Discrete Geometry and Convexity in Honour of Imre Bárány

This special volume is contributed by the speakers of the Discrete Geometry and Convexity conference, held in Budapest, June 19–23, 2017. The aim of the conference is to celebrate the 70th birthday and the scientific achievements of professor Imre Bárány, a pioneering researcher of discrete and convex geometry, topological methods, and combinatorics. The extended abstracts presented here are written by prominent mathematicians whose work has special connections to that of professor Bárány. Topics that are covered include: discrete and combinatorial geometry, convex geometry and general convexity, topological and combinatorial methods. The research papers are presented here in two sections. After this preface and a short overview of Imre Bárány’s works, the main part consists of 20 short but very high level surveys and/or original results (at least an extended abstract of them) by the invited speakers. Then in the second part there are 13 short summaries of further contributed talks. We would like to dedicate this volume to Imre, our great teacher, inspiring colleague, and warm-hearted friend

Plant-derived bioactive compounds for inflammatory diseases

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisA Organização Mundial da Saúde qualifica as doenças inflamatórias crónicas como a principal causa de morbilidade e mortalidade no mundo. A inflamação crónica é caracterizada por uma resposta inflamatória anormal e persistente que conduz à disfunção de tecidos e órgãos (p. ex. artrite). Nas últimas décadas, foram observadas melhorias significativas no tratamento destas doenças. No entanto, a contínua administração de fármacos anti-inflamatórios é limitada devido à sua associação com efeitos secundários graves. Assim, terapias mais seguras e eficazes devem ser exploradas. As plantas, sendo a base da medicina tradicional em muitas culturas por milhares de anos, são uma excelente fonte de moléculas bioativas, tornando-se algumas delas marcos na indústria farmacêutica (p. ex. morfina). Duas plantas tradicionalmente utilizadas no tratamento de doenças imunológicas são a Salvia officinalis e a Echinacea purpurea. Todavia, a sua atividade imunomoduladora ainda não foi amplamente estudada de forma a fornecer evidências científicas sólidas acerca da sua eficácia. Neste trabalho foram preparados extratos de diferentes órgãos dessas plantas (flores, folhas e raízes) para explorar o seu potencial como formulações pró- ou anti-inflamatórias. Diferentes solventes e métodos de extração foram usados para preparar extratos com diferentes características. Em particular, os extratos da E. purpurea foram separados em duas frações (fenóis/ácidos carboxílicos e alquilamidas) para permitir identificar a classe de compostos responsável pela maior bioatividade. A composição química dos extratos e das frações foi caracterizada por diferentes técnicas cromatográficas. A atividade antioxidante das diferentes formulações foi avaliada na presença de espécies reativas relevantes. Os efeitos pró- e anti-inflamatórios dos diferentes extratos e frações foram investigados, respetivamente, em macrófagos não estimulados e estimulados com lipopolissacarídeos. Relativamente às propriedades pró-inflamatórias, somente os extratos aquosos de E. purpurea demonstraram bioatividade ao induzir as principais vias de sinalização inflamatória e os mediadores pró-inflamatórios. Considerando as atividades antioxidantes e anti inflamatórias, todos os extratos e frações preparados apresentaram grande eficácia, a qual foi influenciada pelo método de extração, solvente utilizado e órgão da planta selecionado. Posteriormente, o extrato mais promissor foi encapsulado em vesículas unilamelares grandes, funcionalizadas com ácido fólico, com o objetivo de melhorar a sua biodistribuição. Por fim, demonstrou-se a segurança e a eficácia terapêutica desta formulação num modelo experimental de inflamação em ratos. Assim, concluiu-se que os extratos de plantas são formulações com grande potencial para serem posteriormente utilizadas como base no tratamento eficaz de doenças que afetam o sistema imunológico, seja quando este está comprometido ou hiper-reativo.Chronic inflammation-related diseases are ranked by the World Health Organization as the major cause of morbidity and mortality in the world. Chronic inflammation is characterized by a persistent and abnormal inflammatory response that leads to tissue damage and/or dysfunction (e.g., arthritis). There were remarkable improvements in the last decades in the management of chronic inflammatory diseases. However, the constant administration of the clinically available anti-inflammatory drugs is limited due to their association with serious side effects. Therefore, alternative, safer and more effective therapies must be investigated. Plants, being the basis of traditional medicine in many cultures for thousands of years, are a rich source of bioactive molecules. Some of them became landmarks in the pharmaceutical field (e.g., morphine). Two plants traditionally used in the treatment of immune-related diseases are Salvia officinalis and Echinacea purpurea. However, their immunomodulatory activity has not been extensively studied in a scientifically soundness. Therefore, in this work, we obtained extracts from different organs of those plants (flowers, leaves, and roots) to explore their potential as pro- or anti-inflammatory formulations. Different solvents and extraction methods were used to prepare a variety of extracts. Particularly for E. purpurea extracts were fractionated into phenolic/carboxylic acids and alkylamide fractions to identify the class of compounds responsible for the strongest bioactivity. Then, the chemical fingerprint in the extracts and fractions was evaluated by different chromatographic techniques. The antioxidant activity of the different formulations was evaluated against relevant reactive species. The proand anti-inflammatory effects of the different extracts and fractions were evaluated using non-stimulated and lipopolysaccharide-stimulated macrophages, respectively. Regarding pro-inflammatory properties, aqueous E. purpurea extracts were the most promising by the induction of main inflammatory signaling pathways and pro-inflammatory mediators. Considering antioxidant and anti-inflammatory activities, all the developed extracts displayed strong efficacy that was influenced by the extraction method, solvent used, and source organ of the plant. Afterward, the most promising extract was loaded in folic acidfunctionalized large unilamellar vesicles (FLUVs) to improve its therapeutic biodistribution. Finally, it was demonstrated in an experimental rat model of inflammation the safety and enhanced therapeutic efficacy of the most powerful extracts loaded in FLUVs. Therefore, we showed that the plant extracts are promising natural formulations that can be further used as a basis for the effective treatment for disorders in which the immune system is either overactive or impaired.Fundação para a Ciência e Tecnologia (FCT) for my Ph.D. scholarships (PD/BD/135246/2017 and COVID/BD/152012/2021) and the Ph.D. programme in Advanced Therapies for Health (PATH, PD/00169/2013)

Regulation of the floral transition at the shoot apical meristem of Arabidopsis as studied by genetics and next generation sequencing

Der Vorgang bei welchem Angiospermen von vegetativem Wachstum zur Bildung von Blüten übergehen wird als Übergang zur Blüte („floral transition“) bezeichnet. Dieser entwicklungsbiologische Vorgang ist von großer Bedeutung und wird streng durch ein genetisches Netzwerk reguliert, wobei einige Komponenten des Netzwerkes auf Umweltfaktoren reagieren. Arabidopsis thaliana blüht früher unter Langtagbedingungen (LD) des Frühlings als unter den Kurztagbedingungen (SD) des Winters. Die Tageslänge oder Photoperiode ist einer der wichtigsten Umweltfaktoren welcher die Blühantwort beeinflusst. Die Photoperiode wird über die Blätter wahrgenommen während der Übergang zur Blüte im apikalen Sprossmeristem (SAM) stattfindet. Unter Langtagbedingungen wird eine genetische Kaskade im Leitgewebe des Blattes angestoßen, woraufhin ein Schlüsseltranskriptionsregulator mit dem Namen CONSTANS das Gen mit dem Namen FLOWERING LOCUS T (FT) und sein Homolog TWIN SISTER OF FT (TSF) aktiviert. Das FT Protein wird daraufhin durch das Phloem transportiert und erreicht schlussendlich das SAM wo es den Übergang zur Blüte auslöst. Durch Bildung eines Komplexes mit FD, einem bZIP Transkriptionsfaktor, aktiviert FT Zielgene wie SUPPRESSOR OF OVEREXPRESSION OF CO (SOC1), FRUITFULL (FUL) und später APETALA1 (AP1), welche für MADS-Box Transkriptionsfaktoren codieren. Der Übergang zur Blüte erfordert jedoch im SAM eine dramatische Neuprogrammierung der Transkriptionsvorgänge. Ein vollständiges Bild der Genexpression welche spezifisch im SAM stattfindet fehlt bislang. Daher wurden im ersten Teil dieser Arbeit apikale Sprossmeristeme durch Lasersezierung aus Pflanzen ausgeschnitten, welche von SD nach LD überführt worden waren. RNA, welche aus den Meristemen isoliert worden war, wurde in cDNA umgeschrieben und die Genexpression durch Next-Generation Sequencing durch RNA-seq quantifiziert. Die Gene wurden nach gesteigerter oder verringerter Expression sortiert, wobei ein besonderes Augenmerk auf neue Gene gelegt wurde deren Expression ähnlich der von SOC1 und FUL gesteigert wurde. Ein Teil dieser Gene wurde über in situ Hybridisierung in Wildtyp-Apizes getestet um ihre Aktivierung im SAM zu bestätigen und ihr räumliches Expressionsmuster aufzuklären. Für mehrere neue Gene konnte die Induktion durch Transfer der Pflanzen in LD bestätigt werden; auch zeigten sie spezifische räumliche Expressionsmuster in zahlreichen Regionen des apikalen Sprossmeristems. Es wurden auch Apizes von ft tsf Doppelmutanten hybridisiert um aufzudecken ob die neuen Gene an der von der Photoperiode abhängige Kaskade folgend auf FT/TSF beteiligt sind. Während viele Gene ähnlich wie SOC1 nur in Anwesenheit von FT/TSF induziert wurden, fanden sich erstaunlicherweise auch Gene welche auch in ft tsf Doppelmutanten noch auf die Photoperiode reagierten. Dies legt nahe, dass zusätzliche unbekannte Signale eine Rolle in der Antwort auf eine induzierende Tageslänge unabhängig von FT und TSF spielen. In der vorliegenden Arbeit werden auch weitere Untersuchungen neuer Gene beschrieben. Im zweiten Teil der vorliegenden Arbeit wurden genetische Ansätze verwendet um die Funktion von SHORT VEGETATIVE PHASE (SVP), einem Blührepressor aus der MADS-Box Familie, zu untersuchen. Hierbei wurden neue Interaktionen mit die Blüte fördernden Genen und spezifische Rollen des SVP Gens in Blättern und Meristem aufgedeckt

The total synthesis of (−)-nitidasin, synthetic studies toward astellatol and retigeranic acid B, and development of novel photochromic ligands for L-type voltage-gated calcium channels

The first part of present doctoral thesis is dedicated to sesterterpenoid natural products, which constitute a relatively small subclass of the family of terpenoid compounds. In particular, a small number of biosynthetically closely related iso-propyl-substituted trans-hydrindane- based congeners were chosen as targets for a synthetic program that envisioned their chemical construction via a unified approach. In this context, the first total synthesis of the complex tetracarbocyclic sesterterpenoid (−)-nitidasin was disclosed. To this end, a highly convergent installation of its western cyclopentane moiety was demonstrated by use of an unprecedented Li-alkenyl addition to a versatile trans-hydrindanone building block. An efficient closure of the central 8-membered carbocycle was achieved by means of ring-closing metathesis that benefitted from finely tuned conformational parameters of the employed substrate. In summary, the developed protocol provides a scalable and continuously high yielding route for the assembly of the rare carbon skeleton of (−)-nitidasin. Furthermore, advanced synthetic studies toward the pentacyclic natural products retigeranic acid B and astellatol are described. Notably, the latter involved a biomimetic retrosynthetic proposal for the elaboration of its unique fused ring system. Two different strategies were followed in order to access viable precursors for the suggested cationic cascade, of which the latter allowed for the investigation of the mentioned key step experimentally and via computational methods. In the second section of this manuscript, the design and synthesis of the first known photochromic antagonist for L-type voltage-gated calcium channels is presented. The ultimate goal of this project lies in the establishment of a photopharmacological tool that supports the further elucidation of Ca2+-dependent cellular events, especially in neurons. The central pharmacophore of the prepared compounds was derived from the benzothiazepine drug cis-(+)-diltiazem. This parent substance also served as the starting point for the developed short and convergent semisynthetic strategy. Thirteen analogues were synthesized in total and a preliminary lead structure was identified using electrophysiolocigal methods. Based on these findings, two compounds of increased solubility and desirable photochemical properties were prepared. A possible explanation for their photodependent mode of action was proposed, which is currently subject of our ongoing investigations

Das MYCN-Onkogen als Marker für minimale Resterkrankung und therapeutisches Ziel beim Neuroblastom

Neuroblastoma, the most common extracranial solid childhood cancer, arises from precursors of the developing sympathetic nervous system. MYCN oncogene amplification is a determinant of high risk and occurs in ~25% of neuroblastomas. Despite intensive treatment, more than half these patients succumb to their disease, implying persistence of therapy-resistant MYCN-amplified minimal residual neuroblastoma cells. This thesis proposes a comprehensive concept for the specific diagnostic detection of the MYCN amplicon and evaluates new treatment options for MYCN-amplified neuroblastoma. Disease-relevant nucleotide changes, structural gene rearrangements and copy number alterations were detected in tumor material by next-generation sequencing of a customized hybrid capture-based targeted panel. Unique MYCN amplicon breakpoints in the rearranged gene constitute a target sequence for a personalized minimal residual disease (MRD) PCR diagnostic. MYCN amplicon breakpoints in neuroblastoma cell lines and tumors were identified and recovered by individual, semi-quantitative PCR assays and Sanger sequencing. The assay was further developed for highly sensitive, real-time quantitative and droplet digital PCR detection for selected MYCN breakpoints in cell lines. MRD level detected in bone marrow aspirates collected during therapy outlined different disease courses in patients, including MRD persistence until relapse and good response to the first treatment course. Combining multi-agent chemotherapy in current high-risk protocols with indirect MYCN inhibitors provides a potential route to improve poor cure rates for MYCN-amplified neuroblastomas. Different hyperactive biological networks in MYCN-amplified neuroblastoma were tackled using small molecule inhibitors of the bromodomain and extra-terminal (BET) domain-containing protein BRD4, phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (PLK1). BET (JQ1, OTX015 and TEN-010) and kinase (alpelisib, volasertib and rigosertib) inhibitors demonstrated anti-cancer activity by diminishing viability in cell line-based drug screens at nanomolar to low micromolar concentrations. Rigosertib treatment altered PLK1 and PI3K signaling and strongly impaired the cellular ability for wound healing and colony formation. In line with in vitro observations, rigosertib reduced tumor growth in patient-derived neuroblastoma xenografts in mice. Combining OTX015 and volasertib produced synergistic anti-tumor responses in two MYCN-amplified neuroblastoma cell lines. To prevent MYCN-driven proliferation of tumor cells, further indirect MYCN targets are also being considered. This is exemplified by a substrate of PLK1, ASPM, which is elevated in MYCN-amplified primary neuroblastomas. Knockdown of ASPM, a microtubule-associated protein involved in mitotic spindle assembly, in MYCN-amplified neuroblastoma cell lines reduced viability and proliferation, accompanying a neuronal differentiation phenotype with neurite-like outgrowth, cytoskeletal changes and increased expression of differentiation markers. This study presents clinical implementable molecular diagnostics to pinpoint unique MYCN-amplified neuroblastoma cells within non-invasively accessible biopsy material, and proposes indirect small molecule-based MYCN therapies and potentially new drug targets for a personalized treatment.Das Neuroblastom, der häufigste extrakranielle solide Krebs im Kindesalter, entsteht aus Vorläuferzellen des sich entwickelnden sympathischen Nervensystems. Eine Amplifikation des MYCN-Onkogens ist ein bestimmender Faktor für ein hohes Risiko und tritt bei ~25% der Neuroblastome auf. Trotz intensiver Behandlung erliegt mehr als die Hälfte dieser Patienten ihrer Krankheit, was die Persistenz therapieresistenter, MYCN-amplifizierter minimaler Restneuroblastomzellen impliziert. Diese Arbeit stellt ein umfassendes Konzept für den spezifischen, diagnostischen Nachweis des MYCN-Amplikons vor und evaluiert neue Behandlungsoptionen für MYCN-amplifizierte Neuroblastome. Krankheitsrelevante Nukleotidveränderungen, strukturelle Genrearrangements und Kopienzahl-veränderungen wurden im Tumormaterial mit Hilfe eines maßgeschneiderten, zielgerichteten hybrid-capture-basierten Next Generation Sequencing (NGS) Assays nachgewiesen. Einzigartige MYCN-Amplikon-Bruchpunkte im rearrangierten Gen stellen eine Zielsequenz für eine personalisierte PCR-Diagnostik der minimalen Resterkrankung (MRD) dar. MYCN-Amplikon-Bruchpunkte in Neuroblastom-Zelllinien und Tumoren wurden durch individuelle, semi-quantitative PCR-Assays und Sanger Sequenzierung identifiziert und wiedererkannt. Der Assay wurde für den hochsensitiven, quantitativen Echtzeit- und digitalen Tröpfchen-PCR-Nachweis für ausgewählte MYCN-Bruchpunkte in Zelllinien weiterentwickelt. Die MRD Level, die in den während der Therapie gesammelten Knochenmarkaspiraten nachgewiesen wurden, skizzierten die verschiedenen Krankheitsverläufe bei den Patienten, einschließlich der MRD-Persistenz bis zum Rezidiv und des guten Ansprechens auf den ersten Behandlungsabschnitt. Die Kombination der Multi-Wirkstoff-Chemotherapie in den aktuellen Hochrisikoprotokollen mit indirekten MYCN-Inhibitoren stellt einen möglichen Weg dar, die schlechten Heilungsraten für MYCN-amplifizierte Neuroblastome zu verbessern. Verschiedene, hyperaktive biologische Netzwerke in MYCN-amplifizierten Neuroblastomen wurden mit niedermolekularen Inhibitoren der Bromdomäne und des extra-terminalen (BET) domänenhaltigen Proteins BRD4, der Phosphoinositid-3-Kinase (PI3K) und der polo-ähnlichen Kinase 1 (PLK1) behandelt. BET (JQ1, OTX015 und TEN-010) und Kinase-Inhibitoren (Alpelisib, Volasertib und Rigosertib) zeigten eine krebshemmende Wirkung, indem sie die Viabilität in zelllinienbasierten Wirkstoff-Screens bei nanomolaren bis niedrigen mikromolaren Konzentrationen verminderten. Die Behandlung mit Rigosertib veränderte die PLK1- und PI3K-Signalübertragung und beeinträchtigte die zelluläre Fähigkeit zur Wundheilung und Koloniebildung stark. In Übereinstimmung mit In-vitro-Beobachtungen reduzierte Rigosertib das Tumorwachstum in von Patienten stammenden Neuroblastom-Xenografts bei Mäusen. Die Kombination von OTX015 und Rigosertib erzeugte synergistische antitumorale Aktivität in zwei MYCN-amplifizierten Neuroblastom-Zelllinien. Um die MYCN-gesteuerte Proliferation von Tumorzellen zu verhindern, werden weitere indirekte MYCN-Targets in Betracht gezogen. Ein Beispiel hierfür ist ein Substrat von PLK1, ASPM, das in MYCN-amplifizierten, primären Neuroblastomen erhöht ist. Das Herunterregulieren von ASPM, einem Mikrotubuli-assoziierten Protein, das an der mitotischen Spindelanordnung beteiligt ist, führte in MYCN-amplifizierten Neuroblastom-Zelllinien zu einer verminderten Viabilität und Proliferation, was mit einem neuronalen Differenzierungsphänotyp mit neuritenartigem Auswuchs, zytoskelettalen Veränderungen und erhöhter Expression von Differenzierungsmarkern einherging. Diese Studie stellt eine klinisch umsetzbare, molekulare Diagnostik vor, um einzigartige MYCN-amplifizierte Neuroblastomzellen in nicht-invasiv zugänglichem Biopsiematerial zu detektieren, und schlägt indirekte, niedermolekular-basierende MYCN-Therapien und potenziell neue Zielmoleküle für eine personalisierte Krebsbehandlung vor