosubstrate domain of tyrosine kinases and is responsible for localisation and activation of Src family kinases in response to receptor stimulation. The identification of sequence motives similar to the acidic domain of gp130 in other cytokine receptors, receptor tyrosine kinases and adapter proteins by an in silicio motive scan might suggest a general role of such motives. This could be the efficient recruitment of cytoplasmic kinases to signalling complexes at the time of ligand stimulation. Here, the importance of the acidic domain-kinase interaction for the IL-6-signaling pathway is shown by the growth-inhibiting effect of peptide 18AD on myeloma cells. In the second part, a novel sequence and celltype-specific anti-myeloma agent, peptide 1A, is characterised. It was initially designed as the negative control for a "reverse alanine scan" to define the role of the acidic residues within the sequence of peptide 18AD. Unexpectedly it turned out to be at least a 25-fold more potent growth inhibitor of myeloma cells than peptide 18AD. Similar molecular bases of the observed effects of peptide 18AD and peptide 1A are very unlikely, because in contrast to peptide 18AD, peptide 1A efficiently kills IL-6-independent myeloma cells as well. Moreover, an excess of IL-6 fails to rescue the cells from peptide 1A-induced cell death. Peptide 1A shows, as tested so far, no effects on cells derived from non-tumor tissue or tumor cells of various origins other than multiple myeloma. Peptide 1A specifically kills myeloma cells by the induction of apoptosis and severely disturbs cell cycle progression. Apoptotic cell death induction by peptide 1A is shown by peptide 1A-induced caspase-3 activation and the cleavage of PARP, a substrate of effector caspases. Peptide-induced cell death can partly be inhibited by co-treatment with the pan-caspase inhibitor ZVAD-fmk. Major survival pathways like the STAT3, PI3K/Akt and the MAPK pathway are inhibited in peptide 1A treated cells. If a biotinylated version of the peptide is added to the growth medium, it is incorporated into human myeloma cells and localised in defined regions of the cytoplasm of myeloma cells. Here some of the molecular mechanisms of peptide 1A-induced cell death are elucidated. However, the direct molecular target(s) are still unknown. Despite the potential difference in the molecular mechanisms, both peptides 18AD and 1A are expected to prove useful for developing derivatives with possible applications for the treatment of multiple myeloma

Prävention genotoxischer und karzinogener Effekte kosmischer Strahlung durch den Radioprotektor Ortho-Phospho-L-Tyrosin

Zum Schutz vor Zellschädigung durch Strahlenexposition in Umwelt und Beruf, wie etwa in der Raumfahrt, werden radioprotektiv wirksame Substanzen gesucht, wobei der selektive Radioprotektor Ortho-Phospho-L-Tyrosin (pTyr), ein Aminosäurederivat aus der Sojabohne, einen vielversprechenden Kandidaten darstellt. In dieser Arbeit konnte gezeigt werden, dass pTyr sowohl bei niederenergetischer γ-Strahlung als auch bei hochenergetischer α-Teilchen-Strahlung das Zellüberleben verbessert, indem es bereits vor Schädigung der DNA nötige Reparaturproteinkaskaden initiiert und den Zellmetabolismus kurzfristig ändert, sodass die Zellen optimal auf die strahleninduzierte Schädigung des Erbguts vorbereitet sind und diese schneller und effizienter reparieren können. Somit spricht vieles dafür, dass pTyr präventiv eingenommen einen Schutz gegen verschiedene Arten von Strahlung bieten kann, denen man im (Arbeits-) Alltag ungewollt ausgesetzt ist

Smoking cessation in workplace setting: quit rates and determinants in a group behaviour therapy programme

To capitalise on the opportunities that the smoking ban legislation in Switzerland offers for the prevention of tobacco-related diseases, a smoking cessation programme in a workplace setting was developed and implemented in companies across the language and cultural regions of Switzerland. Our goal was to identify factors associated with relapse into smoking that may be overcome during training sessions or that should be considered for the optimisation of future interventions.; Between 2006 and 2012, 1287 smokers aged 16 to 68 years voluntarily attended smoking cessation training at their workplace. The intervention was based on a cognitive behavioural group therapy combined with individual proactive telephone counselling. The evaluation consisted of three anonymised questionnaires (pre- and postintervention, and 12-month follow-up). In this prospective cohort study, we investigated the association of smoking quit rates with training and participant characteristics, including withdrawal symptoms, by use of multilevel logistic regression analysis with a random intercept for training courses.; The self-reported abstinence rate was 72.4% at the end of the training, and 18.6% 1 year later. The risk of relapse during the training was positively associated with the number of years and daily cigarettes smoked, and negatively with increased appetite, sleeping troubles and satisfaction with learned techniques. Failed abstinence within the first year was associated with younger age, higher numbers of daily smoked cigarettes and unsuccessful recent quit attempts.; Our evaluation suggests that younger and more addicted smokers attending smoking cessation trainings may need additional support to achieve long lasting abstinence rates. Offering smoking cessation training in a workplace setting can achieve reasonable long-term quit rates, but a subset of employees needs additional support at the group or personal level.; Group behaviour therapy could be an effective method to achieve long-term smoking abstinence. The workplace is an important setting to reach and encourage a large number of smokers to participate in a smoking cessation programme, but only few studies investigated its potential. The findings of this study of a modern real-life workplace-based smoking cessation programme endorse the effectiveness and viability of cognitive behavioural group therapy. This group-level intervention at the workplace may be insufficient for young and heavy smokers, as well as women with increased appetite after cessation, who potentially benefit from re-intervention and additional individual level counselling

Noninvasive Analysis of Synthetic and Decellularized Scaffolds for Heart Valve Tissue Engineering

Microcomputed tomography (mu-CT) is a nondestructive, high-resolution, three-dimensional method of analyzing objects. The aim of this study was to evaluate the feasibility of using mu-CT as a noninvasive method of evaluation for tissue-engineering applications. The polyurethane aortic heart valve scaffold was produced using a spraying technique. Cryopreserved/thawed homograft and biological heart valve were decellularized using a detergent mixture. Human endothelial cells and fibroblasts were derived from saphenous vein segments and were verified by immunocytochemistry. Heart valves were initially seeded with fibroblasts followed by colonization with endothelial cells. Scaffolds were scanned by a mu-CT scanner before and after decellularization as well as after cell seeding. Successful colonization was additionally determined by scanning electron microscopy (SEM) and immunohistochemistry (IHC). Microcomputed tomography accurately visualized the complex geometry of heart valves. Moreover, an increase in the total volume and wall thickness as well as a decrease in total surface was demonstrated after seeding. A confluent cell distribution on the heart valves after seeding was confirmed by SEM and IHC. We conclude that mu-CT is a new promising noninvasive method for qualitative and quantitative analysis of tissue-engineering processes. ASAIO Journal 2013;59:169-177

Microfluidic Synthesis of Cell-Type-Specific Artificial Extracellular Matrix Hydrogels

Droplet microfluidic technology is applied for the high-throughput synthesis via Michael-type addition of reactive, micrometer-sized poly(ethylene glycol) (PEG) hydrogels (“microgels”) with precisely controlled dimension and physicochemical properties. A versatile chemical scheme is used to modify the reactive PEG microgels with tethered biomolecules to tune their bioactive properties for the bioreactor culture and manipulation of various (stem) cell types

Micropatterning neuronal networks

Spatially organised neuronal networks have wide reaching applications, including fundamental research, toxicology testing, pharmaceutical screening and the realisation of neuronal implant interfaces. Despite the large number of methods catalogued in the literature there remains the need to identify a method that delivers high pattern compliance, long-term stability and is widely accessible to neuroscientists. In this comparative study, aminated (polylysine/polyornithine and aminosilanes) and cytophobic (poly(ethylene glycol) (PEG) and methylated) material contrasts were evaluated. Backfilling plasma stencilled PEGylated substrates with polylysine does not produce good material contrasts, whereas polylysine patterned on methylated substrates becomes mobilised by agents in the cell culture media which results in rapid pattern decay. Aminosilanes, polylysine substitutes, are prone to hydrolysis and the chemistries prove challenging to master. Instead, the stable coupling between polylysine and PLL-g-PEG can be exploited: Microcontact printing polylysine onto a PLL-g-PEG coated glass substrate provides a simple means to produce microstructured networks of primary neurons that have superior pattern compliance during long term (>1 month) cultur

Antimicrobial susceptibility in E. coli and Pasteurellaceae at the beginning and at the end of the fattening process in veal calves: Comparing 'outdoor veal calf' and conventional operations.

Animal husbandry requires practical measures to limit antimicrobial resistance (AMR). Therefore, a novel management and housing concept for veal calf fattening was implemented on 19 intervention farms (IF) and evaluated regarding its effects on AMR in Escherichia (E.) coli, Pasteurella (P.) multocida and Mannheimia (M.) haemolytica in comparison with 19 conventional control farms (CF). Treatment intensity (-80%) and mortality (-50%) were significantly lower in IF than in CF, however, production parameters did not differ significantly between groups. Rectal and nasopharyngeal swabs were taken at the beginning and the end of the fattening period. Susceptibility testing by determination of the minimum inhibitory concentration was performed on 5420 isolates. The presence of AMR was described as prevalence of resistant isolates (%), by calculating the Antimicrobial Resistance Index (ARI: number of resistance of one isolate to single drugs/total number of drugs tested), by the occurrence of pansusceptible isolates (susceptible to all tested drugs, ARI=0), and by calculating the prevalence of multidrug (≥3) resistant isolates (MDR). Before slaughter, odds for carrying pansusceptible E. coli were higher in IF than in CF (+65%, p=0.022), whereas ARI was lower (-16%, p=0.003), and MDR isolates were less prevalent (-65%, p=0.001). For P. multocida, odds for carrying pansusceptible isolates were higher in IF before slaughter compared to CF (+990%, p=0.009). No differences between IF and CF were seen regarding the prevalence of pansuceptible M. haemolytica. These findings indicate that easy-to-implement measures to improve calf management can lead to a limitation of AMR in Swiss veal fattening farms

Effect of temporal onsets of mechanical loading on bone formation inside a tissue engineering scaffold combined with cell therapy.

Several approaches to combine bone substitutes with biomolecules, cells or mechanical loading have been explored as an alternative to the limitation and risk-related bone auto- and allo-grafts. In particular, human bone progenitor cells seeded in porous poly(L-lactic acid)/tricalcium phosphate scaffolds have shown promising results. Furthermore, the application of mechanical loading has long been known to be a key player in the regulation of bone architecture and mechanical properties. Several in vivo studies have pointed out the importance of its temporal offset. When an early mechanical loading was applied a few days after scaffold implantation, it was ineffective on bone formation, whereas a delayed mechanical loading of several weeks was beneficial for bone tissue regeneration. No information is reported to date on the effectiveness of applying a mechanical loading in vivo on cell-seeded scaffold with respect to bone formation in a bone site. In our study, we were interested in human bone progenitor cells due to their low immunogenicity, sensitivity to mechanical loading and capacity to differentiate into osteogenic human bone progenitor cells. The latest capacity allowed us to test two different bone cell fates originating from the same cell type. Therefore, the general aim of this study was to assess the outcome on bone formation when human bone progenitor cells or pre-differentiated osteogenic human bone progenitor cells are combined with early and delayed mechanical loading inside bone tissue engineering scaffolds. Scaffolds without cells, named cell-free scaffold, were used as control. Surprisingly, we found that (1) the optimal solution for bone formation is the combination of cell-free scaffolds and delayed mechanical loading and that (2) the timing of the mechanical application is crucial and dependent on the cell type inside the implanted scaffolds

Datenerhebung pandemiebedingte, kostenlose Mahlzeiten-, Lebensmittel- und Gutscheinabgaben in der Stadt Zürich

Qualitative SozialforschungAusgangslage Die Covid-19-Pandemie und die damit einhergehenden Massnahmen treffen verschiedene Bevölkerungskreise hart, zahlreiche öffentliche und private Institutionen leisten Unterstützung, wozu auch die Abgabe von Lebensmittelgutscheinen und verbilligten oder kostenlosen Nahrungsmitteln und Mahlzeiten gehört. Während dem Lockdown 2020 entstanden nebst den Angeboten der bisherigen Anbieter, die ihre Leistungen teilweise diversifizierten und stark ausbauten, auch neue Angebote mit Lebensmittelabgabe, die rasch grossen Zulauf erhielten. Gleichzeitig fehlte das Wissen darüber, wer diese Nutzer*innen sind und durch welche kurz- und längerfristigen Bedarfe sie sich auszeichnen. Vor diesem Hintergrund beauftragte das Sozialdepartement Stadt Zürich die ZHAW-Soziale Arbeit damit, diese Wissenslücken im Rahmen einer systematischen Untersuchung möglichst zu beheben. Ziele der Untersuchung Das Ziel der Untersuchung bestand in einer empirisch fundierten Einschätzung aktueller und künftiger Herausforderungen im Hinblick auf die Nahrungsmittelabgabe durch verschiedene Anbieter. Dabei galt es insbesondere zu klären, welche unterschiedlichen Problemkonstellationen zum Bezug von Nahrungsmitteln führen und wie sich die damit einhergehenden Bedarfe kurz- und längerfristig entwickeln werden. Design und Methode Die Untersuchung gliederte sich in drei Phasen. In einer ersten Phase dienten Gespräche mit Geschäftsleitenden sowie Mitarbeitenden der involvierten Stellen (N=35) dazu, die wichtigsten Problemkonstellationen zu identifizieren und zu typisieren. Die zweite Phase galt der Schärfung und detaillierten Beschreibung der einzelnen Problemkonstellationen (in Bezug auf Lebenssituationen, Bedarfe, Nutzungsstrategien, Dynamiken und Perspektiven). Dazu dienten 36 Interviews mit betroffenen Personen aus den verschiedenen Problemkonstellationen sowie bereits vorhandene Daten der Anbieter. In einer dritten Projektphase wurden die Ergebnisse integriert, interpretiert und an einem Workshop mit Vertretenden aus den beteiligten Organisationen sowie der Auftraggeberin validiert. Ergebnisse In der Untersuchung liessen sich neun unterschiedliche Problemkonstellationen identifizieren, die sich im Wesentlichen entlang von zwei Dimensionen bestimmen lassen, der finanziellen Situation bzw. sozialversicherungsrechtlicher Absicherung und des ausländerrechtlichen Aufenthaltsstatus: 1. «Klassische Randständige» mit psychischen Erkrankungen und/oder einer Suchtproblematik und teilweise obdachlos; in der Regel mit sozialhilfe- oder sozialversicherungsrechtlicher Unter-stützung 2. Ältere, einsame Menschen, in der Regel mit sozialhilfe- oder sozialversicherungsrechtlicher Unterstützung 3. Sozialhilfebezüger*innen 4. Armutsbetroffene ohne Sozialhilfebezug 5. Beziehende von Asylfürsorge 6. Wanderarbeiter*innen und sogenannte «Europawander*innen» ohne Anspruch auf sozialhilfe- oder sozialversicherungsrechtliche Unterstützung 7. Sexarbeiter*innen, teilweise mit, teilweise ohne Anspruch auf sozialhilfe- oder sozialversiche-rungsrechtliche Unterstützung 8. Beziehende von asylrechtlicher Nothilfe 9. Sans-Papiers ohne Anspruch auf sozialhilfe- oder sozialversicherungsrechtliche Unterstützun

Human bone progenitor cells for clinical application: what kind of immune reaction does fetal xenograft tissue trigger in immuno-competent rats?

The potential of human fetal bone cells for successful bone regeneration has been shown in vivo. In particular, it has been demonstrated that the seeding of these cells in porous poly-(L-lactic acid)/ ß-tricalcium phosphate scaffolds improved the bone formation compared to cell-free scaffolds in skulls of rats. However, even if the outcome is an improvement of bone formation, a thorough analysis concerning any immune responses, due to the implantation of a xenograft tissue, is not known. As the immune response and skeletal system relationship may either contribute to success or failure of an implant, we were interested in evaluating the presence of any immune cells and specific reactions of human fetal cells (also called human bone progenitor cells) once implanted in femoral condyles of rats. For this purpose (1) cell-free scaffolds, (2) human bone progenitor cells or (3) osteogenic human bone progenitor cells within scaffolds were implanted over 3, 7, 14 days and 12 weeks. The key finding is that human bone progenitor cells and osteogenic human bone progenitor cells do not trigger any particular specific immune reactions in immuno-competent rats, but are noted to delay some bone formation