Reproductive diseases, mainly those resulting in the infertility affect the chances of human being to reproduce. On the contrary, the heart disease, cancer and degenerative diseases currently account for majority of deaths in the world. Usually, these lifestyle diseases need longer lifespan to become the cause of death. The proteins secreted by cells carry important information about the cell's well-being, as well as about the condition of the tissues formed by these cells. Once secreted, these proteins may also be transferred throughout the body by means of body fluids, many of which are easily accessible for further 'in-depth' studies. Cellular and secreted proteins are often a focus of studies using proteomic means and the revelation of protein alterations can lead us to new ideas about the molecular mechanisms of diseases as well as possible identification of proteins that may be used as new targets for pharmaceutical intervention or molecules that could be used for diagnostic or prognostic purposes. Taking into consideration the above aspects, this research was undertaken to find proteins that could: (a) characterise the human follicular fluid as microenvironment of the maturing oocyte, to increase understanding of reproductive processes to improve the techniques of assisted repro- duction;...

Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...

Der Bedarf an modernen Konzepten der regenerativen Medizin für die Therapie von Knochensubstanzdefekten steigt zunehmend angesichts unserer sich demographisch wandeln-den Gesellschaft und der damit einhergehenden, steigenden Zahl altersrelevanter orthopädisch-unfallchirurgischer Erkrankungen. Die wissenschaftlichen Erkenntnisse der letzten Jahrzehnte erlauben es, grundlegende biologische Prozesse der Knochenregeneration nicht nur besser zu verstehen, sondern diese auch durch gezielte Einflussnahme zu nutzen. Auf Grundlage dieser Erkenntnisse fokussiert die Forschung die Entwicklung moderner bioaktiver Biomaterialien mit dem intrinsischen Potenzial, die körpereigene Geweberegeneration lokal im Defektbereich – in situ – zu stimulieren. Die Stimulation zellulärer Regenerationsmechanismen kann entweder direkt durch Zell-Material-Interaktion induziert werden (in situ Regeneration), oder durch chemotaktische Attraktion von Zellen mit regenerativem Potenzial aus dem umliegenden Gewebe, welche ihrerseits die Geweberegeneration induzieren (in situ Tissue Engineering). Ohne extrakorporale Besiedlung der Scaffolds und deren in vitro-Kultivierung vor der Implantation umgehen diese in situ-Strategien mehrere Limitationen und Herausforderungen des klassischen Tissue Engineering-Konzepts. Dem in situ-Konzept folgend wurden im Rahmen dieser Dissertation zwei Strategien zur gezielten Funktionalisierung eines Knochenersatzmaterials aus mineralisiertem Kollagen unter-sucht: I) Zum einen wurde mineralisiertes Kollagen mit dem osteoanabol wirksamen Erdalkalimetall Strontium modifiziert, um lokal die Osteogenese zu stimulieren. II) Zum anderen wurden poröse Scaffolds aus mineralisiertem Kollagen mit einem zentralen Depot funktionalisiert, welches mit einem Wirkstoffgemisch beladen wurde, welches aus dem Sekretom Hypoxie-konditionierter hBMSC (HCM) generiert wurde. Durch gezielte Attraktion von Zellen mit regenerativem Potenzial und gleichzeitiger Stimulation der Vaskularisierung soll dieses Scaffoldsystem gezielt die Knochendefektheilung induzieren. Für die Strontium-Modifikation wurde während der Scaffoldsynthese das Calcium der Mineralphase sukzessive durch Strontium substituiert und die hergestellten Scaffolds eingehend materialwissenschaftlich charakterisiert (Publikation 2.1; Quade et al., 2018a). Die simultane Fibrillierung und Mineralisierung von Kollagen führte zur Bildung von Nanokompositen, wobei die Mineralphasen von nanokristallinem Hydroxylapatit (Sr0), über schwach kristalline Strontium-reiche Phasen zu einer gemischten Mineralphase (Sr100) aus amorphem Strontiumphosphat und hochkristallinem Strontiumhydroxylapatit verschoben wurde. Freisetzungsversu-che über 28 Tage zeigten, dass die getesteten Varianten Sr50 und Sr100 anhaltend Sr2+-Ionen in einem Konzentrationsbereich freisetzten, in dem sowohl die Knochenneubildung stimuliert, als auch die zelluläre Knochenresorption gehemmt wird. In vitro zeigte sich der osteoanabole Effekt Strontium-modifizierter mineralisierter Kollagenscaffolds durch eine signifikant gesteigerte Proliferation und osteogene Differenzierung von hBMSC. In vivo – als Knochenersatzmaterial im murinen segmentalen FemurdefektModell – zeigten Strontium-modifizierte Scaffolds aus mineralisiertem Kollagen zwar ein tendenziell gesteigertes Knochenvolumen mit erhöhter Osteoblastenzahl, signifikant weniger Osteoklasten und signifikant gesteigerter Vaskularisierung, jedoch war der Effekt verhältnismäßig schwach und allein nicht ausreichend für eine knöcherne Überbrückung des Defektbereiches (Publikation 2.2; Quade et al., 2020a). Durch Kombination von Sr-Modifikation und BMP-2-Funktionalisierung konnte die Qualität des neugebildeten Knochens signifikant gesteigert werden. Um das Konzept des neuartigen Wirkstoffdepot-basierten Scaffoldsystems zu testen, wurde das zentrale Depot zunächst mit dem Modell-Wachstumsfaktor VEGF beladen. Der Einsatz der Biopolymere Alginat, Hyaluronsäure und Heparin als Depotbildner erlaubt die Modulation der Wirkstofffreisetzung. Während die Hydrogele Alginat und Hyaluronsäure dabei als physikalischen Barriere fungieren, ermöglichte die hohe ionische Bindungsaffinität von Heparin und VEGF dessen nahezu lineare Freisetzung über den Versuchszeitraum von 28 Tagen. Im Migrationsversuch bewirkte die retardierte VEGF-Freisetzung und damit die Stabilisierung des Wirkstoffgradienten die gerichtete Migration von HDMEC in den Scaffold. Je verzögerter die VEGF-Freisetzung – und damit je steiler der Wirkstoffgradient – desto tiefer migrierten HDMEC in die Scaffolds (Publikation 2.3; Quade et al., 2017a). Entscheidend für eine effiziente Knochenregeneration ist sowohl die Attraktion von Zellen mit regenerativem Potenzial, als auch die Stimulation der Vaskularisierung, um den Gasaustausch, die Nähstoffversorgung und den Abtransport metabolischer Nebenprodukte der Zel-len im Defektbereich zu gewährleisten. Im Sinne des in situ Tissue Engineering-Konzepts wurde das zentrale Wirkstoffdepot mit einem natürliche Wirkstoffgemisch, welches aus dem Sekretom Hypoxie-konditionierter hBMSC (HCM) gewonnen wurde, beladen (Publikation 2.4; (Quade et al., 2020b). Unter hypoxischen Bedingungen sezernieren hBMSC einen Wirkstoffcocktail, der unter anderem Wachstumsfaktoren, Chemokine, Hormone und Exosomen enthält und ein starkes angiogenes und chemotaktisches Potenzial gegenüber hBMSC zeigt. Um das Wirkstoffdepot möglichst effizient zu beladen, wurde zum einen die Wirkstoffausbeute von HCM durch Anpassung der Herstellungsparameter optimiert. Gemessen am Wachstumsfaktor VEGF konnte so die Ausbeute bis zu 100-fach gesteigert werden. Des Weiteren konnte durch Dialyse, Gefriertrocknung und Resuspension mit dem Depot-bildenden Biopolymer zusätzlich eine bis zu 50-fache Konzentrierung des Wirkstoffgemisches erreicht wer-den ohne Verlust der Bioaktivität. Mit steigender HCM-Konzentration im Depot konnte sowohl eine tiefere Migration von hBMSC, als auch eine Verbesserung der Angiogenese erzielt wer-den. Zusätzlich bewirkte die retardierte HCM-Freisetzung aus Alginat-basierten Depots eine signifikante Steigerung von Länge, Dichte und Einsprosstiefe prävaskulärer Strukturen. Zwar ist die Charakterisierung und standardisierte Herstellung des natürlichen HCM-basierten Wirkstoffgemisches eine Herausforderung, jedoch ist davon auszugehen, dass dessen be-deutendes therapeutisches Potenzial gerade durch die Komplexität der sezernierten Faktoren und deren synergistisches Zusammenspiel bedingt wird. Die Standardisierung der HCM-basierten Wirkstoffherstellung, sowie eine bessere Quantifizierung und Charakterisierung der sezernierten Proteine sollte in zukünftigen Studien forciert werden. Beide Strategien, die im Rahmen dieser Dissertation untersucht wurden, haben das Potenzial, als zellfreie „ready-to-use“-Knochenersatzmaterialien zu erschwinglichen Kosten, bei geringerer regulatorischer Komplexität und mit gleichbleibend hoher Qualität produziert zu wer-den. Während Strontium-modifiziertes mineralisiertes Kollagen allein in vivo nur ein schwaches osteoinduktives Potenzial zeigte, konnte die Qualität und Festigkeit des neugebildeten Knochengewebes in Kombination mit BMP-2 signifikant verbessert werden. Als leicht-osteogenes Biomaterial könnte dieses zur Unterstützung etablierter therapeutischer Konzepte eingesetzt werden – vor allem bei Patienten mit systemischen Knochenerkrankungen wie beispielsweise Osteoporose. Das komplexere Depot-basierte Scaffoldsystem hingegen hat ein großes Potenzial für die klinische Translation. Zum einen kann das Depot je nach Bedarf mit verschiedensten Wirkstoffen beladen werden, deren Freisetzung in Abhängigkeit des Depot-bildenden Biopolymers manipuliert werden kann. Beladen mit dem natürlichen HCM-basierten Wirkstoffgemisch zeigte das Scaffoldsystem ein beeindruckendes chemotaktisches und angiogenes Potenzial. Gegenüber etablierten rhBMP-2-Applikationen, stellt dieser in situ Tissue Engineering-Ansatz damit eine vielversprechende Alternative für die Knochen-defekt-Therapie dar, bei gleichzeitig deutlich reduzierten Kosten und Nebenwirkungen. Zu-künftige in vivo-Studien im Großtiermodell sollten das regenerative Potenzial des Depot-basierten Scaffoldsystems umfassend verifizieren. / Our demographically changing society causes a rising number of age-related orthopaedic and trauma surgical diseases. Modern approaches following the concept of regenerative medicine are needed for the therapeutical treatment of bone defects. Scientific findings of the last decades not only allow for a better understanding of fundamental biological processes in the field of bone regeneration, but also to use this knowledge for effective therapeutic concepts. Therefore, research is focusing on the development of modern bioactive biomaterials with the intrinsic potential to locally stimulate the body's own regeneration capacity - in situ. The stimulation of tissue regeneration can either be induced directly by cell-material interaction (in situ regeneration), or by chemotactic attraction of cells with regenerative potential from the surrounding tissue, which would in turn induce local tissue regeneration (in situ tissue engineering). Since these in situ strategies forgo the extracorporeal seeding and in vitro cultivation of scaffolds prior implantation, several limitations and challenges of the classical tissue engineering concept can be circumvented. Within the scope of this dissertation two strategies were investigated. Following the in situ concept, scaffolds based on mineralized collagen were specifically functionalized in order to locally induce bone defect healing: I) On the one hand, mineralised collagen was modified with strontium to locally stimulate osteogenesis. II) On the other hand, porous scaffolds of mineralised collagen were functionalised with a central depot loaded with a cocktail of signalling factors generated from the secretome of hypoxia-conditioned hBMSC (HCM). By specifically attracting cells with regenerative potential and simultaneously stimulating vascularisation, this scaffold-system could actively induce bone defect healing. For the strontium modification, the calcium of the mineral phase was successively substituted by strontium during the scaffold synthesis. The generated scaffolds were characterised in detail from a material science perspective (publication 2.1; Quade et al.et al., 2018a). In all tested approaches simultaneous collagen fibrillation and mineralisation led to the formation of nanocomposites. With rising strontium substitution, the mineral phases shifted from nanocrystalline hydroxylapatite (Sr0), via weakly crystalline strontium-rich phases to a mixed mineral phase of amorphous strontium phosphate and highly crystalline strontium hydroxylapatite (Sr100). Release experiments showed that the scaffold variants Sr50 and Sr100 released Sr2+-ions continuously over 28 days in a range, which is known to exploit the dual effect of strontium by simultaneously promoting proliferation and osteogenic differentiation as well as inhibiting the osteoclastic bone resorption without impairing the osteoclastogenesis. In vitro, the osteoanabolic effect of strontium-modified mineralised collagen scaffolds was demonstrated by significantly increased proliferation and osteogenic differentiation of hBMSC. In vivo - in the murine segmental femoral defect model - strontium-modified scaffolds made of mineralised collagen showed a tendency to increase bone volume with an increased number of osteoblasts, significantly reduced osteoclasts and significantly increased vascularisation. However, the effect was relatively weak and not sufficient to cause a bridging of the defect area (publication 2.2; Quade et al., 2020a). By combining Sr modification and BMP-2 functionalisation, the quality of the newly formed bone was significantly improved. To test the concept of the novel depot-based scaffold system, the central depot was loaded with the model growth factor VEGF. The use of the biopolymers alginate, hyaluronic acid and heparin as depot-forming agents allowed the modulation of drug release. While the hydrogels alginate and hyaluronic acid act as a physical barrier, the high ionic binding affinity of heparin and VEGF facilitated an almost linear VEGF-release over the experimental period of 28 days. In migration experiments, the retarded VEGF release and thus the stabilisation of the VEGF- gradient caused the directed migration of HDMEC into the scaffolds. The slower the VEGF release - and thus the steeper the drug gradient - the deeper HDMEC migrated into the scaffolds (publication 2.3; Quade et al., 2017). Crucial for an efficient bone regeneration is both the attraction of cells with regenerative potential and the stimulation of vascularisation to ensure gas exchange, nutrient supply and removal of metabolic by-products in the defect area. In line with the in situ tissue engineering concept, the central depot was loaded with a natural factor mix obtained from the secretome of hypoxia-conditioned hBMSC (HCM) (publication 2.4; (Quade et al., 2020b). Under hypoxic conditions, hBMSC secrete a cocktail of active substances that contains, among others, growth factors, chemokines, hormones and exosomes. This factor mix shows a strong angiogenic potential and is highly chemo-attractive to hBMSC. In order to load the scaffold depot as efficiently as possible, the signalling factor-yield of HCM was optimised by adjusting the cultivation settings for HCM-generation. Measured by VEGF as a model growth factor, the yield was increased up to 100 times. In addition, dialysis, freeze-drying and resuspension with the depot-forming biopolymer made it possible to achieve another 50-fold concentration without loss of bioactivity. With increasing HCM-concentration in the depot, both a deeper migration of hBMSC and an improvement in angiogenesis could be achieved. In addition, the retarded release of HCM from alginate-based depots resulted in a significant increase in length, density and sprouting depth of prevascular structures. Although the characterisation and standardised production of the natural HCM-based signalling factor cocktail is challenging, it can be assumed that its significant therapeutic potential relies particularly on that complexity of the secreted factors and their synergistic interaction. The standardized production of HCM-derived signalling factor cocktails, as well as a better quantification and characterisation of the secreted proteins should be focused by future studies. Both strategies investigated in this dissertation have the potential to be produced as cell-free 'ready-to-use' bone substitute materials at affordable costs, with less regulatory complexity and with consistently high quality. While strontium-modified mineralised collagen alone showed only a weak osteoinductive potential in vivo, the quality and strength of the newly formed bone tissue was significantly improved in combination with BMP-2. This light-osteogenic biomaterial could be used to support established therapeutic concepts - especially in patients with systemic bone diseases such as osteoporosis. The more complex depot-based scaffold system on the other hand has great potential for clinical translation. Depending on the application, the depot can be loaded with a wide variety of active substances – their release kinetics in turn can be manipulated depending on the depot-forming biopolymer. Loaded with the natural HCM-derived cocktail of signalling molecules, the scaffold system showed an impressive chemotactic and angiogenic potential. Compared to established rhBMP-2 applications, this in situ tissue engineering approach represents a promising alternative for bone defect therapy, at significantly reduced costs and side effects. Future in vivo studies in large animal models should verify the regenerative potential of the herewith developed depot-based scaffold system.

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

No description available.

As previously shown, transplantation of Wharton's jelly-derived mesenchymal stem cells in the experimental model of spinal cord injury leads to the motor and sensory functions improvement, supports neuroregeneration, angiogenesis and provides immunomodulation. On the other hand, these cells have limited migration and survival capacity, and their therapeutic effect is mediated mostly by their secretome. Therefore, application of mesenchymal stem cells derived conditioned medium is studied as an alternative option for cell therapy. In this thesis, therapeutic effect of repeated intrathecal delivery of human Wharton's jelly-derived mesenchymal stem cells and their conditioned media in the treatment of spinal cord injury was compared. After induction of a balloon ischemic compression lesion, stem cells or conditioned media were administered weekly in three doses. Behavioral analyses were carried out up to nine weeks after spinal cord injury and revealed significant improvement of both treated groups compared to the untreated saline control. Application of stem cells and conditioned media also resulted in a higher amount of spared tissue and enhanced expression of genes related to neuroregeneration, although the size of glial scar was not reduced. Compared to application of stem cells, application of...

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.