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The goal of the project VascuBone is to develop a tool box for bone regeneration, which on one hand fulfills basic requirements (e.g. biocompatibility, properties of the surface, strength of the biomaterials) and on the other hand is freely combinable with what is needed in the respective patient's situation. The tool box will include a variation of biocompatible biomaterials and cell types, FDA-approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging-based in vivo diagnostics, which can be combined for the specific medical need. This tool box will be used to develop translational approaches for regenerative therapies of different types of bone defects. This project receives funding from the European Union's Seventh Framework Program (VascuBone 2010).
The present study is embedded into this EU project. The intention of this study is to assess the changes of the global gene expression patterns of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) after direct cell-cell contact as well as the influence of conditioned medium gained from MSCs on EPCs and vice versa. EPCs play an important role in postnatal vasculogenesis. An intact blood vessel system is crucial for all tissues, including bone. Latest findings in the field of bone fracture healing and repair by the use of tissue engineering constructs seeded with MSCs raised the idea of combining MSCs and EPCs to enhance vascularization and therefore support survival of the newly built bone tissue. RNA samples from both experimental set ups were hybridized on Affymetrix GeneChips® HG-U133 Plus 2.0 and analyzed by microarray technology. Bioinformatic analysis was applied to the microarray data and verified by RT-PCR.
This study gives detailed information on how EPCs and MSCs communicate with each other and therefore gives insights into the signaling pathways of the musculoskeletal system. These insights will be the base for further functional studies on protein level for the purpose of tissue regeneration. A better understanding of the cell communication of MSCs and EPCs and subsequently the targeting of relevant factors opens a variety of new opportunities, especially in the field of tissue engineering.
The second part of the present work was to develop an ELISA (enzyme-linked immunosorbent assay) for a target protein from the lists of differentially expressed genes revealed by the microarray analysis. This project was in cooperation with Immundiagnostik AG, Bensheim, Germany. The development of the ELISA aimed to have an in vitro diagnostic tool to monitor e.g. the quality of cell seeded tissue engineering constructs. The target protein chosen from the lists was klotho. Klotho seemed to be a very promising candidate since it is described in the literature as anti-aging protein. Furthermore, studies with klotho knock-out mice showed that these animals suffered from several age-related diseases e.g. osteoporosis and atherosclerosis. As a co-receptor for FGF23, klotho plays an important role in bone metabolism. The present study will be the first one to show that klotho is up-regulated in EPCs after direct cell-cell contact with MSCs. The development of an assay with a high sensitivity on one hand and the capacity to differentiate between secreted and shedded klotho on the other hand will allow further functional studies of this protein and offers a new opportunity in medical diagnostics especially in the field of metabolic bone disease.
Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.
Zur Wahrung der Genomstabilität entwickelten sich verschiedene Reparaturmechanismen, deren Defekte zu diversen Erkrankungen führen. Der 1927 erstmals beschriebenen Fanconi Anämie (FA) (Fanconi 1927) liegt eine fehlerhafte Reparatur der DNA-Doppelstrang-Quervernetzung zugrunde. Als Ursache wurden Defekte innerhalb des FA/BRCA-Weges lokalisiert, welche zur Chromosomeninstabilität führen. Das Krankheitsbild der autosomal rezessiven oder X-chromosomalen Erkrankung wird meist von kongenitalen Fehlbildungen, progressivem Knochenmarkversagen sowie bereits im jugendlichen Alter erhöhten Tumor-raten und Anämien geprägt. Bisher wurden Defekte in 19 verschiedenen Genen als ursächlich für diese Erkrankung diskutiert. Anhand des betroffenen Gens können nur begrenzt Rückschlüsse auf die Ausprä-gung des Phänotyps geschlossen werden, vielmehr scheinen die Art der Mutation und deren Position im Gen mit der Schwere der Erkrankung zu korrelieren. Im Laufe der Zeit wurden immer mehr Patienten mit mild ausgeprägtem Erkrankungsbild beobachtet. Eine mögliche Erklärung hierfür liefern milde Mutationen, eine weitere das Vorhandensein von Mosaiken blutbildender Zellen. Zu letzterem führt die Reversion einer der beiden Mutationen. Diese Art der „natürlichen Gentherapie“ wurde bei 10-30% der FA-Patienten beobachtet. Um die Entwicklung von Reversionen besser zu verstehen, erfolgte im Rahmen dieser Arbeit die Untersuchung verschiedener Zelllinien von 5 Patienten im Alter von 11 (Pat. 5) bis 33 (Pat. 4) Jahren. Die FA-A-Patienten 1 und 2 wurden bereits von Gross et al. 2002 als Mosaikpatienten beschrieben. Für die weiteren Patienten führten unterschiedliche Aspekte, wie normale Blutwerte, MMC-tolerante lympho-blastoide Zelllinien und gDNA-Analysen des Blutes zum Mosaikverdacht. Nähere Analysen bestätigten für die FA-D2-Patienten (Pat. 4, 5) ebenfalls das Vorliegen einer Reversion in den Blutzellen. Allen Patienten gemein war die Reversion in Form einer Rückmutation (Pat. 1: c.971T>G, Pat. 2: c.856 C>T, Pat. 4: c.3467-2A>G, Pat. 5: c.3707G>A), welche meist in einem oder in der Nähe eines Mutationsmotives vorlag. Zur Einschätzung des Mosaikstatus in den Patientenblutzellen wurden, neben der meist mehrjährigen Be-obachtung der Blutwerte (Thrombo-, Mono-, Granulo-, Lymphozyten, Hämoglobin), gDNA-, Chromoso-menbruch- und Zellzyklusanalysen durchgeführt. Chromosomenbruchanalysen von Metaphasen der T-Lymphozyten der Patienten 4 und 5 zeigten nach MMC-Behandlung die mosaik-typische bimodale Vertei-lung der Chromosomenbruchraten. Die nur moderat erhöhten Bruchraten in Metaphasen des Patienten 1 sprachen für eine starke Reversion. Zur besseren Abschätzung des Mosaikstatus wurden Zellzyklusanaly-sen an Mischungsreihen aus FA- und nicht FA- Blut durchgeführt. Die Detektionsgrenze für FA-Mosaike lag bei einem Anteil von 30% Zellen mit spontanem/MMC-induziertem G2-Phasen-Arrest. In Anlehnung an Mischungskurven wurden für die vier Patienten Reversionen von 0% (Pat. 4) bis 90-95% (Pat. 2) ange-nommen. Die gDNA-Analyse MACS-sortierter T-/B-Lympho-, Mono- und Granulozyten sowie von Fib-roblasten und lymphoblastoiden Zelllinien ermöglichte einen detaillierten Einblick in die Mosaikstatus auf molekularer Ebene. Wir fanden bei allen Patienten einen unterschiedlich stark ausgeprägten Mosaikstatus ihrer Blutzellreihen. Tendenziell scheinen die Reversionsgrade mit der Zell-Lebensdauer korrelieren, hier-bei zeigen kurzlebige Zellen (Mono-, Granulo-, B-Lymphozyten) höhere Reversionsgrade als langlebige T-Lymphozyten. Das Auftreten von gleichen Reversionen in allen Zelllinien lässt eine Reversion in einer gemeinsamen Vorläuferzelle vermuten. Als Besonderheit fanden wir, unseren Erachtens erstmalig, eine komplette Reversion einer Knochenmark-Fibroblastenzelllinie (Pat. 1). Häufig in Kultur stattfindende Re-versionen in lymphoblastoiden Zelllinien beobachteten wir für alle vier Patienten. Die Mosaikentstehung im Patientenblut konnte mit allen Methoden bestätigt werden. Jede Methode wies Vor- und Nachteile auf. Zur Abschätzung der Mosaikstatus empfiehlt sich deshalb eine Kombination der Methoden.
Ein weiteres Projekt beschäftigte sich mit Interaktionen des FANCO (RAD51C) innerhalb der RAD51 Paraloge (RAD51B, -C, -D, XRCC2, XRCC3) und mit RAD51. Die Analysen erfolgten im Mammalian Two- und Three-Hybrid (M2H/M3H) System. Die Untersuchungen bestätigten die meisten der bisher detektierten Interaktionen, welche zur Ausbildung des RAD51C-XRCC3 Komplexes und des, aus den Subkomplexen RAD51B-RAD51C (BC) und RAD51D-XRCC2 (DX2) bestehenden, BCDX2-Komplex führen. Die M3H-Analysen weisen auf eine wichtige Rolle des RAD51B-Proteins bei der Ausprägung dieses Komplexes hin. Es scheint die Ausbildung der RAD51C-RAD51D-Interaktion erst zu ermöglichen und zusätzlich, anders als bisher beobachtet, auch mit XRCC2 zu interagieren. Diese Interaktion wiederum wird durch die Anwesenheit von RAD51D stark gefördert. Unsere M2H-/M3H-Beobachtungen weisen darauf hin, dass die Ausbildung der Subkomplexe für die Entstehung des BDCX2-Komplexes wichtig ist und dieser vermutlich als Ringstruktur vorliegt. Zusätzlich fanden wir Hinweise auf mögliche Wechselwir-kungen zwischen den BCDX2- und den XRCC3-Komplexproteinen. Aufgrund der Beteiligung der Protei-ne an der Doppelstrangläsionsreparatur wurde die Auswirkung von MMC-induzierten DNA-Schäden un-tersucht. Diese führten innerhalb der Subkomplexe zu gegensätzlichen Änderungen der Interaktionsinten-sität. Während die Substanz im DX2-Komplex zum Sinken der Interaktionsstärke führte, erhöhte sich diese im BC-Komplex. Die in der Literatur beschriebene und charakterisierte RAD51C-FANCN-Interation war im M2H-Test nicht darstellbar. Möglicherweise würde diese jedoch durch die Anwesenheit eines drit-ten Proteins gefördert werden. Zusätzlich wurde ein RAD51C-Protein, welches die Patientenmutation R258H enthielt, überprüft. Es zeigte nur in der M3H-Analyse, mit pMRAD51D und nativem RAD51B, nach Behandlung mit MMC eine reduzierte Interaktionsstärke im Vergleich zum Wildtyp. Dies unter-streicht einmal mehr die als hypomorph beschriebene Mutation des Proteins.
Das dritte Projekt, die angestrebte Strukturaufklärung des RAD51C-Proteins erwies sich als schwierig. Eine für eine Kristallisation ausreichende Proteinmenge konnte, weder im E. coli-System noch in Insektenzellen oder in Co-Expression mit seinem Interaktionspartner XRCC3, isoliert und aufgereinigt werden. Elektro-phoretische Mobility Shift Assays des CX3-Proteinkomplexes mit DNA-Strukturen (ssDNA, Open Fork, 3‘-/ 5‘-Überhang-Struktur), zeigten eine Bevorzugung des 3‘-Überhang-DNA-Substrates. Diese Art der Analyse könnte in weiterführenden Analysen zur Abschätzung der Auswirkung von Patientenmutationen herangezogen werden. bb
To rapidly process biologically relevant stimuli, sensory systems have developed a broad variety of coding mechanisms like parallel processing and coincidence detection. Parallel processing (e.g., in the visual system), increases both computational capacity and processing speed by simultaneously coding different aspects of the same stimulus. Coincidence detection is an efficient way to integrate information from different sources. Coincidence has been shown to promote associative learning and memory or stimulus feature detection (e.g., in auditory delay lines). Within the dual olfactory pathway of the honeybee both of these mechanisms might be implemented by uniglomerular projection neurons (PNs) that transfer information from the primary olfactory centers, the antennal lobe (AL), to a multimodal integration center, the mushroom body (MB). PNs from anatomically distinct tracts respond to the same stimulus space, but have different physiological properties, characteristics that are prerequisites for parallel processing of different stimulus aspects. However, the PN pathways also display mirror-imaged like anatomical trajectories that resemble neuronal coincidence detectors as known from auditory delay lines. To investigate temporal processing of olfactory information, we recorded PN odor responses simultaneously from both tracts and measured coincident activity of PNs within and between tracts. Our results show that coincidence levels are different within each of the two tracts. Coincidence also occurs between tracts, but to a minor extent compared to coincidence within tracts. Taken together our findings support the relevance of spike timing in coding of olfactory information (temporal code).
While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment.
Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells.
Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade.
We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation.
Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse.
Circadianes und Stress-System sind zwei physiologische Systeme, die dem Organismus helfen sich an Veränderungen ihrer Umwelt anzupassen. Während letzteres spontane und schnelle Antworten auf akute, unvorhersehbare Umweltreize liefert, sagt das circadiane System täglich wiederkehrende Ereignisse vorher and bereitet den Organismus so vorzeitig auf diese nahende Umweltveränderung vor. Dennoch, trotz dieser unterschiedlichen Reaktionsmechanismen agieren beide Systeme nicht komplett autonom. Studien der vergangen Jahre belegen vielmehr eine Interaktion beider Systeme. So postulieren sie zum einem Unterschiede in der Stressantwort in Abhängigkeit von der Tageszeit zu der der Reiz auftritt und weisen zugleich auf eine Zunahme von gestörten biologischen Tagesrhythmen, wie zum Beispiel Schlafstörungen, in Folge von unkontrollierten oder exzessiven Stress hin. Ebenso liefern kürzlich durchgeführte Studien an Vertebraten und Pilzen Hinweise, dass mit p38, eine Stress-aktivierte Kinase, an der Signalweiterleitung zur inneren Uhr beteiligt ist (Hayashi et al., 2003), sogar durch dieses endogene Zeitmesssystem reguliert wird (Vitalini et al., 2007; Lamb et al., 2011) und deuten damit erstmals eine mögliche Verbindung zwischen Stress-induzierten und regulären rhythmischen Anpassungen des Organismus an Umweltveränderungen an. Molekulare und zelluläre Mechanismen dieser Verknüpfung sind bisher noch nicht bekannt.
Während die Rolle von p38 MAPK bei der Stress- und Immunantwort in Drosophila melanogaster gut charakterisiert ist, wurden Expression und Funktion von p38 in der inneren Uhr hingegen bislang nicht untersucht. Die hier vorliegende Arbeit hatte daher zum Ziel mittels immunhistochemischer, verhaltensphysiologischer und molekularer Methoden eine mögliche Rolle der Stress-aktivierten Kinase im circadianen System der Fliege aufzudecken. Antikörperfärbungen sowie Studien mit Reporterlinien zeigen deutliche Färbesignale in den s-LNv, l-LNv und DN1a und erbringen erstmals einen Nachweis für p38 Expression in den Uhrneuronen der Fliege. Ebenso scheint die Aktivität von p38 MAPK in den DN1a uhrgesteuert zu sein. So liegt p38 vermehrt in seiner aktiven Form in der Dunkelphase vor und zeigt, neben seiner circadian regulierten Aktivierung, zusätzlich auch eine Inaktivierung durch Licht. 15-Minuten-Lichtpulse in der subjektiven Nacht führen zu einer signifikanten Reduktion von aktivierter, phosphorylierter p38 MAPK in den DN1a von Canton S Wildtypfliegen im Vergleich zu Fliegen ohne Lichtpuls-Behandlung. Aufzeichnungen der Lokomotoraktivität offenbaren zusätzlich die Notwendigkeit von p38 MAPK für wildtypisches Timing der Abendaktivität sowie zum Erhalt von 24-Stunden-Verhaltensrhythmen unter konstanten Dauerdunkel-Bedindungen. So zeigen Fliegen mit reduzierten p38 Level in Uhrneuronen einen verzögerten Beginn der Abendaktivität und stark verlängerte Freilaufperioden. In Übereinstimmung mit Effekten auf das Laufverhalten scheint darüber hinaus die Expression einer dominant-negativen Form von p38b in Drosophila’s wichtigsten Uhrneuronen eine verspätete nukleäre Translokation von Period zur Folge zu haben. Westernblots legen zusätzlich einen Einfluss von p38 auf den Phosphorylierungsgrad von Period nahe und liefern damit einen mögliche Erklärung für den verspäteten Kerneintritt des Uhrproteins. Abschließende Stützung der Westernblotergebnisse bringen in vitro Kinasenassays und deuten auf p38 als eine potentielle „Uhrkinase“ hin, welche auch in vivo Period an Serin 661 sowie weiteren potentiellen Phosphorylierungsstellen phosphorylieren könnte.
Zusammengenommen deuten die Ergebnisse der hier vorliegenden Arbeit eindeutig auf eine bedeutende Rolle von p38, neben dessen Funkion im Stress-System, auch im circadianen System der Fliege hin und offenbaren damit die Möglichkeit, dass p38 als Schnittstelle zwischen beider Systeme fungiert.
Das Multiple Myelom (MM) ist eine durch monoklonale Vermehrung terminal differenzierter Antikörper-produzierender B-Lymphozyten (Plasmazellen) im Knochenmark charakterisierte maligne Krankheit, die sich v.a. in osteolytischen Knochendestruktionen, hämatopoetischer und Niereninsuffizienz äußert. Verbesserte Therapieansätze wie die Hochdosis-Chemotherapie mit Melphalan und anschließender autologer Stammzelltransplantation sowie die Einführung neuer pharmakologischer Substanzklassen (Proteasom-Inhibitoren, Cereblon-bindende Thalidomidderivate) führten zu einer Verlängerung der durchschnittlichen Überlebenszeit, für die meisten der Patienten ist die Erkrankung jedoch derzeit unheilbar. Die Erforschung neuer potenzieller therapeutischer Angriffspunkte auf Grund pathobiologischer Erkenntnisse bleibt daher unabdingbar. Ein Ansatz zur Verbesserung des Verständnisses der Pathogenese ist die funktionelle, molekulare und genetische Analyse des Signalnetzwerkes im MM. Im Zusammenhang mit diesem Konzept wurde entdeckt, dass wachstums-regulierende Signalwege in MM Zellen aktiviert oder dereguliert sind und zum Überleben und der Proliferation des Tumors beitragen. So konnte beispielsweise von unserer Arbeitsgruppe bereits gezeigt werden, dass onkogenes Ras essentiell zum Überleben der MM Zellen beiträgt. Da Ras derzeit mangels spezifischer Inhibitoren pharmakologisch nicht angreifbar ist, stellen weitere funktionelle Bestandteile des Signalweges eine potenzielle therapeutische Zielstruktur dar. Während die Blockade von MEK1/2 in MM Zellen keinen Einfluss auf das Überleben hatte, konnte durch die Blockade von Raf in ersten Tests unserer Arbeitsgruppe Apoptose hervorgerufen werden. Aus diesem Grund habe ich in der vorliegenden Arbeit zur Evaluation eines neuen Therapieansatzes die Rolle der Raf-abhängigen Signaltransduktion eingehend untersucht. Als Grundlage diente dabei die Hypothese, dass die Raf-Kinasen entscheidende Effektoren der durch onkogenes Ras vermittelten apoptotischen Effekte darstellen. In einem ersten Schritt konnte ich nachweisen, dass alle drei Raf-Isoformen (A-, B- und C-Raf) in humanen MM Zelllinien und in primären MM Zellen aktiviert sind. Mittels shRNA-vermittelter, Isoform-spezifischer Raf-Knockdown-Experimente konnte ich zeigen, dass nur ein simultaner Knockdown aller Isoformen, d.h. ein Pan-Raf-Knockdown, zu einer De-Phosphorylierung von MEK1/2 und ERK1/2 führte. Dieser Versuch ließ sich mittels pharmakologischer Raf-Inhibition, bei der ebenfalls nur eine Pan-Raf-Blockade zu einer Herunterregulation von MEK1/2 und ERK1/2 in MM Zellen führte, bestätigen. Das MEK/ERK-Modul stellte somit einen hervorragenden Surrogat- und Biomarker für die Pan-Raf-Aktivität dar. Im Gegensatz zur Blockade des MEK/ERK-Moduls führte eine Hemmung der Pan-Raf-Aktivität mittels shRNA oder pharmakologischer Inhibitoren in allen untersuchten Zelllinien und in der Mehrheit der primären MM Zellen zu einer starken Induktion von Apoptose. Da das Ansprechen auf eine Pan-Raf-Blockade nicht mit dem Ras-Mutationsstatus korrelierte, könnten die Raf-Kinasen eine von onkogenem Ras unabhängie Qualität als therapeutische Zielstruktur aufweisen. Zur Untersuchung möglicher MEK/ERK-unabhängiger Effektormechanismen der Pan-Raf-Inhibition habe ich die mRNA-basierten Genexpressionsprofile von INA-6 Zellen nach pharmakologischer Pan-Raf- oder MEK-Inhibition verglichen. Dabei führte die Pan-Raf-Inhibition zu einer Regulation von wesentlich mehr Genen, wobei sich auch die Art der regulierten Gene unterschied, darunter Gene mit tumorrelevanten Funktionen wie Regulation von Proliferation, Zellzyklus und Apoptose. Für eine dieser Gengruppen, die Gruppe der PI3K-abhängigen, mTOR-assoziierten Gene, konnte ich eine Regulation auch auf der Proteinebene nachweisen: die Phosphorylierungen von mTOR, p70S6K, Rb und AKT und die Expression von CyclinD1 und PDK1 waren nach Pan-Raf-Inhibition, nicht jedoch nach MEK-Blockade herunterreguliert. Dieses Ergebnis deutet auf eine Ko-Regulation der PI3K-abhängigen Signaltransduktion durch die Raf-kinasen hin. Mittels spezifischer PI3K-Inhibitoren ließ sich sowohl bei der Regulation der untersuchten Proteine als auch bei der Induktion von Apoptose eine deutliche Verstärkung der Pan-Raf-Inhibition in HMZL und in primären Zellen erzielen. Zusammengefasst zeigt diese Arbeit, dass die Pan-Raf-Blockade eine neue Therapiemöglichkeit darstellt, die durch Kombination mit einer PI3K/AKT-Inhibition noch verstärkt werden kann.
LINC complexes are evolutionarily conserved nuclear envelope bridges, physically connecting the nucleus to the peripheral cytoskeleton. They are pivotal for dynamic cellular and developmental processes, like nuclear migration, anchoring and positioning, meiotic chromosome movements and maintenance of cell polarity and nuclear shape. Active nuclear reshaping is a hallmark of mammalian sperm development and, by transducing cytoskeletal forces to the nuclear envelope, LINC complexes could be vital for sperm head formation as well. We here analyzed in detail the behavior and function of Sun4, a bona fide testis-specific LINC component. We demonstrate that Sun4 is solely expressed in spermatids and there localizes to the posterior nuclear envelope, likely interacting with Sun3/Nesprin1 LINC components. Our study revealed that Sun4 deficiency severely impacts the nucleocytoplasmic junction, leads to mislocalization of other LINC components and interferes with the formation of the microtubule manchette, which finally culminates in a globozoospermia-like phenotype. Together, our study provides direct evidence for a critical role of LINC complexes in mammalian sperm head formation and male fertility.
Immunity, Inflammation and Cancer: The role of Foxp3, TLR7 and TLR8 in gastrointestinal cancer
(2015)
Regulatory T cells (Treg) expressing the transcription factor forkhead box protein P3 (Foxp3) have been demonstrated to mediate evasion from anti-tumor immune responses during tumor progression. Moreover, Foxp3 expression by tumor cells themselves may allow them to counteract effector T cell responses, resulting in a survival benefit of the tumor. For gastrointestinal cancers, in particular pancreatic and colorectal cancer (CRC), the clinical relevance of Foxp3 is not clear to date. Therefore the aim of this study was to analyze its impact in CRC and pancreatic cancer. To determine the relevance of Foxp3 for tumor progression and patient survival, gene and protein analysis of human pancreatic and colon cancer cell lines as well as tumor tissues from patients with CRC was performed. The results derived from the patients with CRC were correlated with clinicopathological parameters and patients’ overall survival. Cancer cell mediated Foxp3 expression in vitro was demonstrated in human pancreatic cancer cell lines PANC1, PaCa DD 135, PaCa DD 159 and PaCa DD 185 as well as in human colon cancer cell lines SW480 and SW620. Additionally, Foxp3 expressing cancer cells were found in ex vivo tumor tissue samples of patients with CRC. The percentage of Foxp3+ cancer cells increased from stages UICC I/II to UICC III/IV compared to normal tissue. Moreover, high tumor cell mediated Foxp3 expression was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in patients’ overall survival. Correlation analysis demonstrated a significant association of Foxp3 cancer cell expression with the expression of immunosuppressive cytokines IL-10 and TGF-β. These findings suggest that Immunosuppressive cytokines such as IL-10 and TGF-β released by rather Foxp3+ cancer cells than Foxp3+ Treg cells may inhibit the activation of naive T cells, hence limiting antitumor immune responses and favoring tumorigenesis and progression.
Chronic inflammation has been shown to be an important epigenetic and environmental factor in numerous tumor entities. Recent data suggest that tumorigenesis and tumor progression may be associated with inflammation-triggered activation of Toll-like receptors (TLR). In this study, the specific impact of both TLR7 and TLR8 expression and signaling on tumor cell proliferation and chemoresistance is analyzed in inflammation linked CRC and pancreatic cancer. By gene and protein expression analysis of human pancreatic and colon cancer cell lines TLR7 and TLR8 expression was determined in vitro. Additionally, expression of TLR7/TLR8 in UICC stage I-IV pancreatic cancer, chronic pancreatitis and normal pancreatic tissue was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of TLR expression and stimulation on tumor cell proliferation and chemoresistance. Cancer cell mediated TLR7 and TLR8 expression in vitro was demonstrated in human colon cancer cell lines SW480, SW620 and HT-29 as well as in primary pancreatic cancer cell lines PaCa DD 135, PaCa DD 159 and PaCa DD 185. Additionally, TLR7 and TLR8 expressing tumor cells were found in ex vivo tissue samples of patients with pancreatic cancer and chronic pancreatitis. Significantly elevated expression levels of TLR7 and TLR8 were found in advanced tumor stages (UICC III) compared to early tumor stages (UICC II) and chronic pancreatitis. No or occasionally low expression was detected in normal pancreatic tissue. In contrast to the tissues from patients with pancreatic cancer or chronic pancreatitis, established pancreatic tumor cell lines express only very low levels of TLR7 and TLR8. Therefore, for in vitro and xenograft studies TLR7 or TLR8 overexpressing PANC1 cells were generated. Proliferation promoting effects of TLR7 and TLR8 expression and stimulation with R848 were detected in vitro. Additionally, increased tumor growth of TLR expressing PANC1 cells was demonstrated in subcutaneously injected Balb/c nude mice. Interestingly, activation of TLR7 or TLR8 induced not only an increase in tumor cell proliferation but also a strong chemoresistance of PANC1 cells against 5-fluorouracil (5-FU). Moreover, treatment with R848 resulted in elevated expression levels of NF-κB, COX-2 and inflammatory cytokines IL-1β, IL-8 and TNF-α, suggesting TLR7/8 signaling to contribute to an inflammatory, anti-apoptotic and proliferation promoting tumor microenvironment. These findings emphasize the particular role of TLR7 and TLR8 in inflammation related cancers and their relevance as potential targets for cancer therapy.
Tumor angiogenesis is a process which is traditionally regarded as the tumor’s response to low nutrient supply occurring under hypoxic conditions. However, hypoxia is not a pre-requisite for angiogenesis. The fact that even single tumor cells or small tumor cell aggregates are capable of attracting blood vessels reveals the early metastatic capability of tumor cells. This review sheds light on the hypoxia-independent mechanisms of tumor angiogenesis in melanoma.
Background
Myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. They function as transcription factors that (in vertebrates) control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation.
Results
We combine chromatin immunoprecipitation-sequencing (ChIPseq) and RNAseq approaches in Drosophila tissue culture cells to identify a core set of less than 500 Myc target genes, whose salient function resides in the control of ribosome biogenesis. Among these genes we find the non-coding snoRNA genes as a large novel class of Myc targets. All assayed snoRNAs are affected by Myc, and many of them are subject to direct transcriptional activation by Myc, both in Drosophila and in vertebrates. The loss of snoRNAs impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors.
Conclusions
This work shows that Myc acts as a master regulator of snoRNP biogenesis. In addition, in combination with recent observations of snoRNA involvement in human cancer, it raises the possibility that Myc’s transforming effects are partially mediated by this class of non-coding transcripts.
Background
Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing.
Results
We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase).
Conclusions
This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.
The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.
Long-term behavioral changes related to learning and experience have been shown to be associated with structural remodeling in the brain. Leaf-cutting ants learn to avoid previously preferred plants after they have proved harmful for their symbiotic fungus, a process that involves long-term olfactory memory. We studied the dynamics of brain microarchitectural changes after long-term olfactory memory formation following avoidance learning in Acromyrmex ambiguus. After performing experiments to control for possible neuronal changes related to age and body size, we quantified synaptic complexes (microglomeruli, MG) in olfactory regions of the mushroom bodies (MBs) at different times after learning. Long-term avoidance memory formation was associated with a transient change in MG densities. Two days after learning, MG density was higher than before learning. At days 4 and 15 after learning—when ants still showed plant avoidance—MG densities had decreased to the initial state. The structural reorganization of MG triggered by long-term avoidance memory formation clearly differed from changes promoted by pure exposure to and collection of novel plants with distinct odors. Sensory exposure by the simultaneous collection of several, instead of one, non-harmful plant species resulted in a decrease in MG densities in the olfactory lip. We hypothesize that while sensory exposure leads to MG pruning in the MB olfactory lip, the formation of long-term avoidance memory involves an initial growth of new MG followed by subsequent pruning.
Background
Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor.
Results
Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.
Conclusions
Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.
The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.
Background
Defence mechanisms of organisms are shaped by their lifestyle, environment and pathogen pressure. Carpenter ants are social insects which live in huge colonies comprising genetically closely related individuals in high densities within nests. This lifestyle potentially facilitates the rapid spread of pathogens between individuals. In concert with their innate immune system, social insects may apply external immune defences to manipulate the microbial community among individuals and within nests. Additionally, carpenter ants carry a mutualistic intracellular and obligate endosymbiotic bacterium, possibly maintained and regulated by the innate immune system. Thus, different selective forces could shape internal immune defences of Camponotus floridanus.
Results
The immune gene repertoire of C. floridanus was investigated by re-evaluating its genome sequence combined with a full transcriptome analysis of immune challenged and control animals using Illumina sequencing. The genome was re-annotated by mapping transcriptome reads and masking repeats. A total of 978 protein sequences were characterised further by annotating functional domains, leading to a change in their original annotation regarding function and domain composition in about 8 % of all proteins. Based on homology analysis with key components of major immune pathways of insects, the C. floridanus immune-related genes were compared to those of Drosophila melanogaster, Apis mellifera, and other hymenoptera. This analysis revealed that overall the immune system of carpenter ants comprises many components found in these insects. In addition, several C. floridanus specific genes of yet unknown functions but which are strongly induced after immune challenge were discovered. In contrast to solitary insects like Drosophila or the hymenopteran Nasonia vitripennis, the number of genes encoding pattern recognition receptors specific for bacterial peptidoglycan (PGN) and a variety of known antimicrobial peptide (AMP) genes is lower in C. floridanus. The comparative analysis of gene expression post immune-challenge in different developmental stages of C. floridanus suggests a stronger induction of immune gene expression in larvae in comparison to adults.
Conclusions
The comparison of the immune system of C. floridanus with that of other insects revealed the presence of a broad immune repertoire. However, the relatively low number of PGN recognition proteins and AMPs, the identification of Camponotus specific putative immune genes, and stage specific differences in immune gene regulation reflects Camponotus specific evolution including adaptations to its lifestyle.
Detailed Analysis of the Human Mitochondrial Contact Site Complex Indicate a Hierarchy of Subunits
(2015)
Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.
More than 100 years ago, Karl von Frisch showed that honeybee workers learn and discriminate colors. Since then, many studies confirmed the color learning capabilities of females from various hymenopteran species. Yet, little is known about visual learning and memory in males despite the fact that in most bee species males must take care of their own needs and must find rewarding flowers to obtain food. Here we used the proboscis extension response (PER) paradigm to study the color learning capacities of workers and drones of the bumblebee, Bombus terrestris. Light stimuli were paired with sucrose reward delivered to the insects’ antennae and inducing a reflexive extension of the proboscis. We evaluated color learning (i.e. conditioned PER to color stimuli) in absolute and differential conditioning protocols and mid-term memory retention was measured two hours after conditioning. Different monochromatic light stimuli in combination with neutral density filters were used to ensure that the bumblebees could only use chromatic and not achromatic (e.g. brightness) information. Furthermore, we tested if bees were able to transfer the learned information from the PER conditioning to a novel discrimination task in a Y-maze. Both workers and drones were capable of learning and discriminating between monochromatic light stimuli and retrieved the learned stimulus after two hours. Drones performed as well as workers during conditioning and in the memory test, but failed in the transfer test in contrast to workers. Our data clearly show that bumblebees can learn to associate a color stimulus with a sugar reward in PER conditioning and that both workers and drones reach similar acquisition and mid-term retention performances. Additionally, we provide evidence that only workers transfer the learned information from a Pavlovian to an operant situation.
To trigger innate behavior, sensory neural networks are pre-tuned to extract biologically relevant stimuli. Many male-female or insect-plant interactions depend on this phenomenon. Especially communication among individuals within social groups depends on innate behaviors. One example is the efficient recruitment of nest mates by successful bumblebee foragers. Returning foragers release a recruitment pheromone in the nest while they perform a ‘dance’ behavior to activate unemployed nest mates. A major component of this pheromone is the sesquiterpenoid farnesol. How farnesol is processed and perceived by the olfactory system, has not yet been identified. It is much likely that processing farnesol involves an innate mechanism for the extraction of relevant information to trigger a fast and reliable behavioral response. To test this hypothesis, we used population response analyses of 100 antennal lobe (AL) neurons recorded in alive bumblebee workers under repeated stimulation with four behaviorally different, but chemically related odorants (geraniol, citronellol, citronellal and farnesol). The analysis identified a unique neural representation of the recruitment pheromone component compared to the other odorants that are predominantly emitted by flowers. The farnesol induced population activity in the AL allowed a reliable separation of farnesol from all other chemically related odor stimuli we tested. We conclude that the farnesol induced population activity may reflect a predetermined representation within the AL-neural network allowing efficient and fast extraction of a behaviorally relevant stimulus. Furthermore, the results show that population response analyses of multiple single AL-units may provide a powerful tool to identify distinct representations of behaviorally relevant odors.
Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach.
This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching.
Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect.
The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature.
Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.
Quantifying the spatio-temporal distribution of arthropods in tropical rainforests represents a first step towards scrutinizing the global distribution of biodiversity on Earth. To date most studies have focused on narrow taxonomic groups or lack a design that allows partitioning of the components of diversity. Here, we consider an exceptionally large dataset (113,952 individuals representing 5,858 species), obtained from the San Lorenzo forest in Panama, where the phylogenetic breadth of arthropod taxa was surveyed using 14 protocols targeting the soil, litter, understory, lower and upper canopy habitats, replicated across seasons in 2003 and 2004. This dataset is used to explore the relative influence of horizontal, vertical and seasonal drivers of arthropod distribution in this forest. We considered arthropod abundance, observed and estimated species richness, additive decomposition of species richness, multiplicative partitioning of species diversity, variation in species composition, species turnover and guild structure as components of diversity. At the scale of our study (2km of distance, 40m in height and 400 days), the effects related to the vertical and seasonal dimensions were most important. Most adult arthropods were collected from the soil/litter or the upper canopy and species richness was highest in the canopy. We compared the distribution of arthropods and trees within our study system. Effects related to the seasonal dimension were stronger for arthropods than for trees. We conclude that: (1) models of beta diversity developed for tropical trees are unlikely to be applicable to tropical arthropods; (2) it is imperative that estimates of global biodiversity derived from mass collecting of arthropods in tropical rainforests embrace the strong vertical and seasonal partitioning observed here; and (3) given the high species turnover observed between seasons, global climate change may have severe consequences for rainforest arthropods.
Wasps of the genus Polistes comprise over 200 species and are nearly cosmopolitan. They show a lack of physiological caste differentiation and are therefore considered as primitively eusocial. Furthermore, paper wasps are placed between the solitary living Eumenidae and the highly social organized Vespinae. Hence, they are often called a “key genus” for understanding the evolution of sociality. Particularly, Polistes dominula, with its small easy manageable nests and its frequent occurrence and wide distribution range is often the subject of studies.
In Europe, the invasion of this species into northern regions is on the rise. Since little was known about the nesting behaviour of P. dominula in Central Europe, the basic principles about nesting were investigated in Würzburg, Germany (latitude 49°) by conducting a comprehensive field-study spanning three consecutive years. Furthermore, the thermoregulation of individual wasps in their natural habitat had not yet been investigated in detail. Therefore, their ability to respond to external hazards with elevated thorax temperatures was tested. In addition, different types of nest thermoregulation were investigated using modern methods such as infrared thermography and temperature data logger.
In the present work, the investigation of basic nesting principles revealed that foundress groups (1-4 foundresses) and nests are smaller and that the nesting season is shorter in the Würzburg area than in other regions. The mean size of newly founded nests was 83 cells and the average nesting season was around 4.6 months. The queens neither preferred single (54%) nor multiple founding (46%) in this study. The major benefit of multiple founding is an increased rate of survival. During the three years of observation, only 47% of single-foundress colonies survived, whereas 100% of colonies that were built by more than two queens, survived. However, an influence of the number of foundresses on the productivity of colonies in terms of number of cells and pupae per nest has not shown up. However, the length of the nesting season as well as the nest sizes varied strongly depending on the climatic conditions of the preceding winter during the three consecutive years.
In order to investigate the thermoregulatory mechanisms of individual adult P. dominula wasps, I presented artificial threats by applying smoke or carbon dioxide simulating fire and predator attacks, respectively, and monitored the thorax temperature of wasps on the nest using infrared thermography. The results clearly revealed that P. dominula workers recognized smoke and CO2 and reacted almost instantaneously and simultaneously with an increase of their thorax temperature. The maximal thorax temperature was reached about 65 s after the application of both stressors, but subsequently the wasps showed a different behaviour pattern. They responded to a longer application of smoke with moving to the exit and fled, whereas in case of CO2 the wasps started flying and circling the nest without trying to escape. No rise of the thorax temperature was detectable after an air blast was applied or in wasps resting on the nest. Additionally, the thorax temperatures of queens were investigated during dominance battles. I found that the thorax temperature of the dominant queens rose up to 5°C compared to that of subordinate queens that attacked the former.
The study of active mechanisms for nest thermoregulation revealed no brood incubation or clustering behaviour of P. dominula. Furthermore, I found out that wing fanning for cooling the nest was almost undetectable (4 documented cases). However, I could convincingly record that water evaporation is most effective for nest cooling. By the direct comparison of active (with brood and adults) and non-active (without brood and adults) nests, the start of cooling by water evaporation was detected above maximum outside temperatures of 25°C or at nest temperatures above 35°C. The powerful role of water in nest cooling was manifested by an average decrease of temperature of a single cell of about 8°C and a mean duration of 7 min until the cell reached again its initial temperature. The investigation of passive thermoregulatory mechanisms revealed that the nest site choice as well as nest orientation appears to be essential for P. dominula wasps. Furthermore, I was able to show that the architecture of the nests plays an important role. Based on the presented results, it can be assumed that the vertical orientation of cells helps maintaining the warmth of nests during the night, whereas the pedicel assists in cooling the nest during the day.
Ovarian cancer (OvCa) is the tumor with the most unfavourable prognosis among all gynaecological malignancies causing more than 6000 deaths per year in Germany alone. Patients with OvCa show symptoms at very advanced stages of tumor progression when the only available treatments consist on tumor debulking surgery and administration of platinum based chemotherapeutics and anthracyclins. There is an urgent need to develop new therapeutical strategies since the actual 5 year survival rate of OvCa patients does not exceed 20-40%.
Immunotherapy is a promising approach for treatment of ovarian cancer, since it has been observed that immunological parameters can influence the outcome of the patient. The aim of our research is to overcome tumor immune escape by counteracting the immunosuppressive mechanisms developed by the tumor. In particular, this work studies the influence of adenosine generated by the ectonucleotidases CD39 and CD73 in the tumor microenvironment. Cellular expression of CD39 and CD73 contributes to immunosupression as these ectonucleotidases convert immune-stimulatory extracellular ATP into immunosuppressive adenosine. This was primarily described as effector mechanism for regulatory T cells, but may also be important in the tumor microenvironment.
Having found that tumor cells from OvCa-patients express high levels of ATP-depleting ectonucleotidases CD39 and CD73 we set out to investigate a potential immunosuppressive mechanism via adenosine production in the tumor microenvironment. We could measure 30-60 times higher adenosine production by OvCa cell lines and ascites-derived cancer cells as compared to physiological normal conditions. To confirm this putative immune escape mechanism we investigated its effect on several immune cell populations. CFSE-based assays, for example, showed an inhibition of CD4+ T cell proliferation by OvCA cell-derived adenosine. In this context, we have further established an in-vitro assay, where OvCa cells modulate the function of macrophages towards a M2 or tumor associated (TAM) phenotype. Together with the phenotype modulation adenosine exerts chemotactic effects on human monocytes and is thus likely to attract myeloid precursor cells towards the tumor tissue. Moreover, in a microenvironment that is shaped by OvCa cells, human monocytes differentiate into M2 macrophages or TAMs which themselves express significant levels of the adenosine-generating ectonucleotidases CD39 and CD73.
Investigating the regulation of ectonucleotidase expression, we also observed that approaches clinically used to treat OvCa (namely application of doxorubicine or irradiation) influence CD73 and CD39 levels of OvCa and immune cells in vitro. In this study we show how this treatment-induced change in the ATP/adenosine ratio modulates the effector function of different immune cells. Furthermore, we investigate the potential benefit of clinically available small molecule inhibitors for CD39 and CD73 that could relieve immunosuppression in the tumor microenvironment especially in combination with common treatment regimes.
Im Neuroblastom ist die Amplifikation des MYCN-Gens, das für den Transkriptionsfaktor N-Myc kodiert, der klinisch bedeutendste Faktor für eine schlechte Prognose. Als Mitglied der onkogenen Myc-Familie induziert N-Myc die Expression von Genen, die in vielen biologischen Prozessen wie Metabolismus, Zellzyklusprogression, Zellwachstum und Apoptose eine wichtige Rolle spielen. Die Deregulation der MYCN-Expression führt zu einem charakteristischen Genexpressionsprofil und einem aggressiven Phenotyp in den Tumorzellen.
In normalen neuronalen Vorläuferzellen wird N-Myc gewöhnlich sehr schnell proteasomal abgebaut. Während der Mitose wird N-Myc an Serin 62 phosphoryliert. Diese Phosphorylierung dient als Erkennungssignal für die Kinase GSK3β, die die Phosphorylierung an Threonin 58 katalysiert. Das Phosphodegron wird von Fbxw7, einer Komponente des E3-Ubiquitinligase-Komplex SCFFbxw7, erkannt. Die anschließende Ubiquitinierung induziert den proteasomalen Abbau des Proteins. Die Reduktion der N-Myc–Proteinlevel ermöglicht den neuronalen Vorläuferzellen den Austritt aus dem Zellzyklus und führt zu einer terminalen Differenzierung.
In einem shRNA Screen konnte AURKA als essentielles Gen für die Proliferation MYCN-amplifizierter Neuroblastomzellen identifiziert werden. Eine Aurora-A–Depletion hatte jedoch keinen Einfluss auf das Wachstum nicht-amplifizierter Zellen.
Während dieser Doktorarbeit konnte gezeigt werden, dass Aurora-A speziell den Fbxw7-vermittelten Abbau verhindert und dadurch N-Myc stabilisiert. Für die Stabilisierung ist zwar die Interaktion der beiden Proteine von entscheidender Bedeutung, überraschenderweise spielt die Kinaseaktivität von Aurora-A jedoch keine Rolle.
Zwei spezifische Aurora-A–Inhibitoren, MLN8054 und MLN8237, sind allerdings in der Lage, nicht nur die Kinaseaktivität zu hemmen, sondern auch die N-Myc-Proteinlevel zu reduzieren. Beide Moleküle induzieren eine Konformationsänderung in der Kinasedomäne von Aurora-A. Diese ungewöhnliche strukturelle Veränderung hat zur Folge, dass der N-Myc/Aurora-A–Komplex dissoziiert und N-Myc mit Hilfe von Fbxw7 proteasomal abgebaut werden kann. In MYCN-amplifizierten Zellen führt diese Reduktion an N-Myc zu einem Zellzyklusarrest in der G1-Phase. Die in vitro Daten konnten in einem transgenen Maus-Modell für das MYCN-amplifizierte Neuroblastom bestätigt werden. Die Behandlung mit MLN8054 und MLN8237 führte in den Tumoren ebenfalls zu einer N-Myc-Reduktion. Darüber hinaus konnte ein prozentualer Anstieg an differenzierten Zellen, die vollständige Tumorregression in der Mehrzahl der Neuroblastome und eine gesteigerte Lebenserwartung beobachtet werden.
Insgesamt zeigen die in vitro und in vivo Daten, dass die spezifischen Aurora-A–Inhibitoren ein hohes therapeutisches Potential gegen das MYCN-amplifizierte Neuroblastom besitzen.
Mimicking female insects to attract male pollinators is an important strategy in sexually deceptive orchids of the genus Ophrys, and some species possess flowers with conspicuous labellum patterns. The function of the variation of the patterns remains unresolved, with suggestions that these enhance pollinator communication. We investigated the possible function of the labellum pattern in Ophrys heldreichii, an orchid species in which the conspicuous and complex labellum pattern contrasts with a dark background. The orchid is pollinated exclusively by males of the solitary bee, Eucera berlandi. Comparisons of labellum patterns revealed that patterns within inflorescences are more similar than those of other conspecific plants. Field observations showed that the males approach at a great speed and directly land on flowers, but after an unsuccessful copulation attempt, bees hover close and visually scan the labellum pattern for up to a minute. Learning experiments conducted with honeybees as an accessible model of bee vision demonstrated that labellum patterns of different plants can be reliably learnt; in contrast, patterns of flowers from the same inflorescence could not be discriminated. These results support the hypothesis that variable labellum patterns in O. heldreichii are involved in flower-pollinator communication which would likely help these plants to avoid geitonogamy.
Introduction The fast, precise, and accurate measurement of the new generation of oral anticoagulants such as dabigatran and rivaroxaban in patients' plasma my provide important information in different clinical circumstances such as in the case of suspicion of overdose, when patients switch from existing oral anticoagulant, in patients with hepatic or renal impairment, by concomitant use of interaction drugs, or to assess anticoagulant concentration in patients' blood before major surgery. Methods Here, we describe a quick and precise method to measure the coagulation inhibitors dabigatran and rivaroxaban using ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry in multiple reactions monitoring (MRM) mode (UPLC-MRM MS). Internal standards (ISs) were added to the sample and after protein precipitation; the sample was separated on a reverse phase column. After ionization of the analytes the ions were detected using electrospray ionization-tandem mass spectrometry. Run time was 2.5 minutes per injection. Ion suppression was characterized by means of post-column infusion. Results The calibration curves of dabigatran and rivaroxaban were linear over the working range between 0.8 and 800 mu g/L (r > 0.99). Limits of detection (LOD) in the plasma matrix were 0.21 mu g/L for dabigatran and 0.34 mu g/L for rivaroxaban, and lower limits of quantification (LLOQ) in the plasma matrix were 0.46 mu g/L for dabigatran and 0.54 mu g/L for rivaroxaban. The intraassay coefficients of variation (CVs) for dabigatran and rivaroxaban were < 4% and 6%; respectively, the interassay CVs were < 6% for dabigatran and < 9% for rivaroxaban. Inaccuracy was < 5% for both substances. The mean recovery was 104.5% (range 83.8-113.0%) for dabigatran and 87.0%(range 73.6-105.4%) for rivaroxaban. No significant ion suppressions were detected at the elution times of dabigatran or rivaroxaban. Both coagulation inhibitors were stable in citrate plasma at -20 degrees C, 4 degrees C and even at RT for at least one week. A method comparison between our UPLC-MRM MS method, the commercially available automated Direct Thrombin Inhibitor assay (DTI assay) for dabigatran measurement from CoaChrom Diagnostica, as well as the automated anti-Xa assay for rivaroxaban measurement from Chromogenix both performed by ACL-TOP showed a high degree of correlation. However, UPLC-MRM MS measurement of dabigatran and rivaroxaban has a much better selectivity than classical functional assays measuring activities of various coagulation factors which are susceptible to interference by other coagulant drugs. Conclusions Overall, we developed and validated a sensitive and specific UPLC-MRM MS assay for the quick and specific measurement of dabigatran and rivaroxaban in human plasma.
Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y enterocolitica 8081 rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up regulation of both Th1 (INF-\(\alpha\), IFN-\(\gamma\), IL 2, and IL 12) and Th2 (IL 4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens.
Deregulated MYC expression contributes to cellular transformation as well as progression and
maintenance of human tumours. Interestingly, in the absence of additional genetic alterations,
potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced
apoptosis has long been recognised as a major barrier against cancer development.
However, it is largely unknown how cells discriminate physiological from supraphysiological levels
of MYC in order to execute an appropriate biological response.
The experiments described in this thesis demonstrate that induction of apoptosis in mammary
epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene
expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required
to invade low-affinity binding sites and repress target genes of the serum response factor SRF.
These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely
needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1-
dependent gene repression and increases AKT phosphorylation and downstream function.
Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s
transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive
role for the MYC/MIZ1 complex in epithelial cells.
Habitat fragmentation and destruction due to anthropogenic land use are the major causes of the increasing extinction risk of many species and have a detrimental impact on animal populations in numerous ways. The long-term survival and stability of spatially structured populations in fragmented landscapes largely depends on the colonisation of habitat patches and the exchange of individuals and genes between patches. The degree of inter-patch dispersal, in turn, depends on the dispersal ability of a species (i.e. the combination of physiological and morphological factors that facilitate dispersal) and the landscape structure (i.e. the nature of the landscape matrix or the spatial configuration of habitat patches). As fragmentation of landscapes is increasing and the number of species is continuously declining, a thorough understanding of the causes and consequences of dispersal is essential for managing natural populations and developing effective conservation strategies.
In the context of animal dispersal, movement behaviour is intensively investigated with capture-mark-recapture studies. For the analysis of such experiments, the influence of marking technique, handling and translocation of marked animals on movement pattern is of crucial importance since it may mask the effects of the main research question. Chapter 2 of this thesis presents a capture-mark-recapture study investigating the effect of translocation on the movement behaviour of the blue-winged grasshopper Oedipoda caerulescens. Transferring individuals of this grasshopper species to suitable but unfamilliar sites has a significant influence on their movement behaviour. Translocated individuals moved longer distances, showed smaller daily turning angles, and thus their movements were more directed than those of resident individuals. The effect of translocation was most pronounced on the first day of the experiment, but may persist for longer. On average, daily moved distances of translocated individuals were about 50 % longer than that of resident individuals because they have been transferred to an unfamiliar habitat patch. Depending on experiment duration, this leads to considerable differences in net displacement between translocated and resident individuals. In summary, the results presented in chapter 2 clearly point out that translocation effects should not be disregarded in future studies on arthropod movement, respectively dispersal. Studies not controlling for possible translocation effects may result in false predictions of dispersal behaviour, habitat detection capability or habitat preferences.
Beside direct field observations via capture-mark-recapture methods, genetic markers can be used to investigate animal dispersal. Chapter 3 presents data on the genetic structure of populations of Metrioptera bicolor, a wing-dimorphic bush cricket, in a spatially structured landscape with patches of suitable habitat distributed within a diverse matrix of different habitat types. Using microsatellite markers, the effects of geographic distance and different matrix types on the genetic differentiation among 24 local populations was assessed. The results of this study clearly indicate that for M. bicolor the isolation of local populations severely depends on the type of surrounding matrix. The presence of forest and a river running through the study area was positively correlated with the extent of genetic differentiation between populations. This indicates that both matrix types severely impede gene flow and the exchange of individuals between local populations of this bush cricket. In addition, for a subsample of populations which were separated only by arable land or settlements, a significant positive correlation between pairwise genetic and geographic distances exists. For the complete data set, this correlation could not be found. This is most probably due to the adverse effect of forest and river on gene flow which dominates the effect of geographic distance in the limited set of patches investigated in this study. The analyses in chapter 3 clearly emphasize the differential resistance of different habitat types on dispersal and the importance of a more detailed view on matrix ‘quality’ in metapopulation studies. Studies that focus on the specific dispersal resistance of different matrix types may provide much more detailed information on the dispersal capacity of species than a mere analysis of isolation by distance. Such information is needed to improve landscape oriented models for species conservation.
In addition to direct effects on realised dispersal (see chapter 3), landscape structure on its own is known to act as an evolutionary selection agent because it determines the costs and benefits of dispersal. Both morphological and behavioural traits of individuals and the degree to which a certain genotype responds to environmental variation have heritable components, and are therefore expected to be able to respond to selection pressures. Chapter 4 analyses the influence of patch size, patch connectivity (isolation of populations) and sand dynamics (stability of habitat) on thorax- and wing length as proxies for dispersal ability of O. caerulescens in coastal grey dunes. This study revealed clear and sex-specific effects of landscape dynamics and patch configuration on dispersal-related morphology. Males of this grasshopper species were smaller and had shorter wings if patches were larger and less connected. In addition, both sexes were larger in habitat patches with high sand dynamics compared to those in patches with lower dynamics. The investments in wing length were only larger in connected populations when sand dynamics were low, indicating that both landscape and patch-related environmental factors are of importance. These results are congruent with theoretical predictions on the evolution of dispersal in metapopulations. They add to the evidence that dispersal-related morphology varies and is selected upon in recently structured populations even at small spatial scales.
Dispersal involves different individual fitness costs like increased predation risk, energy expenditure, costs of developing dispersal-related traits, failure to find new suitable habitat as well as reproductive costs. Therefore, the decision to disperse should not be random but depend on the developmental stage or the physiological condition of an individual just as on actual environmental conditions (context-dependent dispersal, e.g. sex- and wing morph-biased dispersal). Biased dispersal is often investigated by comparing the morphology, physiology and behaviour of females and males or sedentary and dispersive individuals. Studies of biased dispersal in terms of capture-mark-recapture experiments, investigating real dispersal and not routine movements, and genetic proofs of biased dispersal are still rare for certain taxa, especially for orthopterans. However, information on biased dispersal is of great importance as for example, undetected biased dispersal may lead to false conclusions from genetic data. In chapter 5 of this thesis, a combined approach of morphological and genetic analyses was used to investigate biased dispersal of M. bicolor. The presented results not only show that macropterous individuals are predestined for dispersal due to their morphology, the genetic data also indicate that macropters are more dispersive than micropters. Furthermore, even within the group of macropterous individuals, males are supposed to be more dispersive than females. To get an idea of the flight ability of M. bicolor, the morphological data were compared with that of Locusta migratoria and Schistocerca gregaria, which are proved to be very good flyers. Based on the morphological data presented here, one can assume a good flight ability for macropters of M. bicolor, although flying individuals of this species are seldom observed in natural populations.
The honeybee Apis mellifera is a social insect well known for its complex behavior and the ability to learn tasks associated with central place foraging, such as visual navigation or to learn and remember odor-reward associations. Although its brain is smaller than 1mm² with only 8.2 x 105 neurons compared to ~ 20 x 109 in humans, bees still show amazing social, cognitive and learning skills. They express an age – related division of labor with nurse bees staying inside the hive and performing tasks like caring for the brood or cleaning, and foragers who collect food and water outside the hive. This challenges foragers with new responsibilities like sophisticated navigation skills to find and remember food sources, drastic changes in the sensory environment and to communicate new information to other bees. Associated with this plasticity of the behavior, the brain and especially the mushroom bodies (MBs) - sensory integration and association centers involved in learning and memory formation – undergo massive structural and functional neuronal alterations. Related to this background my thesis on one hand focuses on neuronal plasticity and underlying molecular mechanisms in the MBs that accompany the nurse – forager transition.
In the first part I investigated an endogenous and an internal factor that may contribute to the nurse - forager phenotype plasticity and the correlating changes in neuronal network in the MBs: sensory exposure (light) and juvenile hormone (JH). Young bees were precociously exposed to light and subsequently synaptic complexes (microglomeruli, MG) in the MBs or respectively hemolymph juvenile hormone (JH) levels were quantified. The results show that light input indeed triggered a significant decrease in MG density, and mass spectrometry JH detection revealed an increase in JH titer. Interestingly light stimulation in young bees (presumably nurse bees) triggered changes in MG density and JH levels comparable to natural foragers. This indicates that both sensory stimuli as well as the endocrine system may play a part in preparing bees for the behavioral transition to foraging.
Considering a connection between the JH levels and synaptic remodeling I used gene knockdown to disturb JH pathways and artificially increase the JH level. Even though the knockdown was successful, the results show that MG densities remained unchanged, showing no direct effect of JH on synaptic restructuring.
To find a potential mediator of structural synaptic plasticity I focused on the calcium-calmodulin-dependent protein kinase II (CaMKII) in the second part of my thesis. CaMKII is a protein known to be involved in neuronal and behavioral plasticity and also plays an important part in structural plasticity reorganizing synapses. Therefore it is an interesting candidate for molecular mechanisms underlying MG reorganization in the MBs in the honeybee. Corresponding to the high abundance of CaMKII in the learning center in vertebrates (hippocampus), CaMKII was shown to be enriched in the MBs of the honeybee. Here I first investigated the function of CaMKII in learning and memory formation as from vertebrate work CaMKII is known to be associated with the strengthening of synaptic connections inducing long term potentiation and memory formation. The experimental approach included manipulating CaMKII function using 2 different inhibitors and a specific siRNA to create a CaMKII knockdown phenotype. Afterwards bees were subjected to classical olfactory conditioning which is known to induce stable long-term memory. All bees showed normal learning curves and an intact memory acquisition, short-term and mid-term memory (1 hour retention). However, in all cases long-term memory formation was significantly disrupted (24 and 72 hour retention). These results suggests the necessity of functional CaMKII in the MBs for the induction of both early and late phases of long-term memory in honeybees. The neuronal and molecular bases underlying long-term memory and the resulting plasticity in behavior is key to understanding higher brain function and phenotype plasticity. In this context CaMKII may be an important mediator inducing structural synaptic and neuronal changes in the MB synaptic network.
Innerhalb des letzten Jahrzehnts entstanden zahlreiche neue Anreicherungs- und Sequenzier-technologien der zweiten (und dritten) Generation, die in rasantem Tempo weiterentwickelt und schon jetzt in vielen Bereichen als neuer Goldstandard für molekulargenetische For-schung und Diagnostik angesehen werden. Als Hochdurchsatz-Verfahren ermöglichen diese Next Generation Sequencing-Methoden (NGS) in immer kürzerer Zeit die parallele Analyse zahlreicher Proben und immer größerer Zielregionen bis hin zum ganzen Genom und führten in der Humangenetik dadurch zu Forschungsansätzen in neuen Dimensionen.
In dieser Doktorarbeit, die im molekulargenetischen Diagnostik-Labor der Humangenetik Würzburg durchgeführt wurde, wurden in fünf Projekten NGS-Ansätze unterschiedlicher Stufen bzw. Größenordnungen für verschiedene erblich bedingte Erkrankungen konzipiert und etabliert und in Forschungsprojekten sowie der Routinediagnostik eingesetzt. Dabei wurden verschiedene Methoden zur Anreicherung der Zielsequenzen und zur NGS-Sequenzierung erprobt und auf ihre Effizienz beurteilt. Die Ergebnisse des NGS und darauf basierender Nachweis-Experimente wurden in sieben Veröffentlichungen dokumentiert, auf denen diese Dissertation aufbaut.
In den drei ersten Projekten wurden das Access Array-System (Fluidigm) zur Anreicherung der Zielsequenzen und der GS Junior (Roche) zur Erzeugung der Sequenzen verwendet.
In Projekt 1 wurde COL4A6 als neues Kandidatengen für nicht-syndromale Hörstörungen identifiziert. Um mögliche weitere Mutationsträger zu detektieren, wurde erfolgreich ein kleiner NGS-Ansatz für das zügige Screening dieses Gens bei knapp 100 weiteren Patienten etabliert. Diese und weitere Ergebnisse bestätigten die Kausalität der COL4A6-Mutation eines Index-Patienten mit schwerer, X-chromosomal-rezessiver Hörstörung.
Ein geeigneter NGS-Ansatz für die Analyse des großen RYR1-Gens wurde in Projekt 2 ge-sucht. Der erste Ansatz mit Access Array-System und GS Junior führte zwar bei 39 von 87 Patienten mit Maligner Hyperthermie und/oder Central Core Disease zu dem Auffinden einer (potentiell) pathogenen Variante, allerdings mit hohen Ausfallquoten. Mit der zweiten Methode (Anreicherung: SureSelect-System custom design, Agilent; Sequenzierung: HiSeq, Illumina) wurden neben RYR1 noch 63 weitere Gene analysiert, was zu deutlich besseren Ergebnissen und vier Mutationsfunden führte.
Projekt 3 beinhaltete die Etablierung zwei kleiner Panels für Muskelkrankheiten. Ein Panel für drei Gene für Gliedergürteldystrophien wurde sogar erfolgreich in die akkreditierte Rou-tinediagnostik übernommen. Mit dem zweiten Panel für acht Kandidatengene myofibrillärer Myopathien (MFM) wurde u.a. eine neue Mutation im BAG3-Gen identifiziert.
Das Exom eines MFM-Patienten wurde in Projekt 4 nach Anreicherung mit dem SureSelect-System (Agilent) auf dem HiSeq (Illumina) sequenziert. Nach Auswertung und Beurteilung der identifizierten Varianten wurde ein neuer Erbgang für Myotilinopathien entdeckt. Verschiedene Nachweisexperimente bestätigten die Kausalität der Mutation im Myotilin-Gen.
In Projekt 5 wurde die komplette genomische Sequenz des F8-Gens nach tiefen intronischen Mutationen bei Hämophilie-Patienten abgesucht (Anreicherung SureSelect custom design, Agilent; Sequenzierung MiSeq, Illumina). Bei jedem der analysierten Patienten konnte min-destens eine verdächtige Variante identifiziert werden, die zu verändertem Spleißverhalten führen könnte. Drei Mutationen waren schon durch Publikationen bekannt, bei einer weite-ren konnten in vitro-Spleißanalysen die Kausalität bestätigen.
Die Ergebnisse dieser Arbeit zeigen, dass die zur Verfügung stehenden Methoden zur An-reicherung von Zielsequenzen aus dem menschlichen Genom und zu deren Sequenzierung je nach Komplexität der Fragestellung, d.h. der Anzahl und Größe der Gene sowie der Anzahl der zu untersuchenden Proben, sinnvoll und effizient kombiniert werden können. Im Verlauf der Arbeit haben sich die NGS-Techniken rasant weiterentwickelt. So sind PCR-basierte Ansätze zur Anreicherung der Zielsequenzen für die meisten Anwendungen von hybridisierungs-basierten Methoden verdrängt worden. Von den ursprünglich drei konkur-rierenden Verfahren zur Hochdurchsatzsequenzierung hat sich die Methode des „sequen-cing-by-synthesis“ (Illumina) weitgehend durchgesetzt. Diese Entwicklung spiegelt sich auch in den während dieser Arbeit erhobenen Daten wider.
Pilze sind in unserer Umwelt allgegenwärtig und besiedeln im Fall von Candida albicans (C. albicans) sogar bei über 50% der Menschen die Schleimhäute, während Sporen von Aspergillus fumigatus (A. fumigatus) täglich über die Atmung in die Lunge des Menschen gelangen. Dennoch sind Erkrankungen, die durch diese zwei Pilze ausgelöst werden, bei gesunden Menschen selten. Ist jedoch das Immunsystem beeinträchtigt, können diese Pilze zu systemischen und damit lebensbedrohlichen Erkrankungen wie der invasiven Aspergillose und der systemischen Candidiasis führen. Für eine Verbesserung der Behandlung solcher Infektionen ist das genaue Verständnis der Immunabwehrmechanismen entscheidend. Da A. fumigatus über die Lunge in den Körper gelangt, wurden in dieser Arbeit die häufigsten Immunzellen der Lunge, die Makrophagen, und deren Immunantwort auf A. fumigatus untersucht. Parallel hierzu wurden dendritische Zellen (DCs) verwendet, die als Brücke zwischen dem angeborenen und adaptiven Immunsystem wirken. Ein besonderes Augenmerk wurde hierbei auf A. fumigatus induzierte Genexpressionsänderungen und deren Regulationsmechanismen gelegt. Dabei wurden kurze, regulatorische RNAs, die sogenannten miRNAs, untersucht, die eine wichtige Rolle in der post-transkriptionalen Genregulation spielen. Bislang ist nur wenig über die miRNA-abhängigen Genregulationen in DCs, die auf eine Infektion mit A. fumigatus oder C. albicans reagieren, bekannt. Um alle durch A. fumigatus und C. albicans regulierten miRNAs zu identifizieren, wurden DCs mit A. fumigatus und C. albicans ko-kultiviert und anschließend eine Komplettsequenzierung der kurzen RNAs durchgeführt. Die Pilz-spezifische Induktion der miRNA-Regulation wurde zudem mit der miRNA-Regulation durch den bakteriellen Zellwandbestandteil Lipopolysaccharid verglichen. Durch die Stimulation mit Keimschläuchen von A. fumigatus wurden die miRNAs miR-132-3p/5p, miR-155-5p, miR129-2-3p, miR-129-5p, miR-212-3p/5p und miR-9-5p in DCs induziert. Diese wurden ebenfalls durch C. albicans induziert, zudem noch die miRNAs miR-147a und miR-147b. Spezifisch für A. fumigatus war die Regulation der miR-129-2-3p. Neben dem miRNA-Profiling wurde auch das mRNA-Transkriptom über Microarrays analysiert und dadurch 18 potentielle Zielgene der Pilz-induzierten miRNAs identifiziert.
Neben den Elementen der Translationsregulation wurden auch die Transkriptionsfaktoren untersucht. Als einziger unter den 60 regulierten Transkriptionsfaktoren zeigte KLF4 eine veränderte Expressionsrichtung in DCs, die mit Pilzen oder LPS behandelt waren. Während die Stimulation mit LPS die Expression von KLF4 induzierte, wurde es durch die Pilze A. fumigatus und C. albicans reprimiert. In einer Untersuchung der unterschiedlichen A. fumigatus-Rezeptoren, wurde deren Einfluss auf die KLF4-Regulation gezeigt. Während TLR4-Liganden KLF4 induzierten, führten Liganden, die an die Rezeptoren TLR2/TLR1 und Dectin-1 binden, zu einer Reduktion von KLF4. Nach einem erfolgreich etablierten KLF4-knock-down mittels RNA-Interferenz wurden KLF4-Zielgene untersucht. Während kein bzw. nur ein geringer Effekt auf die Genexpression von CCL2, RANTES, CXCL10 und TNF beobachtet wurde, sorgte der KLF4 knock-down für eine hoch signifikante Reduktion der IL6-Genexpression in LPS-stimulierten DCs.
Um die KLF4-Regulation weiter zu untersuchen, wurde zudem eine weitere Zellpopulation des angeborenen Immunsystems, die Makrophagen, verwendet. Auch hier wurde die Immunantwort gegen A. fumigatus analysiert. Zudem wurde die Rolle der Thrombozyten als Immunmediatoren betrachtet. Zuerst wurde ein Zytokinprofil des plättchenreichen Plasmas (PRP), das mit A. fumigatus stimuliert wurde, erstellt. In diesem konnte nur RANTES in hoher Konzentration nachgewiesen werden. Daraufhin wurde der Einfluss von PRP auf die Reifung von DCs, die Phagozytosefähigkeit von Makrophagen und DCs sowie der Einfluss von DCs und Makrophagen auf die metabolische Aktivität von A. fumigatus in An- und Abwesenheit von plättchenreichem Plasma untersucht. Es konnte eine gering verstärkte Reifung der DCs durch PRP gezeigt werden. Isolierte Thrombozyten konnten die Phagozytose von DCs steigern, während Makrophagen durch PRP verstärkt Konidien phagozytierten. In einem genomweiten Transkriptomprofiling wurde die Immunantwort von DCs und Makrophagen verglichen. Zudem wurde untersucht, wie PRP die Immunantwort dieser Immunzellen beeinflusst. Es wurden 2 bzw. 24 Gene identifiziert, die signifikant in A. fumigatus-stimulierten DCs und Makrophagen reguliert waren. Hierbei wurde gezeigt, dass KLF4 durch die Zugabe von PRP herabreguliert wurde. Das zuvor beschriebene Zielgen IL6 wurde durch PRP in A. fumigatus-stimulierten DCs gegenüber stimulierten DCs ohne PRP deutlich reduziert, wodurch sich eine immunmodulatorische Fähigkeit des PRP zeigte.
Die Induktion von IL-6, weiteren Zytokinen und der Reifemarker durch A. fumigatus in DCs wurden zudem in einem Booleschen Modell simuliert. Dieses Modell soll in Zukunft Vorhersagen über experimentelle Ergebnisse und dadurch eine optimale Versuchsvorbereitung ermöglichen.
Der Kehlkopf ist ein stimmerzeugendes knorpelhaltiges Organ und spielt eine wichtige Rolle in der Atemfunktion und beim aspirationsfreien Schluckakt. Funktionsstörungen des Kehlkopfs wie Stimmbandlähmungen werden durch Schädigungen des Kehlkopfnervs nach operativen Eingriffen und Halsverletzungen hervorgerufen. Des Weiteren führen durch Traumen, Teil- und komplette Resektionen verursachte Substanzdefekte des Kehlkopfs zu Funktionsverlusten. Die hierfür notwendigen und komplexen Rekonstruktionen werden durch das schlechte Regenerationspotential von Knorpelgewebe eingeschränkt und können nur bedingt durch synthetische Ersatzmaterialen oder körpereigenes Ersatzgewebe bewerkstelligt werden. Ist es möglich, mit Hilfe des Tissue Engineerings aus körpereigenen Stammzellen und biokompatiblen Trägermaterialien implantierbares Knorpelersatzgewebe herzustellen, welches zur dauerhaften Wiederherstellung der Kehlkopffunktionen eingesetzt werden kann? Die zusätzliche Markierung von Stammzellen mit superparamagnetischen Eisenoxidnanopartikeln (VSOP) als Zellmarker bietet die Möglichkeit der Detektion und der Verfolgung der Zellen mittels nicht-invasiver Nachweismethoden nach deren Implantation. Ist die Verwendung dieser Nanopartikel ohne negative Folgen für die Stammzellen möglich und sind diese für den Einsatz in der Laryngologie geeignet?
Fettgewebsstammzellen (ASC) wurden aus humanem Liposuktionsmaterial und Kaninchen-Nackenfett isoliert und expandiert. Die Zellen wurden in Hydrogelkombinationen aus Kollagen Typ-I, Agarose, Fibrin und Hyaluronsäure eingebettet und mit den chondrogenen Wachstumsfaktoren TGF-β3, BMP-6 und IGF-I über 14 Tage differenziert. Anschließend wurden diese Zell-Hydrogelkonstrukte bezüglich Morphologie, extrazellulärer Matrixanreicherung und knorpelspezifischer Genexpression histologisch, immunhistochemisch und molekularbiologisch analysiert. In einem weiteren Schritt wurden die Integration der Zell-Hydrogelkonstrukte in natives Knorpelgewebe sowie die Defektdeckung in einem in vitro- und einem in vivo-Knorpeldefektmodell mit vor- und nicht-vordifferenzierten Zell-Hydrogelkonstrukten untersucht. Die Analyse möglicher zyto- und genotoxischer Effekte von VSOP sowie des Einflusses der Markierung von ASC mit VSOP auf die Proliferation, Migration und das Multidifferenzierungspotential erfolgte nach der Markierung der Zellen mit unterschiedlichen VSOP-Konzentrationen. Außerdem wurden VSOP-markierte ASC in Kaninchenstimmlippen injiziert und die Nachweisbarkeit dieser Zellen im Injektionsareal histologisch und mittels Magnetresonanztomographie (MRT) untersucht.
Nach 14-tägiger chondrogener Differenzierung wurde in den Zell-Hydrogelkonstrukten eine knorpelähnliche Morphologie, die Anreicherung knorpelspezifischer Matrixproteine und die Expression chondrogener Markergene nachgewiesen. Die Kombination der chondrogenen Wachstumsfaktoren zeigte keinen verstärkenden Einfluss auf die Chondrogenese von ASC. Hydrogele aus Kollagen Typ I und Hyaluronsäure wiesen die stärkste extrazelluläre Matrixanreicherung auf. Bei den agarosefreien Hydrogelen war eine ausgeprägte Gelschrumpfung auffällig. In den beiden Knorpeldefektmodellen konnte weder eine Integration der Zell-Hydrogelkonstrukte in den Nativknorpel noch eine vollständige Defektdeckung nachgewiesen werden. Nach der Markierung von ASC mit VSOP zeigte sich bei der höchsten Konzentration von 1,5 mM eine genotoxische Wirkung. Zytotoxische Effekte sowie Einflüsse der Markierung auf die Proliferation, Migration und das Multidifferenzierungspotential von ASC waren nicht nachweisbar. VSOP-markierte ASC konnten nach deren Injektion in Kaninchenstimmlippen im Injektionsareal nur vereinzelt mittels MRT und histologisch nachgewiesen werden.
Es ist möglich, mit Hilfe des Tissue Engineerings aus körpereigenen Stammzellen und biokompatiblen Trägermaterialien implantierbares knorpelähnliches Gewebe herzustellen. Dabei begünstigen agarosefreie Trägermaterialien die chondrogene Differenzierung von ASC. Diese könnte durch die jeweilige Erhöhung der Zelldichte und Wachstumsfaktorkonzentrationen sowie die Verlängerung der Induktionszeit verstärkt werden. Eine mögliche klinische Anwendung dieser knorpelähnlichen Gewebe in der Laryngologie ist jedoch durch deren Schrumpfung wie auch mangelnde Integration und Defektdeckung noch weit entfernt. Aufgrund ihrer genotoxischen Wirkung kann eine Verwendung von VSOP als Zellmarker auch unterhalb von 1,5 mM ohne negative Folgen für den Organismus nicht sicher ausgeschlossen werden. Der inhomogene Gewebekontrast im Kehlkopf, die schlechte Auflösung im MRT und die geringe Größe von VSOP erschweren die Nachweisbarkeit und Verfolgung markierter Zellen mittels MRT. Daher sind andere nicht-invasive Nachweismethoden für die Verwendung von VSOP im Kehlkopf zu evaluieren. Der möglichen Anwendung dieser knorpelähnlichen Gewebe und VSOP in der rekonstruktiven Laryngologie muss eine erfolgreiche Optimierung und ausführliche positive Validierung in klinischen Tests vorausgehen.
Microbial species (bacteria and archaea) in the gut are important for human health in various ways. Not only does the species composition vary considerably within the human population, but each individual also appears to have its own strains of a given species. While it is known from studies of bacterial pan-genomes, that genetic variation between strains can differ considerably, such as in Escherichia coli, the extent of genetic variation of strains for abundant gut species has not been surveyed in a natural habitat. This is mainly due to the fact that most of these species cannot be cultured in the laboratory. Genetic variation can range from microscale genomic rearrangements such as small nucleotide polymorphism (SNP) to macroscale large genomic rearrangements like structural variations. Metagenomics offers an alternative solution to study genetic variation in prokaryotes, as it involves DNA sequencing of the whole community directly from the environment. However, most metagenomic studies to date only focus on variation in gene abundance and hence are not able to characterize genetic variation (in terms of presence or absence of SNPs and genes) of gut microbial strains of individuals.
The aim of my doctorate studies was therefore to study the extent of genetic variation in the genomic sequence of gut prokaryotic species and its phenotypic effects based on: (1) the impact of SNP variation in gut bacterial species, by focusing on genes under selective pressure and (2) the gene content variation (as a proxy for structural variation) and their effect on microbial species and the phenotypic traits of their human host.
In the first part of my doctorate studies, I was involved in a project in which we created a catalogue of 10.3 million SNPs in gut prokaryotic species, based on metagenomes. I used this to perform the first SNP-based comparative study of prokaryotic species evolution in a natural habitat. Here, I found that strains of gut microbial species in different individuals evolve at more similar rates than the strains within an individual. In addition, I found that gene evolution can be uncoupled from the evolution of its originating species, and that this could be related to selective pressure such as diet, exemplified by galactokinase gene (galK). Despite the individuality (i.e. uniqueness of each individual within the studied metagenomic dataset) in the SNP profile of the gut microbiota that we found, for most cases it is not possible to link SNPs with phenotypic differences. For this reason I also used gene content as a proxy to study structural variation in metagenomes.
In the second part of my doctorate studies, I developed a methodology to characterize the variability of gene content in gut bacterial species, using metagenomes. My approach is based on gene deletions, and was applied to abundant species (demonstrated using a set of 11 species). The method is sufficiently robust as it captures a similar range of gene content variability as has been detected in completely sequenced genomes. Using this procedure I found individuals differ by an average of 13% in their gene content of gut bacterial strains within the same species. Interestingly no two individuals shared the same gene content across bacterial species. However, this variation corresponds to a lower limit, as it is only accounts for gene deletion and not insertions. This large variation in the gene content of gut strain was found to affect important functions, such as polysaccharide utilization loci (PULs) and capsular polysaccharide synthesis (CPS), which are related with digestion of dietary fibers.
In summary, I have shown that metagenomics based approaches can be robust in characterizing genetic variation in gut bacterial species. I also illustrated, using examples both for SNPs and gene content (galK, PULs and CPS), that this genetic variation can be used to predict the phenotypic characteristics of the microbial species, as well as predicting the phenotype of their human host (for example, their capacity to digest different food components). Overall, the results of my thesis highlight the importance of characterizing the strains in the gut microbiome analogous to the emerging variability and importance of human genomics.
Protected areas are the central strategy for preserving biodiversity in the face of overexploitation and global change. To ensure their long-term survival, however, these areas may not be regarded as last havens of wilderness, but as complex social-ecological systems. Modern approaches of protected area (PA) management support this view by balancing conservation and development issues in a sustainable way and adapted to the local context. However, success of these strategies in achieving their aims so far remains limited.
This study therefore aimed at analysing processes and outcomes of PA co-management approaches implemented in a large transfrontier conservation area in West Africa. The W-Arly-Pendjari (WAP) complex spans over more than 30.000 square km in Benin, Burkina Faso and Niger and is composed of approximately 20 subunits. Due to national legal and administrative variety as well as a high diversity of local (project) implementation approaches, the general setting resembled a quasi-experimental design facilitating comparative studies.
A mix of quantitative (e.g. survey of 549 households) and qualitative (e.g. expert interviews, literature review) methods was used to evaluate the institutional and organisational differences of PA management approaches implemented in the different parts of WAP belonging to Benin and Burkina Faso. I included an analysis of contextual factors (e.g. land-cover-change) and ecological data, but concentrated on the role of local resource users within the co-management arrangements and the effectiveness of governance regimes to deliver positive socio-economic outputs. Exploring the question whether promotion of development in PA surroundings indeed stipulates conservation success (and vice versa) remained challenging: the lack of sound ecological data, a general mismatch of spatial scale in existing data sets, as well as the high complexity of realities on the ground made me refrain from using simplified proxy indicators and (statistical) modelling approaches.
I found that the Sudano-Sahelian context is a very difficult one for the implementation of effective participation approaches in the short-term. Political, demographic, socio-economic as well as ecological factors generated a very dynamic situation characterized by limited financial and natural resources as well as weak institutional and organisational settings.
Arenas of interaction were often marked rather by a high degree of distrust and competition than by cooperation among actors. Amid all rhetoric, participation in most cases was hence limited to the transfer of (sparse) information, regulated resource access and financial funds. Options for participation of local resource users in decision-making arenas were generally scarce. Underlying processes were dominated by opacity and often low accountability of actors on all levels. Negative, but also positive affection of local residents by PA existence and management hence was high.
Governance regimes of the complex performed very differently with regard to their ability of effectively empowering local village participatory bodies (vpb), generating and distributing benefits to individuals and village communities as well as providing mechanisms of conflict resolution. People around Pendjari enjoyed a relative wealth of high value benefits, while negative impacts caused by human-wildlife conflicts were widespread around the complex. Autochthonous farmers usually were better integrated in incentive schemes than were newcomers or herders.
While there was functional separation of actors’ roles in all parts of WAP, these roles differed significantly between blocks. Existence and functioning of village participatory bodies ameliorated the situation for local resource users fundamentally, as they acted as cut-points between different networks (governmental hierarchies, private concessionaires and local resource users). Vpbs in the Pendjari region proved to be most advanced in their capacity to push resource users’ claims in action arenas on the micro-level. Via their union, these associations also managed to impact arenas on the meso- and the macro scale.
Project interventions often had catalyst functions to empower local resource users and their vbps. However, they also contributed to social imbalance and intra-organisational competition.
My results represent a snapshot of an ongoing process to establish effective co-governance regimes in the WAP-area. Though I identified a large scope of shortcomings, there were also very promising initiatives underway. This work is therefore meant to foster future research and further positive development by giving guidance scholars and decision-makers form the local to the global level alike.
Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection
(2015)
Staphylococcus aureus is both a human commensal and a pathogen. 20%-30% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options.
In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen.
In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death.
In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger.
All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys.
Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades.
1) Modern European agricultural landscapes form a patchy mosaic of highly fragmented natural and semi-natural habitat remnants embedded in a matrix of intensively managed agricultural land. In those landscapes many organism frequently cross habitat borders including the crop – non-crop boundary, hereby connecting the biotic interactions of multiple habitat types. Therefore biodiversity and ecosystem functions within habitats are expected to depend on adjacent habitat types and the surrounding landscape matrix. In this thesis the biodiversity of non-crop habitats, and ecosystem services and disservices in crop habitats were studied in the human-dominated agricultural landscape in the district Lower Franconia, Bavaria, Germany. First we examined the effect of adjacent habitat type on species composition, diversity and ecosystem functions in semi-natural calcareous grasslands, a biodiversity-rich habitat of high conservation value (chapter 2 and 3). Second we studied the effect of habitat composition in the landscape on herbivory, biological pest control and yield in oilseed rape fields (chapter 4).
2) We examined the effect of adjacent habitat type on the diversity of carabid beetles in 20 calcareous grasslands using pitfall traps. Half of the grasslands were adjacent to a coniferous forest and half to a cereal crop field. We found different species compositions of carabid beetles depending on adjacent habitat type. In addition calcareous grasslands adjacent to crop fields harboured a higher species richness and activity density but a lower evenness of carabid beetles than calcareous grasslands adjacent to forests. These differences can be explained by the spillover of carabid beetles from the adjacent habitats. After crop harvest carabid beetle activity density in crop fields decreased while in parallel the activity density in the calcareous grasslands adjacent to the crop fields increased, indicating an unidirectional carabid beetle spillover. Our results underline that type and management of adjacent habitats affect community composition and diversity in calcareous grasslands. Therefore nature conservation measures, which focused on the improvement of local habitat quality so far, additionally need to consider adjacent habitat type.
3) In addition to carabid beetle communities we also surveyed predation rates of ground-dwelling predators on the same calcareous grasslands in two study periods (June and late August). As ground-dwelling predators of forests or crop fields can move into adjacent calcareous grasslands we expected different predation rates depending on adjacent habitat type. We exposed in total 32.000 lady bird eggs as prey items on the calcareous grasslands in distances of 5 and 20m from the habitat border. We found higher predation rates on calcareous grasslands adjacent to forests than on calcareous grasslands adjacent to crop fields, but only on cool days. On warm days a very high extent (often 100%) of the exposed prey items were consumed adjacent to both habitat types, which did not allow the detection of possible differences between the adjacent habitat types. Predation rates differed not between the two study periods or the two distances to the habitat edge. The higher predation rates adjacent to forests can be explained by the spillover of ground-dwelling predators from forests into calcareous grasslands. Our results show, that spillover into semi-natural habitats affects ecosystem functioning in addition to species composition and diversity.
4) In chapter 4 of this thesis we examined the effect of spatiotemporal changes in crop cover on pest - natural enemy interactions and crop yields. During two study years we surveyed the abundance of adult and larval pollen beetles, parasitism of pollen beetle larvae by a hymenopteran parasitoid and oilseed rape yields of 36 oilseed rape fields. The surrounding landscape of the fields (1 km radius) differed in the oilseed rape proportion and in the inter-annual change in the oilseed rape proportion since the previous year. We found a dilution effect, i.e. a decreasing abundance with increasing oilseed rape proportions, for pollen beetle larvae and parasitoids in both study years and for adult pollen beetles in one study year. Oilseed rape yields increased with increasing oilseed rape proportions. Inter-annual changes in oilseed rape proportions led to inter-annual crowding and dilution effects for pollen beetles, but had no effect on parasitism or yield. Our results indicate the potential to reduce pest loads and increase yields in intensively managed oilseed rape fields by a coordinated management of the spatiotemporal oilseed rape cover in the landscape.
5) In summary, we showed in this thesis that the biodiversity and functioning of crop and non-crop habitats within agricultural landscapes is affected by the spillover of organisms and thus by the habitat composition in the close surrounding and in the broader landscape context. Spillover affects also ecosystem services and disservices and therefore crop productivity. Thereby the spatial and temporal variation of specific crop types in the landscape can be of particular importance for crop yields. Thus a coordinated landscape wide management can help to optimize both biodiversity conservation and the delivery of ecosystem services and thus crop yields. Future studies integrating landscape effects across several ecosystem functions, multiple taxonomic groups and different crop types are necessary to develop definite landscape management schemes.
Cancer is one of the leading causes of death. 90% of all deaths are caused by the effects of metastases. It is of major importance to successfully treat the primary tumor and metastases. Tumors and metastases often differ in their properties and therefore, treatment is not always successful. In contrast, those therapeutic agents can even promote formation and growth of metastases. Hence, it is indispensable to find treatment options for metastatic disease. One promising candidate represents the oncolytic virus therapy with vaccinia viruses.
The aim of this work was to analyze two cell lines regarding their metastatic abilities and to investigate whether oncolytic vaccinia viruses are useful therapy options. The cell lines used were the human cervical cancer cell line C33A implanted into immune-compromised mice and the murine melanoma cell line B16F10, implanted into immune-competent mice.
The initial point of the investigations was the observation of enlarged lumbar und renal lymph nodes in C33A tumor-bearing mice 35 days post implantation of C33A cells subcutaneously into immune-compromised nude mice. Subsequently, the presence of human cells in enlarged lymph nodes was demonstrated by RT-PCR. To facilitate the monitoring of cancer cell spreading, the gene encoding for RFP was inserted into the genome of C33A cells. In cell culture experiments, it was possible to demonstrate that this insertion did not negatively affect the susceptibility of the cells to virus infection, replication and virus-mediated cell lysis. The analysis of the metastatic process in a xenografted mouse model revealed the continuous progression of lumbar (LN) and renal (RN) lymph node metastasis after C33A-RFP tumor cell implantation. The lymph node volume and the amount of RFP-positive LNs and RNs was increasing from week to week in accordance with the gain of the primary tumor volume. Moreover, the metastatic spread of cancer cells in lymph vessels between lumbar and renal lymph nodes was visualized. Additionally, the haematogenous way of cancer cell migration was demonstrated by RFP positive cancer cells in blood vessels. The haematogenous route of spreading was confirmed by detecting micrometastases in lungs of tumor bearing mice.
The next step was to investigate whether the recombinant oncolytic vaccinia virus GLV-1h68 is a suitable candidate to cure the primary tumor and metastases. Therefore, GLV-1h68 was systemically injected into C33A-RFP tumor bearing mice 21 days after tumor cell implantation. It was demonstrated that the volume of the primary tumor was drastically reduced, and the volume and the amount of RFP positive lumbar and renal lymph nodes were significantly decreasing compared to the untreated control group. Subsequently, this process was analyzed further by investigating the colonization pattern in the C33A-RFP model. It was shown that first the primary tumor was colonized with highest detectable virus levels, followed by LN and RN lymph nodes. Histological analyses revealed the proliferative status of tumor cells in the tumor and lymph nodes, the amount of different immune cell populations and the vascular permeability in primary tumors and lymph nodes having an influence on the colonization pattern of the virus. Whereby, the vascular permeability seems to have a crucial impact on the preferential colonization of tumors compared to lymph node metastases in this tumor model.
C33A turned out to be a useful model to study the formation and therapy of metastases. However, a metastatic model in which the influence of the immune system on tumors and especially on tumor therapy can be analyzed would be preferable. Therefore, the aim of the second part was to establish a syngeneic metastatic mouse model.
Accordingly, the murine melanoma cell line B16F10 was analyzed in immunocompetent mice. First, the highly attenuated GLV 1h68 virus was compared to its parental strain LIVP 1.1.1 concerning infection, replication and cell lysis efficacy in cell culture. LIVP 1.1.1 was more efficient than GLV-1h68 and was subsequently used for following mouse studies. Comparative studies were performed, comparing two different implantation sites of the tumor cells, subcutaneously and footpad, and two different mouse strains, FoxN1 nude and C57BL/6 mice. Implantation into the footpad led to a higher metastatic burden in lymph nodes compared to the subcutaneous implantation site. Finally, the model of choice was the implantation of B16F10 into the footpad of immune-competent C57BL/6 mice. Furthermore, it was inevitable to deliver the virus as efficient as possible to the tumor and metastases. Comparison of two different injection routes, intravenously and intratumorally, revealed, that the optimal injection route was intratumorally. In summary, the murine B16F10 model is a promising model to study the effects of the immune system on vaccinia virus mediated therapy of primary tumors and metastases.
Glioblastoma multiforme (GBM) is one of the most frequent and malignant forms of brain cancer in adults. The prognosis is poor with a median survival time of 12-15 months. There is a broad range of alternative treatment options studied in preclinical and clinical trials for GBM. One alternative treatment option is oncolytic virotherapy, defined as the use of replication‐competent viruses that selectively infect and destroy cancer cells while leaving, non‐transformed cells unharmed. Vaccinia virus (VACV) is one favorable candidate. Although oncolytic viruses can kill tumor cells grown in vitro with high efficiency, they often exhibit reduced replication capacity in vivo suggesting that physiological aspects of the tumor microenvironment decrease the virus’ therapeutic potential. The percentage and composition of immune cells varies between cancer types and patients and is investigated as a biomarker in several studies. Making oncolytic virotherapy successful for GBM, it is necessary to understand the individual tumor biology, the interaction with the microenvironment and immune system.
It was demonstrated that the attenuated VACV wild-type (wt) isolate LIVP 1.1.1 replicate and lyse the murine GL261 glioma cell line in vitro. In the following, the replication efficacy was characterized in a comparative approach in vivo. Immunocompetent C57BL/6 (wt) mice and immunodeficient mouse strains of different genetic background C57BL/6 athymic and Balb/c athymic mice were used. In addition, subcutaneous and intracranial locations were compared. The results revealed viral replication exclusively in Balb/c athymic mice with subcutaneous tumors but in none of the other models.
In the following, the tumor microenvironment of the subcutaneous tumor models at the time of infection was performed. The study showed that implantation of the same tumor cells in different mouse strains resulted in a different tumor microenvironment with a distinct composition of immune cells. Highest differences were detected between immunodeficient and immunocompetent mice. The study showed major differences in the expression of MHCII with strongest expression in C57BL/6 wt and weakest in Balb/c athymic tumors. In the following, the influence of the phenotypic change associated with the upregulation of MHCII on GL261 tumor cells on viral replication was analyzed. Comparison of C57BL/6 wt and C57BL/6 IFN-γ knockout mice revealed endogenous IFN-γ levels to upregulate MHCII on GL261 tumor cells and to reduce viral replication in C57BL/6 wt mice. Analysis of single cell suspensions of tumor homogenates of C57BL/6 and Balb/c athymic mice showed that the IFN-γ-mediated anti-tumor effect was a reversible effect. Furthermore, reasons for inhibition of virus replication in orthotopic glioma models were elucidated. By immunohistochemical analysis it was shown that intratumoral amounts of Iba1+ microglia and GFAP+ astrocytes in Gl261 gliomas was independent from intratumoral VACV injection. Based on these findings virus infection in glioma, microglia and astrocytes was compared and analyzed in cell culture. In contrast to the GL261 glioma cells, replication was barely detectable in BV-2 microglia and IMA2.1 astrocytic cells. Co-culture experiments revealed that microglia compete for virus uptake in cell culture. It was further shown that BV-2 cells showed apoptotic characteristics after VACV infection while GL261 cells showed signs of necrotic cell death. Additionally, in BV-2 cells with M1-phenotype a further reduction of viral replication and inhibition of cell lysis was detected. Infection of IMA 2.1 cells was independent of the M1/M2-phenotype. Application of BV-2 microglia with M1-phenotype onto organotypic slice cultures with implanted GL261 tumors resulted in reduced infection of BV-2 cells with LIVP 1.1.1, whereas GL261 cells were significantly infected.
Taken together, the analyzed GL261 tumors were imprinted by the immunologic and genetic background in which they grow. The experimental approach applied in this thesis can be used as suitable model which reflects the principles of personalized medicine
In an additional project, based on gene expression data and bioinformatic analyses, the biological role and function of the anti-apoptotic factor AVEN was analyzed with regard to oncolytic VACV therapy. Besides a comparison of the replication efficacy of GLV-1h68 and VACV-mediated cell killing of four human tumor cell lines, it was shown that AVEN was expressed in all analyzed cells. Further, shown for HT-29 and 1936-MEL, the knockdown of AVEN by siRNA in cell culture resulted in an increase of apoptotic characteristics and a decrease of VACV infection. These findings provide essential insights for future virus development.
Molecular and cellular cross talk between angiogenic, immune and DNA mismatch repair pathways
(2015)
VEGF is a main driver of tumor angiogenesis, playing an important role not only in the formation of new blood vessels, but also acts as a factor for cell migration, proliferation, survival and apoptosis. Angiogenesis is a universal function shared by most solid tumors and its inhibition was thought to have the potential to work across a broad patient population. Clinical evidence has shown that inhibiting pathological angiogenesis only works in a subset of patients and the identification of those patients is an important step towards personalized cancer care. The first approved antiangiogenic therapy was bevacizumab (Avastin®), a monoclonal antibody targeting VEGF in solid tumors including CRC, BC, NSCLC, RCC and others.
In addition to endothelial cells, VEGF receptors are present on a number of different cell types including tumor cells, monocytes and macrophages. The work presented in this thesis looked at the in vitro cellular changes in tumor cells and leukocytes in response to the inhibition of VEGF signaling with the use of bevacizumab. In the initial experiments, VEGF was induced by hypoxia in tumor cells to evaluate changes in survival, proliferation, migration and changes in gene or protein expression. There was a minimal direct response of VEGF inhibition in tumor cells that could be attributed to bevacizumab treatment, with minor variations in some of the cell lines screened but no uniform or specific response noted.
MMR deficiency often results in microsatellite instability (MSI) in tumors, as opposed to microsatellite stable (MSS) tumors, and accounts for up to 15% of colorectal carcinomas (CRCs). It has been suggested in clinical data that MMR deficient tumors responded better to bevacizumab regimens, therefore further research used isogenic paired CRC tumor cell lines (MMR deficient and proficient). Furthermore, a DNA damaging agent was added to the treatment regimen, the topoisomerase inhibitor SN-38 (the active metabolite of irinotecan). Inhibiting VEGF using bevacizumab significantly inhibited the ability of MMR deficient tumor cells to form anchor dependent colonies, however conversely, bevacizumab treatment before damaging cells with SN-38, showed a significant increase in colony numbers. Moreover, VEGF inhibition by bevacizumab pretreatment also significantly increased the mutation fraction in MMR deficient cells as measured by transiently transfecting a dinucleotide repeat construct, suggesting VEGF signaling may have an intrinsic role in MMR deficient cells. A number of pathways were analyzed in addition to changes in gene expression profiles resulting in the identification of JNK as a possible VEGF targeted pathway. JUN expression was also reduced in these conditions reinforcing this hypothesis, however the intricate molecular mechanisms remain to be elucidated.
In order to remain focused on the clinical application of the findings, it was noted that some cytokines were differentially regulated by bevacizumab between MMR proficient and deficient cells. Treatment regimens employed in vitro attempted to mimic the clinical setting by inducing DNA damage, then allowing cells to recover with or without VEGF using bevacizumab treatment. Inflammatory cytokines, CCL7 and CCL8, were found to have higher expression in the MMR deficient cell line with bevacizumab after DNA damage, therefore the cross talk via tumor derived factors to myeloid cells was analyzed. Gene expression changes in monocytes induced by tumor conditioned media showed CCL18 to be a bevacizumab regulated gene by MMR deficient cells and less so in MMR proficient cells. CCL18 has been described as a prognostic marker in gastric, colorectal and ovarian cancers, however the significance is dependent on tumor type. CCL18 primarily exerts its function on the adaptive immune system to trigger a TH2 response in T cells, but is also described to increase non-specific phagocytosis. The results of this study did show an increase in the phagocytic activity of macrophages in the presence of bevacizumab that was significantly more apparent in MMR deficient cells. Furthermore, after DNA damage MMR deficient cells treated with bevacizumab released a cytokine mix that induced monocyte migration in a bevacizumab dependent manner, showing a functional response with the combination of MMR deficiency and bevacizumab. In summary, the work in this thesis has shown evidence of immune cell modulation that is specific to MMR deficient tumor cells that may translate into a marker for the administration of bevacizumab in a clinical setting.
VEGF ist ein zentraler Regulator der Tumor-Angiogenese, und spielt eine wichtige Rolle nicht nur in der Bildung von neuen Blutgefäßen, sondern ist auch für die Migration, Proliferation, das Überleben und Apoptose von Tumorzellen essentiell. Angiogenese ist eine der universellen Funktionen, welche das Wachstum der meisten soliden Tumoren charakterisiert. Eine der klassischen therapeutischen Ideen wurde auf der Basis entwickelt, dass die spezifische Hemmung der Angiogenese das Potenzial hat in einer breiten Patientenpopulation einen klinischen Effekt zu zeigen. Die klinische Erfahrung und Anwendung hat jedoch gezeigt, dass die Hemmung der pathologischen Angiogenese nur in einem Teil der Patienten einen therapeutischen Nutzen aufweist. Somit stellt die Identifikation derjenigen Patienten, welche von der anti-angiogenen Therapie profitieren, einen wichtiger Schritt zur personalisierten Krebsbehandlung dar. Die erste zugelassene antiangiogene Therapie war Bevacizumab (Avastin®), ein monoklonaler Antikörper gegen VEGF, welcher unter anderem in soliden Tumoren wie CRC, BC, nicht-kleinzelligem Lungenkrebs (NSCLC) und dem Nierenzellkarzinom angewandt wird.
VEGF-Rezeptoren befinden sich nicht nur auf Endothelzellen, sondern sind auch auf einer Anzahl von verschiedenen Zelltypen, einschließlich Tumorzellen, Monozyten und Makrophagen nachweisbar. Die in dieser Arbeit vorgestellten Ergebnisse befassen sich mit den zellulären Veränderungen an Tumorzellen und Leukozyten als Reaktion auf die Hemmung der VEGF-Signalkaskade durch Bevacizumab in-vitro. In den Initialen Experimenten wurde VEGF durch Hypoxie in Tumorzellen induziert und Veränderungen der Überlebensrate, der Proliferation, Migration als auch in der Gen- oder Protein-Expression gemessen. Es konnte eine minimale direkte Reaktion der VEGF-Hemmung auf Tumorzellen beobachtet werden, welche auf die Bevacizumab Behandlung zurückgeführt werden könnte. Es zeigten sich aber auch geringfügige Abweichungen in einigen der verwendeten Zellinien, die keine einheitliche Interpretation erlauben oder auf eine uniformelle Reaktion hinweisen würden.
Das phänotypische Korrelat einer „Mismatch“ Reparatur (MMR)-Defizienz ist die Mikrosatelliteninstabilität im Gegensatz zu mikrosatellitenstabilen Tumoren und findet sich bei bis zu 15% der kolorektalen Karzinomen (CRC) wieder. Klinischen Daten deuten daraufhin, dass Bevacizumab besser in MMR-defizienten Tumoren wirkt. Daher wurden die weiteren Untersuchungen in gepaarten MMR stabilen und MMR instabilen CRC-Tumorzelllinien (MMR defizient und kompetent) durchgeführt. Weiterhin wurde ein DNA-schädigendes Agens, SN-38, ein Topoisomerase-Inhibitor (der aktive Metabolit von Irinotecan) dem Behandlungsschema zugefügt. Es zeigte sich, dass die Hemmung von VEGF mittels Bevacizumab die Fähigkeit der MMR defizienten Tumorzellen Kolonien zu bilden signifikant inhibiert. Im Gegensatz dazu, hatte die Behandlung von Bevacizumab vor der Zugabe des DNA schädigenden Agens zu einer vermehrten Kolonienzahl geführt. Außerdem erhöhte die Vorbehandlung mit Bevacizumab deutlich die Mutationsrate in MMR-defizienten Zellen, was durch die transiente Transfektion eines Dinukleotid-Repeat-Konstrukts nachgewiesen werden konnte. Dies deutete darauf hin, dass VEGF eine intrinsische Rolle in der Signalkaskade des MMR-Systems haben könnte. Deshalb wurde eine Anzahl von Signalalkaskaden zusätzlich zu Veränderungen von Genexpressionsprofilen untersucht und JNK als mögliche Verbindungsstelle der beiden Signalkaskaden, VEGF und MMR, identifiziert. Diese Hypothese wurde zusätzlich unterstützt durch die Tatsache, dass die JUN Expression unter diesen experimentellen Bedingungen reduziert war. Die Aufklärung der komplexen molekularen Mechanismen der potentiellen Interaktion bleibt zukünftigen Untersuchungen vorbehalten.
In Hinblick auf die klinische Konsequenz der erhaltenen Ergebnisse war es auffällig, dass einige Zytokine durch Bevacizumab in den MMR defizienten Zellen im Gegensatz zu den MMR kompetenten Zellen unterschiedlich reguliert wurden. Die in-vitro verwendeten Behandlungsschemata waren den klinisch zur Anwendung kommenden Protokollen nachempfunden. Zuerst wurde ein DNA-Schaden gesetzt, und den Zellen ermöglicht, sich mit oder ohne Bevacizumab zu erholen. Es konnte gezeigt werden, dass die inflammatorischen Zytokine CCL7 und CCL8 eine höhere Expression in der MMR-defiziente Zelllinie in Kombination mit Bevacizumab aufweisen. Daher wurde ein möglicher Crosstalk zwischen von Tumorzellen sezernierten Faktoren und myeloischen Zellen weiter verfolgt. Veränderungen der Genexpression in Monozyten durch Tumorzell- konditionierte Medien zeigte CCL18 als ein Bevacizumab reguliertes Gen in MMR-defizienten Zellen, aber nicht in MMR kompetenten Zellen. CCL18 übt seine Funktion primär im adaptiven Immunsystems aus um eine TH2-Antwort in T-Zellen auszulösen Ausserdem wird eine Erhöhung der nicht-spezifische Phagozytose als weitere Funktion beschrieben. CCL18 wurde bereits als prognostischer Marker in Magen-, Dickdarm- und Eierstockkrebsarten beschrieben; die klinische Bedeutung ist jedoch abhängig von Tumortyp.
Die Ergebnisse dieser Arbeit zeigen, dass eine Erhöhung der phagozytischen Aktivität von Makrophagen in Gegenwart von Bevacizumab wesentlich deutlicher in MMR-defizienten Zellen ausgeprägt war. Weiterhin wurde gefunden, dass nach DNA-Schädigung in Bevacizumab behandelten MMR-defizienten Zellen Zytokine freigesetzt werden, welche eine Monozytenmigration in einer Bevacizumab-abhängigen Weise induzieren. Dies weist auf eine funktionelle Interaktion von MMR-Defizienz und Bevacizumab hin. Zusätzlich zeigen die Ergebnisse dieser Arbeit eine Immunzellmodulation, die spezifisch für Mismatch-Reparatur defiziente Tumorzellen ist und in der klinischen Praxis als Marker für die Verabreichung von Bevacizumab verwendet werden könnte.
Infertilität stellt in unserer heutigen Gesellschaft ein zunehmendes Problem dar. Bei der Suche nach den der Infertilität zugrunde liegenden Ursachen gerät immer mehr die Epigenetik in den Fokus. Epigenetische Prozesse sind nicht nur in die Embryo-nalentwicklung und Wachstumsprozesse des Kindes involviert, sondern auch in korrekte Funktionsweisen von Gameten. Störungen können die Fertilität beeinträch-tigen. Eine besondere Rolle spielen geprägte Gene, die auf einem ihrer Allele, je nach parentaler Herkunft, ein Imprint in Form von DNA-Methylierung tragen. Fehl-regulationen solcher geprägter Gene können zu Imprinting-Erkrankungen führen.
Seit Einführung der In-vitro-Fertilisation (IVF) wurden verschiedene assistierte Re-produktionstechniken (ART) entwickelt, um infertilen Paaren zu helfen. Die Sicher-heit dieser Techniken ist nicht abschließend geklärt. Immer wieder wird von nach ART-Behandlung gehäuft auftretenden Imprinting-Erkrankungen berichtet. Diese Erkrankungen stehen jedoch eher in Zusammenhang mit der zugrunde liegenden Infertilität, als mit ART selbst. Dennoch ist es notwendig zu untersuchen inwieweit sich ART eventuell auf den Gesundheitszustand dieser Kinder auswirken könnte.
In der hier vorgelegten Arbeit wurde der Zusammenhang von Epigenetik, Infertilität und ART von verschiedenen Standpunkten aus beleuchtet.
In humanen Spermien wurde die DNA-Methylierung verschiedener geprägter Gene hinsichtlich Epimutationen untersucht. ICSI (intracytoplasmatic sperm injection) und IMSI (intracytoplasmic morphologically selected sperm injection) sind verschiedene Techniken zur Selektion von Spermien für eine ART-Behandlung. Hier wurde un-tersucht, ob IMSI epigenetisch bessere Spermien selektiert als die konventionelle ICSI-Methode. Außerdem ist bekannt, dass in Spermienköpfen fertiler und infertiler Männer Vakuolen vorkommen können, deren epigenetische Bedeutung jedoch un-bekannt ist. Ob diese Vakuolen in Zusammenhang mit Epimutationen stehen könn-ten, wurde ebenfalls überprüft. Dazu wurde bisulfitkonvertierte DNA weniger Sper-mien (11 je Probe) mithilfe der Limiting Dilution (LD)–Technik und Pyrosequenzie-rung analysiert. Insgesamt standen 880 Spermien für diese Untersuchung zur Ver-fügung. Es konnte kein Unterschied zwischen IMSI- und ICSI-selektierten Spermien gefunden werden. Vorhandene Vakuolen im Spermienkopf beeinträchtigten nicht die DNA-Methylierung der Gene hGTL2, hLIT1 und hPEG3.
Ein weiteres Projekt befasste sich mit der Frage, inwieweit die Technik der In-vitro-Maturation (IVM) DNA-Methylierung in humanen Oocyten beeinflussen könnte. Bisulfitkonvertierte DNA einzelner humaner Oocyten wurde mit LD und Pyrose-quenzierung analysiert. Verglichen wurden IVM und in vivo gereifte Oocyten. Hier-für standen 139 Oocyten zur Verfügung, wovon 90 mittels IVM und 49 in vivo gereift waren. Untersucht wurden vier geprägte Gene (hGTL2, hLIT1, hPEG3 und hSNRPN) und drei nicht geprägte Gene (hDNMT3Lo, hNANOG und hOCT4). Es konnten keine IVM-bedingten Epimutationen gefunden werden.
Im dritten Projekt wurde untersucht, ob sich die DNA-Methylierung normaler Sper-mien von Spermien aus Oligozoospermie-Asthenozoospermie-Teratozoospermie (OAT)–Syndrom-Patienten unterscheidet. Eine weitere Frage war, ob Epimutationen einen Einfluss auf den ART-Ausgang haben. Untersucht wurden 54 Spermienpro-ben von Paaren in ART-Behandlung. Zur Untersuchung der DNA-Methylierungsmuster der geprägten Gene hGTL2 und hPEG3 sowie der beiden nicht geprägten Pluripotenzgene hNANOG und hOCT4 wurde die Methode Deep Bisulfite Sequencing (DBS) verwendet. Dies ist eine Next Generation Sequencing (NGS)–Technik, angewandt an bisulfitkonvertierter DNA. Diese Technik ermöglicht es mehrere Proben sowie Gene gleichzeitig zu analysieren. Es zeigte sich, dass OAT-Spermien, die zu einer Lebendgeburt geführt hatten, sich epigenetisch nicht von normalen Spermien unterschieden. Besonders viele Epimutationen konnten hingegen in OAT-Spermien gefunden werden, die zu keiner Schwangerschaft ge-führt hatten. Zwischen Spermien die zu einer Lebendgeburt oder keiner Schwan-gerschaft geführt hatten, zeigten sich Unterschiede in der Häufigkeit von hGTL2-Epimutationen. Über die Häufigkeit von Epimutationen konnte eine prädiktive Aus-sage zum ART-Ausgang getroffen werden.
Zusammenfassend konnte in dieser Arbeit festgestellt werden, dass sich eine Häu-fung von Epimutationen darauf auswirkt, ob eine Schwangerschaft erreicht werden kann oder nicht. Diese Epimutationen liegen bereits im parentalen Genom vor. Sie werden nicht durch ART verursacht. Allerdings muss man Techniken finden, mit denen man Gameten mit möglichst wenig Epimutationen selektiert, um eine Über-tragung solcher auf das Kind zu verhindern.
Summary (English)
I. Human induced global change threatens biodiversity and trophic interactions. Fragmentation is considered as one of the major threats to biodiversity and can cause reduced species richness, population declines, loss of genetic diversity and disruption of trophic interactions such as predation and parasitism. However forest fragmentation effects can be eclectic due to species specific traits. Specialist species with narrower niches or at higher trophic levels may be in danger of extinction whereas generalist species with less specific habitat requirements may even profit from fragmentation. In the tropics, known as “the” terrestrial biodiversity hotspots, even biodiversity inventories are often lacking, especially in forest canopies. Ongoing deforestation and resulting fragmentation in tropical regions are expected to heavily affect ecosystem functions by changes in biodiversity, community compositions and disruption of trophic interactions. It is even less unknown in what extent different global change drivers for example climate change and fragmentation interact. It is unlikely that deforestation will end, so that small secondary forest fragments will be important habitat elements that must be investigated to optimize their potential contribution to biodiversity conservation.
This dissertation aimed to disentangle the effects of forest fragmentation on trap-nesting bee and wasp communities in small secondary forest fragments addressing the following main questions:
1) Are there interactive effects between microclimate and fragmentation on the abundance of bees and wasps, their mortality - and parasitism rates (Chapter II)?
2) How does fragmentation affect bee biodiversity from canopy to the understory with considerations of single species patterns (Chapter III)?
3) How is fragmentation affecting diversity and community composition of different trophic levels between understory and canopy with emphasis on the host-antagonist relation? (Chapter IV).
II. A variety of global change drivers affect biodiversity and trophic interactions. The combined effects of habitat fragmentation and climate change are poorly understood and with ongoing deforestation and agricultural intensification secondary rainforest fragments might contribute to biodiversity conservation and mitigation of climate warming. This chapter investigated the interactive effects of habitat fragmentation and microclimate on the abundance and biotic interactions of trap-nesting bees and wasps in secondary forest fragments in the Northeastern lowlands of Costa Rica.
Habitat area did not affect hymenopteran abundance, parasitism and mortality rates, but tree location- from the forest border to the forest center- influenced all variables. Interactive effects were found such as in the higher mortality rates at interior locations in larger fragments. Mean temperature at edge and interior locations led to significant effects on all tested variables and interactive effects between temperature and tree locations were found. Abundances at interior locations were significantly higher with increasing temperatures. Mortality rates at interior location increased at lower mean temperatures, whereas higher temperatures at edges marginally increased mortality rates. Our results indicate, that edge effects, mediated by altered microclimatic conditions, significantly change biotic interactions of trap-nesting hymenopterans in small secondary fragments.
III. This chapter focusses on the vertical distribution of bees, their parasitism and mortality rates as well as single species patterns in relation to fragment size and edge effects in secondary rainforest remnants.
No size effects on bee abundance, bee diversity and on parasitism- and mortality rates were found. Bees were least abundant at the intermediate height and were most abundant in the understory; whereas the highest diversity was found in the canopy. Tree location had no effect on bee abundance, but on bee diversity since most species were found in the forest interior. The cuckoo bees Aglaomelissa duckei and Coelioxys sp. 1 only partly followed the patterns of their hosts, two Centris species.
Edge effects greatly influenced the bee community, so that the amount of edge habitat in secondary forest fragments will influence the conservation value for bees.
IV. In this section the effects of habitat fragmentation on biodiversity, on community structure of hosts and natural enemies as well as the relation of hosts and antagonists were investigated from the understory to the canopy. The results stress the importance to monitor biodiversity, community composition and trophic interactions from the understory to the canopy. The higher trophic level of the antagonists was found to be more sensitive to fragment size compared to their hosts. Again edge effects were found to be the dominant driver since both host and antagonist richness, as well as community compositions were strongly affected. Ongoing fragmentation and increased amount of edge habitat could favor few abundant disturbance-adapted species over the rare and more diverse forest-adapted species. A positive-density dependent parasitism rate was demonstrated, as well as an increase of the parasitism rate not only with antagonist abundance but also diversity.
Small secondary forest fragments surely can contribute to the conservation of biodiversity and trophic interactions, but increase of edge habitat will have negative consequences on above-ground nesting Hymenoptera, so that important interactions such as pollination, predation and parasitism could be disrupted. Therefore small forest fragments could contribute to biodiversity conservation but will not be able to compensate for the loss of large areas of primary forests.
V. This dissertation contributes to the understanding of habitat area - and edge effects as well as the interaction of those with microclimatic conditions in small secondary rainforest fragments. As study system trap nests inhabited by solitary above-ground nesting bees, wasps and their natural enemies were chosen because they allow to study trophic interactions along their whole vertical distribution from the understory to the canopy. The effect of fragment size was rather weak, however, larger sizes affected the diversity of natural enemies positively, proofing the hypothesis that higher trophic levels react more sensitive to habitat loss. Edge effects heavily affected the abundance, diversity and community composition of hosts and their natural enemies as well as parasitism and mortality rates. Increased edge conditions resulting from ongoing fragmentation and deforestation will therefore negatively affect bees, wasps and their trophic interactions with natural enemies. Those changes affect important processes such as pollination, predation and parasitism, which could result in changes of ecosystem functioning. This study showed the importance to include all strata in biodiversity monitoring since height did matter for the trap-nesting communities. Diversity was shown to be higher in the canopy and community composition did change significantly. To conclude we could show that secondary forest fragments can sustain a trap-nesting bee and wasp community, but the amount of interior habitat is highly important for the conservation of forest-adapted species. Probably the conservation of large primary forest in combination with a high habitat connectivity, for example with small secondary forest fragments, will help to sustain biodiversity and ecosystem functioning better than the mere presence of small forest fragments.
Freshly cut beech deadwood was enriched in the canopy and on the ground in three cultural landscapes in Germany (Swabian Alb, Hainich-Dun, Schorfheide-Chorin) in order to analyse the diversity, distribution and interaction of wood-inhabiting fungi and beetles. After two years of wood decay 83 MOTUs (Molecular Operational Taxonomic Units) from 28 wood samples were identified. Flight Interception Traps (FITs) installed adjacent to the deadwood enrichments captured 29.465 beetles which were sorted to 566 species. Geographical 'region' was the main factor determining both beetle and fungal assemblages. The proportions of species occurring in all regions were low. Statistic models suggest that assemblages of both taxa differed between stratum and management praxis but their strength varied among regions. Fungal assemblages in Hainich-Dun, for which the data was most comprehensive, discriminated unmanaged from extensively managed and age-class forests (even-aged timber management) while canopy communities differed not from those near the ground. In contrast, the beetle assemblages at the same sites showed the opposite pattern. We pursued an approach in the search for fungus-beetle associations by computing cross correlations and visualize significant links in a network graph. These correlations can be used to formulate hypotheses on mutualistic relationships for example in respect to beetles acting as vectors of fungal spores.
Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.
G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. Our study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. We show that dCirl shapes the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. Our results indicate that aGPCR may generally process and modulate the perception of mechanical signals, linking these important stimuli to the sensory canon of the GPCR superfamily.
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ~17 cGMPs\(^{-1}\)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O\(_2\)/CO\(_2\) sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.
Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
Fungal microorganisms frequently lead to life-threatening infections. Within this group of pathogens, the commensal Candida albicans and the filamentous fungus Aspergillus fumigatus are by far the most important causes of invasive mycoses in Europe. A key capability for host invasion and immune response evasion are specific molecular interactions between the fungal pathogen and its human host. Experimentally validated knowledge about these crucial interactions is rare in literature and even specialized host pathogen databases mainly focus on bacterial and viral interactions whereas information on fungi is still sparse. To establish large-scale host fungi interaction networks on a systems biology scale, we develop an extended inference approach based on protein orthology and data on gene functions. Using human and yeast intraspecies networks as template, we derive a large network of pathogen host interactions (PHI). Rigorous filtering and refinement steps based on cellular localization and pathogenicity information of predicted interactors yield a primary scaffold of fungi human and fungi mouse interaction networks. Specific enrichment of known pathogenicity-relevant genes indicates the biological relevance of the predicted PHI. A detailed inspection of functionally relevant subnetworks reveals novel host fungal interaction candidates such as the Candida virulence factor PLB1 and the anti-fungal host protein APP. Our results demonstrate the applicability of interolog-based prediction methods for host fungi interactions and underline the importance of filtering and refinement steps to attain biologically more relevant interactions. This integrated network framework can serve as a basis for future analyses of high-throughput host fungi transcriptome and proteome data.
Control of genetic regulatory networks is challenging to define and quantify. Previous control centrality metrics, which aim to capture the ability of individual nodes to control the system, have been found to suffer from plausibility and applicability problems. Here we present a new approach to control centrality based on network convergence behaviour, implemented as an extension of our genetic regulatory network simulation framework Jimena (http://stefan-karl.de/jimena). We distinguish three types of network control, and show how these mathematical concepts correspond to experimentally verified node functions and signalling pathways in immunity and cell differentiation: Total control centrality quantifies the impact of node mutations and identifies potential pharmacological targets such as genes involved in oncogenesis (e.g. zinc finger protein GLI2 or bone morphogenetic proteins in chondrocytes). Dynamic control centrality describes relaying functions as observed in signalling cascades (e.g. src kinase or Jak/Stat pathways). Value control centrality measures the direct influence of the value of the node on the network (e.g. Indian hedgehog as an essential regulator of proliferation in chondrocytes). Surveying random scale-free networks and biological networks, we find that control of the network resides in few high degree driver nodes and networks can be controlled best if they are sparsely connected.
How allopolyploids are able not only to cope but profit from their condition is a question that remains elusive, but is of great importance within the context of successful allopolyploid evolution. One outstanding example of successful allopolyploidy is the endemic Iberian cyprinid Squalius alburnoides. Previously, based on the evaluation of a few genes, it was reported that the transcription levels between diploid and triploid S. alburnoides were similar. If this phenomenon occurs on a full genomic scale, a wide functional "diploidization'' could be related to the success of these polyploids. We generated RNA-seq data from whole juvenile fish and from adult livers, to perform the first comparative quantitative transcriptomic analysis between diploid and triploid individuals of a vertebrate allopolyploid. Together with an assay to estimate relative expression per cell, it was possible to infer the relative sizes of transcriptomes. This showed that diploid and triploid S. alburnoides hybrids have similar liver transcriptome sizes. This in turn made it valid to directly compare the S. alburnoides RNA-seq transcript data sets and obtain a profile of dosage responses across the S. alburnoides transcriptome. We found that 64% of transcripts in juveniles' samples and 44% in liver samples differed less than twofold between diploid and triploid hybrids (similar expression). Yet, respectively 29% and 15% of transcripts presented accurate dosage compensation (PAA/PA expression ratio of 1 instead of 1.5). Therefore, an exact functional diploidization of the triploid genome does not occur, but a significant down regulation of gene expression in triploids was observed. However, for those genes with similar expression levels between diploids and triploids, expression is not globally strictly proportional to gene dosage nor is it set to a perfect diploid level. This quantitative expression flexibility may be a strong contributor to overcome the genomic shock, and be an immediate evolutionary advantage of allopolyploids.
The flagellate Trypanosoma brucei, which causes the sleeping sickness when infecting a mammalian host, goes through an intricate life cycle. It has a rather complex propulsion mechanism and swims in diverse microenvironments. These continuously exert selective pressure, to which the trypanosome adjusts with its architecture and behavior. As a result, the trypanosome assumes a diversity of complex morphotypes during its life cycle. However, although cell biology has detailed form and function of most of them, experimental data on the dynamic behavior and development of most morphotypes is lacking. Here we show that simulation science can predict intermediate cell designs by conducting specific and controlled modifications of an accurate, nature-inspired cell model, which we developed using information from live cell analyses. The cell models account for several important characteristics of the real trypanosomal morphotypes, such as the geometry and elastic properties of the cell body, and their swimming mechanism using an eukaryotic flagellum. We introduce an elastic network model for the cell body, including bending rigidity and simulate swimming in a fluid environment, using the mesoscale simulation technique called multi-particle collision dynamics. The in silico trypanosome of the bloodstream form displays the characteristic in vivo rotational and translational motility pattern that is crucial for survival and virulence in the vertebrate host. Moreover, our model accurately simulates the trypanosome's tumbling and backward motion. We show that the distinctive course of the attached flagellum around the cell body is one important aspect to produce the observed swimming behavior in a viscous fluid, and also required to reach the maximal swimming velocity. Changing details of the flagellar attachment generates less efficient swimmers. We also simulate different morphotypes that occur during the parasite's development in the tsetse fly, and predict a flagellar course we have not been able to measure in experiments so far.
The incidence of malignant melanoma continues to increase each year with poor prognosis for survival in many relapse cases. To reverse this trend, whole body response measures are needed to discover collaborative paths to primary and secondary malignancy. Several species of fish provide excellent melanoma models because fish and human melanocytes both appear in the epidermis, and fish and human pigment cell tumors share conserved gene expression signatures. For the first time, we have examined the whole body transcriptome response to invasive melanoma as a prelude to using transcriptome profiling to screen for drugs in a medaka (Oryzias latipes) model. We generated RNA-seq data from whole body RNA isolates for controls and melanoma fish. After testing for differential expression, 396 genes had significantly different expression (adjusted p-value <0.02) in the whole body transcriptome between melanoma and control fish; 379 of these genes were matched to human orthologs with 233 having annotated human gene symbols and 14 matched genes that contain putative deleterious variants in human melanoma at varying levels of recurrence. A detailed canonical pathway evaluation for significant enrichment showed the top scoring pathway to be antigen presentation but also included the expected melanocyte development and pigmentation signaling pathway. Results revealed a profound down-regulation of genes involved in the immune response, especially the innate immune system. We hypothesize that the developing melanoma actively suppresses the immune system responses of the body in reacting to the invasive malignancy, and that this mal-adaptive response contributes to disease progression, a result that suggests our whole-body transcriptomic approach merits further use. In these findings, we also observed novel genes not yet identified in human melanoma expression studies and uncovered known and new candidate drug targets for further testing in this malignant melanoma medaka model.
Sigma factor SigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections
(2015)
Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, \(\Delta\)sigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.
Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ), and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression). Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3' UTR of the aminomethyl transferase (amt) gene of the closely related platyfish (Xiphophorus maculatus). This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.
Swords are exaggerated male ornaments of swordtail fishes that have been of great interest to evolutionary biologists ever since Darwin described them in the Descent of Man (1871). They are a novel sexually selected trait derived from modified ventral caudal fin rays and are only found in the genus Xiphophorus. Another phylogenetically more widespread and older male trait is the gonopodium, an intromittent organ found in all poeciliid fishes, that is derived from a modified anal fin. Despite many evolutionary and behavioral studies on both traits, little is known so far about the molecular mechanisms underlying their development. By investigating transcriptomic changes (utilizing a RNA-Seq approach) in response to testosterone treatment in the swordtail fish, Xiphophorus hellerii, we aimed to better understand the architecture of the gene regulatory networks underpinning the development of these two evolutionary novelties. Large numbers of genes with tissue-specific expression patterns were identified. Among the sword genes those involved in embryonic organ development, sexual character development and coloration were highly expressed, while in the gonopodium rather more morphogenesis-related genes were found. Interestingly, many genes and genetic pathways are shared between both developing novel traits derived from median fins: the sword and the gonopodium. Our analyses show that a larger set of gene networks was co-opted during the development and evolution of the older gonopodium than in the younger, and morphologically less complex trait, the sword. We provide a catalog of candidate genes for future efforts to dissect the development of those sexually selected exaggerated male traits in swordtails.
Dysregulated IGFBP5 expression causes axon degeneration and motoneuron loss in diabetic neuropathy
(2015)
Diabetic neuropathy (DNP), afflicting sensory and motor nerve fibers, is a major complication in diabetes.The underlying cellular mechanisms of axon degeneration are poorly understood. IGFBP5, an inhibitory binding protein for insulin-like growth factor 1 (IGF1) is highly up-regulated in nerve biopsies of patients with DNP. We investigated the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP5 in motor axons and sensory nerve fibers. These mice develop motor axonopathy and sensory deficits similar to those seen in DNP. Motor axon degeneration was also observed in mice in which the IGF1 receptor(IGF1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF1 on IGF1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP5 in diabetic nerves reduces the availability of IGF1 for IGF1R on motor axons, thus leading to progressive neurodegeneration. Inhibition of IGFBP5 could thus offer novel treatment strategies for DNP.
Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO\(^{-}\) mutant and carO\(^{+}\) control strains showed a faster development of light-exposed carO-germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.
In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells
(2015)
Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi-or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS\(^{61K}\) in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.
Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins
Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.
Trypanosoma brucei is a uniflagellated protist and the causative agent of African trypanosomiasis, a neglected tropical disease. The single flagellum of T. brucei is essential to a number of cellular processes such as motility, and has been a longstanding focus of scientific enquiry. A number of cytoskeletal structures are associated with the flagellum in T. brucei, and one such structure—a multiprotein complex containing the repeat motif protein TbMORN1—is the focus of this review. The TbMORN1-containing complex, which was discovered less than ten years ago, is essential for the viability of the mammalian-infective form of T. brucei. The complex has an unusual asymmetric morphology, and is coiled around the flagellum to form a hook shape. Proteomic analysis using the proximity-dependent biotin identification (BioID) technique has elucidated a number of its components. Recent work has uncovered a role for TbMORN1 in facilitating protein entry into the cell, thus providing a link between the cytoskeleton and the endomembrane system. This review summarises the extant data on the complex, highlights the outstanding questions for future enquiry, and provides speculation as to its possible role in a size-exclusion mechanism for regulating protein entry. The review additionally clarifies the nomenclature associated with this topic, and proposes the adoption of the term “hook complex” to replace the former name “bilobe” to describe the complex.
The diploid, polymorphic yeast Candida albicans is one of the most important human pathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within the human host for a long time. However, alterations in the host environment can render C. albicans virulent. In this review, we describe the immunological cross-talk between C. albicans and the human innate immune system. We give an overview in form of pairs of human defense strategies including immunological mechanisms as well as general stressors such as nutrient limitation, pH, fever etc. and the corresponding fungal response and evasion mechanisms. Furthermore, Computational Systems Biology approaches to model and investigate these complex interactions are highlighted with a special focus on game-theoretical methods and agent-based models. An outlook on interesting questions to be tackled by Systems Biology regarding entangled defense and evasion mechanisms is given.
The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knockdowns of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.
The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, "-omics" data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology.
Aphids are a major concern in agricultural crops worldwide, and control by natural enemies is an essential component of the ecological intensification of agriculture. Although the complexity of agricultural landscapes is known to influence natural enemies of pests, few studies have measured the degree of pest control by different enemy guilds across gradients in landscape complexity. Here, we use multiple natural-enemy exclosures replicated in 18 fields across a gradient in landscape complexity to investigate (1) the strength of natural pest control across landscapes, measured as the difference between pest pressure in the presence and in the absence of natural enemies; (2) the differential contributions of natural enemy guilds to pest control, and the nature of their interactions across landscapes. We show that natural pest control of aphids increased up to six-fold from simple to complex landscapes. In the absence of pest control, aphid population growth was higher in complex than simple landscapes, but was reduced by natural enemies to similar growth rates across all landscapes. The effects of enemy guilds were landscape-dependent. Particularly in complex landscapes, total pest control was supplied by the combined contribution of flying insects and ground-dwellers. Birds had little overall impact on aphid control. Despite evidence for intraguild predation of flying insects by ground-dwellers and birds, the overall effect of enemy guilds on aphid control was complementary. Understanding pest control services at large spatial scales is critical to increase the success of ecological intensification schemes. Our results suggest that, where aphids are the main pest of concern, interactions between natural enemies are largely complementary and lead to a strongly positive effect of landscape complexity on pest control. Increasing the availability of seminatural habitats in agricultural landscapes may thus benefit not only natural enemies, but also the effectiveness of aphid natural pest control.
Climate change can alter the phenology of organisms. It may thus lead seasonal organisms to face different day lengths than in the past, and the fitness consequences of these changes are as yet unclear. To study such effects, we used the pea aphid Acyrthosiphon pisum as a model organism, as it has obligately asexual clones which can be used to study day length effects without eliciting a seasonal response. We recorded life-history traits under short and long days, both with two realistic temperature cycles with means differing by 2 °C. In addition, we measured the population growth of aphids on their host plant Pisum sativum. We show that short days reduce fecundity and the length of the reproductive period of aphids. Nevertheless, this does not translate into differences at the population level because the observed fitness costs only become apparent late in the individual's life. As expected, warm temperature shortens the development time by 0.7 days/°C, leading to faster generation times. We found no interaction of temperature and day length. We conclude that day length changes cause only relatively mild costs, which may not decelerate the increase in pest status due to climate change.
Delivery of crop pollination services is an insufficient argument for wild pollinator conservation
(2015)
There is compelling evidence that more diverse ecosystems deliver greater benefits to people, and these ecosystem services have become a key argument for biodiversity conservation. However, it is unclear how much biodiversity is needed to deliver ecosystem services in a cost- effective way. Here we show that, while the contribution of wild bees to crop production is significant, service delivery is restricted to a limited subset of all known bee species. Across crops, years and biogeographical regions, crop-visiting wild bee communities are dominated by a small number of common species, and threatened species are rarely observed on crops. Dominant crop pollinators persist under agricultural expansion and many are easily enhanced by simple conservation measures, suggesting that cost- effective management strategies to promote crop pollination should target a different set of species than management strategies to promote threatened bees. Conserving the biological diversity of bees therefore requires more than just ecosystem-service-based arguments.
Electric shock is a common stimulus for nociception-research and the most widely used reinforcement in aversive associative learning experiments. Yet, nothing is known about the mechanisms it recruits at the periphery. To help fill this gap, we undertook a genome-wide association analysis using 38 inbred Drosophila melanogaster strains, which avoided shock to varying extents. We identified 514 genes whose expression levels and/or sequences covaried with shock avoidance scores. We independently scrutinized 14 of these genes using mutants, validating the effect of 7 of them on shock avoidance. This emphasizes the value of our candidate gene list as a guide for follow-up research. In addition, by integrating our association results with external protein-protein interaction data we obtained a shock avoidance- associated network of 38 genes. Both this network and the original candidate list contained a substantial number of genes that affect mechanosensory bristles, which are hairlike organs distributed across the fly's body. These results may point to a potential role for mechanosensory bristles in shock sensation. Thus, we not only provide a first list of candidate genes for shock avoidance, but also point to an interesting new hypothesis on nociceptive mechanisms.