Effective clinical treatments for volumetric muscle loss resulting from traumatic injury or resection of a large amount of muscle mass are not available to date. Tissue engineering may represent an alternative treatment approach. Decellularization of tissues and whole organs is a recently introduced platform technology for creating scaffolding materials for tissue engineering and regenerative medicine. The muscle stem cell niche is composed of a three-dimensional architecture of fibrous proteins, proteoglycans, and glycosaminoglycans, synthesized by the resident cells that form an intricate extracellular matrix (ECM) network in equilibrium with the surrounding cells and growth factors. A consistent body of evidence indicates that ECM proteins regulate stem cell differentiation and renewal and are highly relevant to tissue engineering applications. The ECM also provides a supportive medium for blood or lymphatic vessels and for nerves. Thus, the ECM is the nature's ideal biological scaffold material. ECM-based bioscaffolds can be recellularized to create potentially functional constructs as a regenerative medicine strategy for organ replacement or tissue repopulation. This article reviews current strategies for the repair of damaged muscle using bioscaffolds obtained from animal ECM by decellularization of small intestinal submucosa (SIS), urinary bladder mucosa (UB), and skeletal muscle, and proposes some innovative approaches for the application of such strategies in the clinical setting.

Small proteins are difficult to study at high resolution with single-particle cryo-electron microscopy (cryo-EM). In general, sample properties such as large size (> 80 kDa), symmetry and rigidity are key to utilize this technology. To facilitate structural studies of small proteins as well, using cryo-EM, this project aims to incorporate a photo-inducible cross-link in a large and symmetric scaffold that is amenable for study, and covalently bind small proteins of interest to this scaffold. The scaffold in this project consists of rabbit muscle aldolase (157 kDa in tetrameric state) with an engineered affibody affinity protein (7 kDa) attached to the N-terminus of each aldolase monomer via a rigid helix fusion. The affibody-domain of the scaffold will be cross-linked to small proteins of structural interest, with a focus on a model target consisting of a second affibody with affinity for the affibody displayed on the aldolase scaffold. Photoconjugation of the affibody Zwt was performed to crosslink both the Fc of IgG and the anti-idiotypic affibody Z963, revealing that a methionine acceptor in the target is preferable but not necessary for UV crosslinking using BPA. Binding of affibodies rigidly displayed on of the scaffold to targets such as affibodies and antibody fragments was demonstrated , using surface plasmon resonance (SPR). / Att studera små protein vid hög upplösning med enpartikelsrekonstruktion i kryo-elektronmikroskopi (kryo-EM) är utmanande. Generellt så krävs stora (> 80 kDa), symmetriska och stabila protein för att använda sig av kryo-EM. Med målet att möjliggöra strukturbestämning och strukturella studier av små protein, så ska detta projekt föra in en foto-aktiverad korslänk i ett stort och symmetriskt bärarprotein. Bäraren består av aldolas från kaninmuskel (157 kDa som tetramer) med en affibody (7 kDa) kopplad till N-terminalen av varje aldolas-monomer via en rigidt fuserad helix. Affibody-domänen av bärarproteinet kan bilda korslänkar till små protein vars struktur sedan kan studeras. Fokus i projektet är ett modellprotein som består av en annan affibody som binder den affibody i bäraren. Fotokonjugering av affibodyn Zwt utfördes för att skapa korslänkar till både Fc av IgG, samt den anti-idiotypiska affibodyn Z963, vilket påvisade att en metionin-mottagare i målproteinet är fördelaktigt för UV korslänkning med BPA, men inte ett krav. Affinitet av affibodies i bärarproteinet till målprotein såsom andra affibodies och antikroppsfragment påvisades.

La necesidad de polímeros bioestables para fabricación de implantes protésicos queda patente, entre otros indicadores, por la proliferación de dispositivos actualmente comercializados. La caracterización físico-química así como la respuesta biológica de un conjunto de materiales poliméricos bioestables es el objetivo último de esta tesis. En este trabajo se han sintetizado diferentes materiales poliméricos de la familia de los acrilatos y metacrilatos variando sutilmente sus características superficiales, como el grado de hidrofilia o la distribución de cargas eléctricas. El procedimiento consistió en la copolimerización via radical de acrilato de etilo, EA, acrilato de 2-hidroxietilo, HEA, y ácido metacrílico, MAAc. Se ha caracterizado los materiales en estado seco y en presencia de diferentes contenidos de agua mediante calorimetría diferencial de barrido, DSC, análisis dinámico-mecánico, DMA, microscopía de fuerza atómica, AFM, análisis dieléctrico, DRS, contenido de agua en equilibrio, EWC, y energía superficial, SE, persiguiendo el objetivo de dilucidar si el agua es capaz de inducir cambios conformacionales en las cadenas poliméricas que den lugar a una separación de fases. Sobre los materiales en forma de scaffold poroso con poros esféricos interconectados se ha cultivado fibroblastos y endoteliales. La compatibilidad de las células endoteliales se midió en términos de viabilidad celular y la adecuada diferenciación endotelial y su funcionamiento. Se han realizado cultivos de células endoteliales humanas primarias, HUVEC, y se ha determinado si su morfología y función se vio afectada por el material. Se examinó la adhesión y proliferación de las mismas, así como un marcador importante de activación endotelial, la E-selectina. Se evaluó si se mantuvieron los fenotipos endoteliales normales y sus funciones observadas in vivo mediante análisis de los contactos célula-célula y la regulación de la expresión génica del marcador de activación E-selectina cuando se añadió un estímulo (LPS). Además, como posible aplicación de estos materiales en una prótesis de córnea artificial, y dado que los fibroblastos del estroma de la córnea (es decir, los queratocitos) son de relevancia en la cicatrización de la córnea se determinó cómo afectaba la hidrofilicidad del substrato a la adhesión celular de la línea de fibroblastos humanos MRC-5, como modelo celular para estudiar la disposición del citoesqueleto tras la adhesión a los diferentes soportes mediante la detección de F-actina. Asimismo, se ha sembrado células epiteliales evaluando su comportamiento/funcionamiento celular ya que uno de los requisitos esenciales para que un implante de queratoprótesis tenga éxito es que se cree y mantenga una capa de células epiteliales que impidan entrar a las bacterias al interior del ojo y permita la difusión la capa lagrimal de manera estable en el tiempo. Así, se han analizado parámetros celulares como adhesión, proliferación y viabilidad de una línea de células epiteliales de conjuntiva humana, NHC, cultivada sobre substratos poliméricos con diferentes grados de hidrofilia y cargas eléctricas superficiales buscando qué grado de hidrofilicidad permite la epitelización del substrato y podría darle al material flexibilidad y la hidrofilicidad necesaria para un mejor contacto con los párpados y lágrima. Los resultados obtenidos se han correlacionado con la adsorción y conformación de una proteína de la matriz extracelular, la fibronectina. / Campillo Fernández, AJ. (2014). Polímeros bioestables para fabricación de implantes protésicos: caracterización físico-química y respuesta biológica in vitro [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/44232 / Premios Extraordinarios de tesis doctorales

There is growing interest in the development of biomimetic matrices that are simultaneously cell-friendly, allow rapid vascularization, exhibit enough mechanical integrity to be comfortably handled and resist mechanical stresses when implanted in the site of interest. Meeting all these requirements with a single component material has proved to be very challenging. The hypothesis underlying this work was that hybrid materials obtained by combining scaffolds with bioactive hydrogels would result in a synergy of their best properties: a construct with good mechanical properties, easily handled and stable thanks to the scaffold; but also, because of the gel, cell-friendly and with enhanced oxygen and nutrients diffusion, and promoter of cell colonization. Moreover, such a composite material would also be useful as a controlled release system because of the gel’s incorporation. Poly (ethyl acrylate) (PEA) scaffolds prepared with two different morphologies were envisaged to provide the mechanical integrity to the system. Both types of scaffolds were physicochemically characterized and the effect of the scaffolds production process on the PEA properties was examined. The scaffolds preparation methods affected the PEA properties; nevertheless, the modifications induced were not detrimental for the PEA biological performance. Two different bioactive gels were studied as fillers of the scaffolds’ pores: hyaluronan (HA), which is a natural polysaccharide, and a synthetic self-assembling peptide, RAD16-I. HA is ubiquitously present in the body and its degradation products have been reported to be angiogenic. RAD16-I is a synthetic polypeptide that mimics the extracellular matrix providing a favourable substrate for cell growth and proliferation. Given the hydrophobic nature of poly(ethyl acrylate), the combination of PEA scaffolds with aqueous gels raised a number of problems regarding the methods to combine such different elements, the ways to gel them inside the pores, and the procedures to seed cells in the new composite materials. Different alternatives to solve these questions were thoroughly studied and yielded protocols to reliably obtain these complex structures and their biohybrids. An extensive physico-chemical characterization of the components’ interaction and the combined systems was undertaken. As these materials were intended for cardiac tissue engineering applications, the mechanical properties and the effect of the fatigue on them were studied. The different composite systems here developed were homogeneously filled or coated with the hydrogels, were easy to manipulate, and displayed appropriate mechanical properties. Interestingly, these materials exhibited a very good performance under fatigue. The use of the composite systems as a controlled release device was based on the possibility of incorporating active soluble molecules in the hydrogel within the pores. A release study of bovine serum albumin (BSA), intended as a model protein, was performed, which served as a proof of concept. The biological performance of the hybrid scaffolds was first evaluated with fibroblasts to discard the materials cytotoxicity and to optimize the cell seeding procedure. Subsequently, human umbilical vein endothelial cells (HUVECs) cultures were performed for their interest in angiogenic and vascularization processes. Finally, co-cultures of HUVECs with adipose-tissue derived mesenchymal cells (MSCs) were carried out. These last cells are believed to play an important role for clinical regenerative medicine, and their cross-talk with the endothelial cells enhances the viability and phenotypic development of HUVECs. Through the different experiments undertaken, hybrid scaffolds exceeded the outcome achieved by bare PEA scaffolds. / Arnal Pastor, MP. (2014). New scaffolding materials for the regeneration of infarcted myocardium [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/46129 / Premios Extraordinarios de tesis doctorales

Yes / The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data. / University of Turin (Ricerca Locale grant 2015-2017) and Prostate Cancer UK grant S12-027.

In the course of this work, compounds of the natural product class of polycyclic polyprenylated acylphloroglucinols (PPAPs) were analysed. They consist of a polycyclic core structure decorated with carbonyl groups and unsaturated sidechains. The modular synthesis platform developed and optimised in the work group, facilitated the creation of a 23 compound PPAP library. This initiated the study of antibacterial properties. Further research into the derivatisation of PPAPs paved the way to a synthetic toolbox for the synthesis of new derivatives. Now a follow-up structure-activity relationship (SAR) study, aided by a novel C acylation strategy developed in the group, expanded the library with 25 new unnatural type B PPAPs. Antibiotic efficacies with minimum inhibitory concentrations (MICs) in the nanomolar range against methicillin-resistant Staphylococcus aureus (MRSA) were observed, identifying new lead compounds with significant therapeutic windows. Transitioning to cancer research, the anticancer activities of PPAPs across leukaemia, glioblastoma (GB), and prostate cancer (PCa) were investigated. A 3 phenylpropanoyl head group turned out to be crucial for proapoptotic efficacies. Optimisation efforts yielded different potent compounds for each cancer type with proapoptotic half maximal inhibitory concentration (IC50) values in the low micromolar and antiproliferative IC50 values in the nanomolar range. Flavonoid-like PPAP scaffolds were explored for enhanced biological activities through the combination of anticancer activities of PPAPs with flavonoids. A one-step synthesis method produced eleven flavonoid-like PPAPs with promising anticancer potential, particularly against GB. They exhibited strong proapoptotic effects with low toxicity on healthy cells. To expand the covalent combination of different active substances, further conjugation strategies were employed for the synthesis of PPAP-containing drug-drug conjugates (DDCs). This resulted in several PPAP–temozolomide- (TMZ) and PPAP–doxorubicin (DOX) conjugates. PPAP–TMZ showed significant anticancer potential against GB, while PPAP–DOX demonstrated substantial antileukemic activity. Challengingly, both DDCs also exhibited significant toxic effects on healthy peripheral blood mononuclear cells (PBMCs). Therefore, targeted drug delivery of PPAPs utilising PPAP hybrid drugs was investigated to enhance the selectivity of PPAP treatments. This involved the successful combination of PPAPs with linkers for antibody-drug conjugates (ADCs). To evaluate the coupling potential of the synthesised linker with monoclonal antibodies, it was reacted with cysteine. The successful reaction showed the potential of future couplings with real antibodies. For PCa specifically, prostate specific membrane antigen (PSMA)-targeting PPAPs were explored. This protein is solely expressed by PCa cells, therefore being an ideal target protein. Employing chemical structures from existing pharmaceuticals led to the design of different PSMA-targeting PPAPS. While in silico screenings indicated extraordinarily high affinity to the PSMA binding site, biological tests revealed very low anticancer activities. As the binding affinity was too strong, the PPAPs were presumably not released in the cell, which could explain the low anticancer efficacies. For this reason, cleavable linker systems were investigated as part of further research. In conclusion, this research significantly advanced the understanding of diverse PPAP derivatives, demonstrating their potential in antibacterial and anticancer applications. The outlined synthesis strategies, SAR studies, and conjugation approaches provide a foundation for future developments. Further optimisations and collaborations are needed to bring PPAPs a step closer to applications as medical treatment option.:Table of Contents Acknowledgements III Table of Contents V List of Abbreviations IX Abstract (English) XIV Abstract (German) XVII I Theoretical Part 1 Introduction 1 1.1 Medical need in modern society 1 1.2 Natural products in drug development 1 1.3 Polycyclic polyprenylated acylphloroglucinols (PPAPs) 2 1.3.1 Classifications 2 1.3.2 Biosynthesis 4 1.3.3 Biological activities 6 1.3.4 PPAP-synthesis strategies 7 1.4 Flavonoids in drug discovery 10 1.5 Hybrid drugs in cancer treatment 11 1.5.1 Antibody-drug conjugates 11 1.5.2 Drug-drug conjugates 12 1.5.3 Prostate cancer and prostate-specific membrane antigen (PSMA) 13 2 Objectives 16 2.1 Extending the PPAP library and improving biological activities 16 2.2 Examination and improvement of anticancer efficacy with unexplored PPAP scaffolds 17 2.3 Improving target selectivity for PCa using PSMA-targeting PPAPs 17 3 Expanding the PPAP library 18 3.1 Evaluation of the existing library 18 3.2 Synthesis of the PPAP22 core (PPAP26, 31) 21 3.3 Acylation of the C3 position (head group) 24 3.3.1 Established acylation method using acyl cyanides 24 3.3.2 Cyanide free acylation by PESLALZ 25 3.4 Structure-activity relationship (SAR) study of PPAP78 (50) derivatives (PPAP generation 2) 26 3.4.1 Designing the SAR study 26 3.4.2 Synthesis of PPAPs for the SAR study 28 3.4.3 Evaluation of antibacterial activity 29 3.4.4 Conclusion 32 3.5 Evaluation of anticancer activity 33 3.5.1 Synthesis of the used PPAPs 33 3.5.2 Blood cancer (leukaemia) 36 3.5.3 Brain cancer (glioblastoma) 39 3.5.4 Prostate cancer (PCa) 42 3.6 Exploring synthesis methods towards new PPAP amides 46 3.6.1 PPAP amide synthesis via isocyanates 46 3.6.2 PPAP amides from carbamates 49 3.7 Summary of the library expansion 51 4 Novel flavonoid-like PPAP scaffold 54 4.1 Exploring approaches to flavonoid-like PPAPs 55 4.1.1 Flavanones in one step from PPAP26 (31) 55 4.1.2 Conversion of flavanones to flavones 58 4.1.3 Flavone and aurone synthesis with propiolic acids 59 4.1.4 First biological activity screening 62 4.1.5 Synthesis of substituted propiolic acids 63 4.1.6 Elaborating the substitution scope with dimedone test substrates 65 4.2 Investigating flavone-like PPAPs 66 4.2.1 Synthesis of flavone-like PPAPs 66 4.2.2 Biological activity investigations 68 4.3 Exploring the synthesis of isoflavanone-like PPAPs 71 4.4 Summary of flavonoid-like PPAPs 74 5 Studies towards PPAP hybrid drugs 76 5.1 PPAP drug-drug conjugates 76 5.1.1 PPAP–TMZ conjugates 77 5.1.2 PPAP–DOX conjugates 92 5.1.3 Summary of PPAP drug-drug conjugates 96 5.2 PPAP antibody-drug conjugates 97 5.2.1 Synthesis of a linker with a Val–Cit recognition sequence 99 5.2.2 Synthesis and coupling of simplified linkers 102 5.2.3 Testing of cysteine coupling potential 106 5.2.4 Summary of PPAP antibody-drug conjugates 107 5.3 Using PSMA to increase PCa specificity of PPAPs 107 5.3.1 In silico studies for PSMA-targeting PPAPs 108 5.3.2 Synthesis of PSMA-targeting PPAPs 115 5.3.3 Biological activity of PSMA-targeting PPAPs 217 – 220 118 5.3.4 Studies towards PSMA-targeting PPAPs with cleavable hydrazide linkers 121 5.3.5 Summary of PSMA-targeting PPAPs 127 6 Summary of results 129 II Experimental Part 7 General Remarks 137 7.1 Depiction of structures 137 7.2 Substance names 137 7.3 Solvents and common chemicals 137 7.4 Analytical methods 138 7.5 Biological tests 140 7.6 Docking studies with Schrödinger© 146 8 General synthesis procedures 147 9 Syntheses expanding the PPAP library 150 9.1 Synthesis of PPAP22-core (PPAP26, 31) 150 9.2 PPAP derivatisation at C3 161 9.2.1 Standard acyl derivatisation 161 9.2.2 Standard PPAP amide synthesis 197 9.2.3 Studies towards a new PPAP amide synthesis 201 9.3 PPAP salts 205 10 Synthesis of flavonoid-like PPAPs 209 10.1 Synthesis of substituted propiolic acids 209 10.1.1 Acetylene precursors 209 10.1.2 Substituted propiolic acids 211 10.2 Synthesis of flavonoid-like dimedone test-substrates 216 10.3 Synthesis of flavonoid-like PPAPs 223 10.3.1 Aurone-like PPAPs 223 10.3.2 Flavanoid- and chalcone-like PPAPs 224 10.3.3 Flavone-like PPAPs 227 10.3.4 Dihydrocoumarin-like PPAPs 240 11 Synthesis of PPAP hybrids 242 11.1 PPAP drug-drug conjugates 242 11.1.1 PPAP-Temozolomide conjugates 242 11.1.2 PPAP-Doxorubicin conjugates 267 11.2 PPAP-antibody conjugates 276 11.2.1 Linker synthesis 276 11.2.2 Conjugate synthesis and modification 284 11.3 PSMA-targeting PPAPs 293 11.3.1 Synthesis of linkable PSMA-targeting units (PSMA-TUs) 293 11.3.2 Synthesis of a linkable PPAP amide 300 11.3.3 Linking PSMA-TUs to PPAPs 301 11.3.4 Deprotection of tBu-esters 312 III Appendix 12 X-Ray crystal structures 317 13 Exact structures of docked ligands 341 14 References 345 Curriculum vitae 355 List of publications 356 / Im Zuge dieser Arbeit wurden Verbindungen der Naturstoffklasse der polyzyklischen polyprenylierten Acylphloroglucinole (PPAPs) untersucht. PPAPs besitzen eine polyzyklische Kernstruktur, die mehrere Carbonylgruppen aufweist und mit ungesättigten Seitenketten dekoriert ist. Die in der Arbeitsgruppe entwickelte und optimierte, modulare Syntheseplattform ermöglichte die Erstellung einer ersten PPAP-Bibliothek mit 23 Verbindungen. Damit wurde die großflächige Untersuchung der antibakteriellen Aktivitäten von PPAPs eingeleitet. Weitere Erforschungen zur Derivatisierung von PPAPs ebneten den Weg zu einem synthetischen Werkzeugkasten für die Erzeugung neuer Derivate. Eine nachfolgende Struktur-Aktivitäts-Beziehungsstudie (engl. structure-activity relationship, SAR), die durch eine neuartige, in der Gruppe entwickelte C-Acylierungsstrategie ermöglicht wurde, erweiterte die Bibliothek um 25 neue nicht natürlich vorkommende Typ B PPAPs. Hierbei konnten antibiotische Wirksamkeiten mit minimalen Hemmkonzentrationen (MICs) im nanomolaren Bereich gegen Methicillin-resistenten Staphylococcus aureus (MRSA) erreicht werden und neue Leitverbindungen mit großen therapeutischen Fenstern wurden identifiziert. Die krebshemmende Wirkung von PPAPs wurde an den verschiedenen Krebsarten Leukämie, Glioblastom (GB) und Prostatakrebs (PCa) untersucht. Hierbei erwies sich eine 3 Phenylpropanoyl-Kopfgruppe als entscheidend für die proapoptotische Wirksamkeit. Strukturoptimierungen zeigten, dass für jede Krebsart jeweils unterschiedliche Verbindungen potente Wirkungen mit proapoptotischen halbmaximal inhibierenden Konzentrationen (IC50) im niedrigen mikromolaren und antiproliferativen IC50-Werten im nanomolaren Bereich aufwiesen. Flavonoid-ähnliche PPAP-Gerüste wurden im Hinblick auf verstärkte biologische Aktivitäten durch die Kombination der krebshemmenden Aktivitäten von PPAPs mit Flavonoiden untersucht. Es konnte eine einstufige Synthesemethode etabliert werden, mit welcher elf Flavonoid-ähnliche PPAPs mit vielversprechendem Krebs-bekämpfungspotenzial, insbesondere gegen GB, hergestellt wurden. Sie zeigten eine starke proapoptotische Wirkung bei geringer Toxizität gegenüber gesunden Zellen. Um die kovalente Verknüpfung verschiedener Wirkstoffe miteinander zu erweitern, wurden zusätzliche Konjugationsstrategien für die Synthese von PPAP-haltigen Wirkstoff-Wirkstoff-Konjugaten (engl.: drug-drug conjugates, DDCs) eingesetzt. Dies führte zu mehreren PPAP–Temozolomid- (TMZ) und PPAP–Doxorubicin (DOX) Konjugaten. PPAP–TMZ zeigte eine signifikante krebshemmende Wirksamkeit hinsichtlich GB, während PPAP–DOX eine beträchtliche antileukämische Aktivität zeigte. Problematisch ist, dass beide DDCs auch erhebliche toxische Wirkungen auf gesunde periphere mononukleare Blutzellen (PBMCs) zeigten. Aus diesem Grund wurden Untersuchungen zur Verbesserung der Selektivität von PPAP-Behandlungen unter Zuhilfenahme von Wirkstoffhybriden für den gezielten Wirkstofftransport (engl.: targeted drug delivery) durchgeführt. Dazu wurden PPAPs mit Linkern für Antikörper-Wirkstoff-Konjugate (engl.: antibody-drug conjugate, ADCs) verknüpft. Um das Kopplungspotenzial des hergestellten Linkers mit Antikörpern zu testen, wurde dieser mit Cystein umgesetzt. Die erfolgreiche Reaktion zeigte das Potenzial von zukünftigen Kopplungen mit echten Antikörpern. Speziell für PCa wurden gegen das prostataspezifische Membranantigen (PSMA) gerichtete PPAPs erforscht. Dieses Protein wird ausschließlich von PCa-Zellen exprimiert und stellt daher eine ideale Zielstruktur dar. Die Verwendung chemischer Strukturen aus bereits eingesetzten Arzneimitteln führte zur Entwicklung verschiedener PSMA-gerichteter PPAPs. Während in-silico-Screenings auf eine außergewöhnlich hohe Affinität zur PSMA-Bindungsstelle hinwiesen, ergaben biologische Tests nur eine sehr geringe krebshemmende Wirkung. Aufgrund der zu starken Bindungsaffinität wurden die PPAPs in der Zelle vermutlich nicht freigesetzt, was die niedrige krebshemmende Wirkung erklären könnte. Deshalb wurden im Rahmen weiterer Forschung spaltbare Linkersysteme untersucht. Insgesamt hat diese Forschungsarbeit das Verständnis für verschiedene PPAP-Derivate erheblich verbessert und ihr Potenzial für antibakterielle und krebsbekämpfende Anwendungen aufgezeigt. Die skizzierten Synthesestrategien, SAR-Studien und Konjugationsansätze bilden eine Grundlage für zukünftige Weiterentwicklungen dieses Forschungsprojektes. Weitere Optimierungen und Kooperationen sind notwendig, um PPAPs einen Schritt näher an die Anwendung als medizinische Behandlungsoption zu bringen.:Table of Contents Acknowledgements III Table of Contents V List of Abbreviations IX Abstract (English) XIV Abstract (German) XVII I Theoretical Part 1 Introduction 1 1.1 Medical need in modern society 1 1.2 Natural products in drug development 1 1.3 Polycyclic polyprenylated acylphloroglucinols (PPAPs) 2 1.3.1 Classifications 2 1.3.2 Biosynthesis 4 1.3.3 Biological activities 6 1.3.4 PPAP-synthesis strategies 7 1.4 Flavonoids in drug discovery 10 1.5 Hybrid drugs in cancer treatment 11 1.5.1 Antibody-drug conjugates 11 1.5.2 Drug-drug conjugates 12 1.5.3 Prostate cancer and prostate-specific membrane antigen (PSMA) 13 2 Objectives 16 2.1 Extending the PPAP library and improving biological activities 16 2.2 Examination and improvement of anticancer efficacy with unexplored PPAP scaffolds 17 2.3 Improving target selectivity for PCa using PSMA-targeting PPAPs 17 3 Expanding the PPAP library 18 3.1 Evaluation of the existing library 18 3.2 Synthesis of the PPAP22 core (PPAP26, 31) 21 3.3 Acylation of the C3 position (head group) 24 3.3.1 Established acylation method using acyl cyanides 24 3.3.2 Cyanide free acylation by PESLALZ 25 3.4 Structure-activity relationship (SAR) study of PPAP78 (50) derivatives (PPAP generation 2) 26 3.4.1 Designing the SAR study 26 3.4.2 Synthesis of PPAPs for the SAR study 28 3.4.3 Evaluation of antibacterial activity 29 3.4.4 Conclusion 32 3.5 Evaluation of anticancer activity 33 3.5.1 Synthesis of the used PPAPs 33 3.5.2 Blood cancer (leukaemia) 36 3.5.3 Brain cancer (glioblastoma) 39 3.5.4 Prostate cancer (PCa) 42 3.6 Exploring synthesis methods towards new PPAP amides 46 3.6.1 PPAP amide synthesis via isocyanates 46 3.6.2 PPAP amides from carbamates 49 3.7 Summary of the library expansion 51 4 Novel flavonoid-like PPAP scaffold 54 4.1 Exploring approaches to flavonoid-like PPAPs 55 4.1.1 Flavanones in one step from PPAP26 (31) 55 4.1.2 Conversion of flavanones to flavones 58 4.1.3 Flavone and aurone synthesis with propiolic acids 59 4.1.4 First biological activity screening 62 4.1.5 Synthesis of substituted propiolic acids 63 4.1.6 Elaborating the substitution scope with dimedone test substrates 65 4.2 Investigating flavone-like PPAPs 66 4.2.1 Synthesis of flavone-like PPAPs 66 4.2.2 Biological activity investigations 68 4.3 Exploring the synthesis of isoflavanone-like PPAPs 71 4.4 Summary of flavonoid-like PPAPs 74 5 Studies towards PPAP hybrid drugs 76 5.1 PPAP drug-drug conjugates 76 5.1.1 PPAP–TMZ conjugates 77 5.1.2 PPAP–DOX conjugates 92 5.1.3 Summary of PPAP drug-drug conjugates 96 5.2 PPAP antibody-drug conjugates 97 5.2.1 Synthesis of a linker with a Val–Cit recognition sequence 99 5.2.2 Synthesis and coupling of simplified linkers 102 5.2.3 Testing of cysteine coupling potential 106 5.2.4 Summary of PPAP antibody-drug conjugates 107 5.3 Using PSMA to increase PCa specificity of PPAPs 107 5.3.1 In silico studies for PSMA-targeting PPAPs 108 5.3.2 Synthesis of PSMA-targeting PPAPs 115 5.3.3 Biological activity of PSMA-targeting PPAPs 217 – 220 118 5.3.4 Studies towards PSMA-targeting PPAPs with cleavable hydrazide linkers 121 5.3.5 Summary of PSMA-targeting PPAPs 127 6 Summary of results 129 II Experimental Part 7 General Remarks 137 7.1 Depiction of structures 137 7.2 Substance names 137 7.3 Solvents and common chemicals 137 7.4 Analytical methods 138 7.5 Biological tests 140 7.6 Docking studies with Schrödinger© 146 8 General synthesis procedures 147 9 Syntheses expanding the PPAP library 150 9.1 Synthesis of PPAP22-core (PPAP26, 31) 150 9.2 PPAP derivatisation at C3 161 9.2.1 Standard acyl derivatisation 161 9.2.2 Standard PPAP amide synthesis 197 9.2.3 Studies towards a new PPAP amide synthesis 201 9.3 PPAP salts 205 10 Synthesis of flavonoid-like PPAPs 209 10.1 Synthesis of substituted propiolic acids 209 10.1.1 Acetylene precursors 209 10.1.2 Substituted propiolic acids 211 10.2 Synthesis of flavonoid-like dimedone test-substrates 216 10.3 Synthesis of flavonoid-like PPAPs 223 10.3.1 Aurone-like PPAPs 223 10.3.2 Flavanoid- and chalcone-like PPAPs 224 10.3.3 Flavone-like PPAPs 227 10.3.4 Dihydrocoumarin-like PPAPs 240 11 Synthesis of PPAP hybrids 242 11.1 PPAP drug-drug conjugates 242 11.1.1 PPAP-Temozolomide conjugates 242 11.1.2 PPAP-Doxorubicin conjugates 267 11.2 PPAP-antibody conjugates 276 11.2.1 Linker synthesis 276 11.2.2 Conjugate synthesis and modification 284 11.3 PSMA-targeting PPAPs 293 11.3.1 Synthesis of linkable PSMA-targeting units (PSMA-TUs) 293 11.3.2 Synthesis of a linkable PPAP amide 300 11.3.3 Linking PSMA-TUs to PPAPs 301 11.3.4 Deprotection of tBu-esters 312 III Appendix 12 X-Ray crystal structures 317 13 Exact structures of docked ligands 341 14 References 345 Curriculum vitae 355 List of publications 356

To investigate and assess bone regeneration in sheep in combination with new implant materials classical histological staining methods as well as immunohistochemistry may provide additional information to standard radiographs or computer tomography. Available published data of bone defect regenerations in sheep often present none or sparely labeled histological images. Repeatedly, the exact location of the sample remains unclear, detail enlargements are missing and the labeling of different tissues or cells is absent. The aim of this article is to present an overview of sample preparation, staining methods and their benefits as well as a detailed histological description of bone regeneration in the sheep tibia. General histological staining methods like hematoxylin and eosin, Masson-Goldner trichrome, Movat’s pentachrome and alcian blue were used to define new bone formation within a sheep tibia critical size defect containing a polycaprolactone-co-lactide (PCL) scaffold implanted for 3 months (n = 4). Special attention was drawn to describe the bone healing patterns down to cell level. Additionally one histological quantification method and immunohistochemical staining methods are described.

L’ingénierie tissulaire vise à l’élaboration de prothèses biologiques par la régénération ou la culture, in vitro ou in vivo, de tissus ou d’organes. Dans la stratégie de culture in vitro, le développement de nouveaux outils, tels que des bioréacteurs, permettant la culture de cellules ou de tissus sous sollicitations mécaniques spécifiques au tissu est primordial. De plus, l’avancée de cette discipline dans la régénération des tissus nécessite de développer, dès à présent, des méthodes d’évaluation mécanique satisfaisantes permettant de comparer ces néo-tissus aux tissus sains selon des critères de sollicitations physiologiques. En effet, pour parvenir à une bonne évaluation de ces matériaux, il est nécessaire de pouvoir les tester sur des chargements représentatifs des sollicitations physiologiques auxquelles ils sont soumis. Nous avons ainsi, dans un premier temps, conçu et développé un bioréacteur de ligaments permettant la culture de cellules stimulées mécaniquement par des sollicitations cycliques de traction-torsion. Ce bioréacteur a été dimensionné afin de pouvoir obtenir des bio-prothèses de taille comparable aux ligaments et tendons à remplacer (4 à 5 cm de long). Nous avons, dans un deuxième temps, développé un modèle du comportement mécanique global de ces tissus à partir du formalisme thermodynamique développé au sein de notre laboratoire et des observations faites sur des tendons d’Achille de lapin. Ce modèle a pour but d’approfondir la compréhension des structures intervenant de façon prépondérante dans la qualité mécanique de ces tissus ainsi que l’évaluation et l’optimisation des matrices de support et des néo-tissus devant s’y substituer / Tissue Engineering aims to fabricate bio-prostheses by regenerating or culture, in vivo or in vitro, tissues or organs. In the in vitro strategy, developing new tools such as bioréactors which allow the culture of cells or tissues under their specific mechanical solicitations is a huge point. Moreover, the last advances of this discipline in regeneration of tissues require new mechanical model allowing their evaluation and comparison to native tissue under physiological loading. Indeed, in order to obtain a good evaluation of their mechanical quality, it is important to be able to applied mechanical solicitations linked to physiological ones. As a first step, a bioreactor of ligament allowing the culture of cells under mechanical solicitations of cyclic traction-torsion was designed and developed. This bioreactor was sized to potentially obtain a bio-prosthesis comparable to native tissue in term of size (4 to 5 cm long). In a second time, a mechanical model was elaborated based on a thermodynamic formalism developed in our laboratory and the observation made on rabbit Achilles tendons. The goals of this model are to improve our knowledge on the mayor structures involved into the mechanical quality of theses tissues and to evaluate and optimise the scaffolds and neo-tissues of substitution

The ERK signalling cascade comprised of protein kinases Raf, MEK and ERK is an evolutionarily conserved member of MAPK family that is activated in response to wide range of extracellular stimuli. The ERK pathway controls fundamental cellular functions including cell proliferation, differentiation, apoptosis or cell motility. To control such a diverse cellular responses by a single pathway cells have evolved regulatory mechanisms that channel the extracellular signals towards the specific biological response. Crucial to this control are non- enzymatic proteins termed scaffolds that associate with and enhance functional interaction of the components of MAPK pathways and can regulate amplitude, timing, specificity and location of signals. Scaffold protein RACK1 associates with several components of cell migration machinery including integrins, FAK, Src and the ERK pathway core protein kinases. RACK1 regulates distinct steps of cell migration such as establishment of cell polarity and focal adhesion turnover, however, the molecular mechanism by which RACK1 regulates these processes remains largely unknown. The main aim of this study was to investigate the functional role of RACK1 in cell motility, in particular to identify new effector proteins utilized by the ERK pathway and RACK1 in the regulation of...

L’ingénierie de la muqueuse orale humaine (MOH) a pour but le comblement des pertes de substances suite à un traumatisme facial ou à la chirurgie des lésions malignes. Elle a aussi des applications en recherche pour élucider les mécanismes biologiques de la MO et en pharmacotoxicologie comme alternative à l’expérimentation animale. L'objectif de cette thèse était de reconstruire une MOH proche du tissu normal. À cette fin, la faisabilité du concept a d'abord été testée par co-culture de fibroblastes de la lamina propria et de cellules épithéliales de MOH dans le substrat de collagène-chitosan glycosaminoglycane, développé pour la production de peaux reconstruites. La caractérisation de la MOH reconstruite par histologie, immunohistochimie et microscopie électronique à transmission a montré la présence d’une LP équivalente avec un épithélium pluristratifié et non kératinisé très proche du tissu d’origine. Grâce à ce modèle, nous avons ensuite démontré que l’origine des fibroblastes (MO, cornée, peau) influence significativement l’épaisseur et l’ultrastructure de l'épithélium obtenu par culture de cellules épithéliales orales. Enfin, afin d'améliorer les propriétés adhésives du substrat à base collagène, nous avons ajouté au collagène, une élastine-like recombinante (ELR) contenant le tri-peptide d’adhésion cellulaire, RGD, et produit un nouveau substrat bicouche, poreux par lyophilisation et recouvert d’une couche fibreuse par électrofilage. Ces substrats ont été caractérisés par porosimétrie au mercure, microscopie électronique à balayage et essais mécaniques. Nous avons démontré l’effet stimulant de ELR sur la prolifération des fibroblastes et des cellules épithéliales / Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering