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Peritonitis is a common disease in man, frequently caused by fungi, such as Candida albicans; however, in seldom cases opportunistic infections with Saccharomyces cerevisiae are described. Resident peritoneal macrophages (prMΦ) are the major group of phagocytic cells in the peritoneum. They express a broad range of surface pattern recognition receptors (PRR) to recognize invaders. Yeast infections are primarily detected by the Dectin-1 receptor, which triggers activation of NFAT and NF-κB pathways.
The transcription of the Nfatc1 gene is directed by the two alternative promoters, inducible P1 and relatively constitutive P2 promoter. While the role of P1-directed NFATc1α-isoforms to promote survival and proliferation of activated lymphocytes is well-established, the relevance of constitutively generated NFATc1β-isoforms, mainly expressed in resting lymphocytes, myeloid and non-lymphoid cells, remains unclear. Moreover, former work at our department indicated different roles for NFATc1α- and NFATc1β-proteins in lymphocytes.
Our data revealed the functional role of NFATc1 in peritoneal resident macrophages. We demonstrated that the expression of NFATc1β is required for a proper immune response of prMΦ during fungal infection-induced acute peritonitis. We identified Ccl2, a major chemokine produced in response to fungal infections by prMΦ, as a novel NFATc1 target gene which is cooperatively regulated through the NFAT- and canonical NF-κB pathways. Consequently, we showed that NFATc1β deficiency in prMΦ results in a decreased infiltration of inflammatory monocytes, leading to a delayed clearance of peritoneal fungal infection.
We could further show that the expression of NFATc1β-isoforms is irrelevant for homeostasis of myeloid and adaptive immune system cells and that NFATc1α- (but not β-) isoforms are required for a normal development of peritoneal B1a cells. In contrast to the situation in myeloid cells, NFATc1β deficiency is compensated by increased expression of NFATc1α-isoforms in lymphoid cells. As a consequence, NFATc1ß is dispensable for activation of the adaptive immune system.
Taken together our results illustrate the redundancy and indispensability of NFATc1-isoforms in the adaptive and innate immune system, indicating a complex regulatory system for Nfatc1 gene expression in different compartments of the immune system and likely beyond that.
In initial experiments, the well characterized VACV strain GLV-1h68 and three wild-type LIVP isolates were utilized to analyze gene expression in a pair of autologous human melanoma cell lines (888-MEL and 1936 MEL) after infection. Microarray analyses, followed by sequential statistical approaches, characterized human genes whose transcription is affected specifically by VACV infection. In accordance with the literature, those genes were involved in broad cellular functions, such as cell death, protein synthesis and folding, as well as DNA replication, recombination, and repair. In parallel to host gene expression, viral gene expression was evaluated with help of customized VACV array platforms to get better insight over the interplay between VACV and its host. Our main focus was to compare host and viral early events, since virus genome replication occurs early after infection. We observed that viral transcripts segregated in a characteristic time-specific pattern, consistent with the three temporal expression classes of VACV genes, including a group of genes which could be classified as early-stage genes. In this work, comparison of VACV early replication and respective early gene transcription led to the identification of seven viral genes whose expression correlated strictly with replication. We considered the early expression of those seven genes to be representative for VACV replication and we therefore referred to them as viral replication indicators (VRIs). To explore the relationship between host cell transcription and viral replication, we correlated viral (VRI) and human early gene expression. Correlation analysis revealed a subset of 114 human transcripts whose early expression tightly correlated with early VRI expression and thus early viral replication. These 114 human molecules represented an involvement in broad cellular functions. We found at least six out of 114 correlates to be involved in protein ubiquitination or proteasomal function. Another molecule of interest was the serine-threonine protein kinase WNK lysine-deficient protein kinase 1 (WNK1). We discovered that WNK1 features differences on several molecular biological levels associated with permissiveness to VACV infection. In addition to that, a set of human genes was identified with possible predictive value for viral replication in an independent dataset. A further objective of this work was to explore baseline molecular biological variances associated with permissiveness which could help identifying cellular components that contribute to the formation of a permissive phenotype. Therefore, in a subsequent approach, we screened a set of 15 melanoma cell lines (15-MEL) regarding their permissiveness to GLV-1h68, evaluated by GFP expression levels, and classified the top four and lowest four cell lines into high and low permissive group, respectively. Baseline gene transcriptional data, comparing low and highly permissive group, suggest that differences between the two groups are at least in part due to variances in global cellular functions, such as cell cycle, cell growth and proliferation, as well as cell death and survival. We also observed differences in the ubiquitination pathway, which is consistent with our previous results and underlines the importance of this pathway in VACV replication and permissiveness. Moreover, baseline microRNA (miRNA) expression between low and highly permissive group was considered to provide valuable information regarding virus-host co-existence. In our data set, we identified six miRNAs that featured varying baseline expression between low and highly permissive group. Finally, copy number variations (CNVs) between low and highly permissive group were evaluated. In this study, when investigating differences in the chromosomal aberration patterns between low and highly permissive group, we observed frequent segmental amplifications within the low permissive group, whereas the same regions were mostly unchanged in the high group. Taken together, our results highlight a probable correlation between viral replication, early gene expression, and the respective host response and thus a possible involvement of human host factors in viral early replication. Furthermore, we revealed the importance of cellular baseline composition for permissiveness to VACV infection on different molecular biological levels, including mRNA expression, miRNA expression, as well as copy number variations. The characterization of human target genes that influence viral replication could help answering the question of host cell response to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.
Cancer-related anemia is prevalent in cancer patients. Anemia negatively affects normal mental and physical function capacity with common symptoms s like fatigue, headache, or depression. Human erythropoietin (hEPO), a glycoprotein hormone regulating red blood cell formation, is approved for the treatment of cancer-related anemia. It has shown benefits in correcting anemia, and subsequently improving health-related quality of life and/or enhancing radio-, and chemotherapy. Several recent clinical trials have suggested that recombinant hEPO (rhEPO) may promote tumor growth that raises the questions concerning the safety of using rhEPO for cancer treatment. However in others, such effects were not indicated. As of today, the direct functional effect of rhEPO in tumor models remains controversial and needs to be further analyzed. Based on the GLV-1h68 backbone, the hEPO-expressing recombinant VACV strains (EPO-VACVs) GLV-1h210, GLV-1h211, GLV-1h212 and GLV-1h213 were generated by replacing the lacZ expression cassette at the J2R locus with hEPO under the control of different vaccinia promoters p7.5, pSE, pSEL, pSL, respectively. Also, GLV-1h209 was generated, which is similar to GLV-1h210 but expresses a mutated non-functinal EPO (R103A). The EPO-VACV strains were characterized for their oncolytic efficacy in lung (A549) cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed hEPO in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. As expected, EPO-VACVs enhanced red blood cell (RBC) formation in xenograft model. The number of RBCs and hemoglobin (Hb) levels were significantly increased in EPO-VACVs-treated mice compared to GLV-1h68-treated or untreated control mice. However, the mean size of RBC or Hb content per RBC remained normal. Furthermore, over-expression of hEPO did not significantly affect numbers of lymphocytes, monocytes, leucocytes or platelets in the peripheral blood stream. The expression of hEPO in colonized tumors of mice treated with EPO-VACVs was demonstrated by immunohistological staining. Interestingly, there were 9 - 10 hEPO isoforms detected either in tumors, cells, or supernatant, while 3-4 basic isoforms were missing in blood serum, where only six hEPO isoforms were found. Tumor-bearing mice after treatment with EPO-VACVs showed enhanced tumor regression compared to GLV-1h68. The virus titers in tumors in EPO-VACVs-treated mice were 3-4 fold higher compared to GLV-1h68-treated mice. Nevertheless, no significant difference in virus titers among EPO-VACVs was found. The blood vessels in tumors were significantly enlarged while the blood vessel density remained unchanged compared to the GLV-1h68 treated mice, indicating that hEPO did not affect endothelial cell proliferation in this model. Meanwhile, rhEPO (Epoetin alfa) alone or in combination with GLV-1h68 did not show any signs of enhanced tumor growth when compared to untreated controls and GLV-1h68 groups, while doses used were clinical relevant (500 U/kg). These findings suggested that hEPO did not promote angiogenesis or tumor growth in the A549 tumor xenograft model. Human EPO has been reported to function as an immune modulator. In this study, however, we did not find any involvement of hEPO in immune cytokine and chemokine expression or innate immune cell infiltration (leucocytes, B cells, macrophages and dendritic cells) into infected tumors. The degree of immune infiltration and cytokine expression was directly correlated to the number of virus particles. Increased virus replication, led to more recruited immune cells and secreted cytokines/chemokines. It was proposed that tumor regression was at least partially mediated through activation of innate immune mechanisms. In conclusion, the novel EPO-VACVs were shown to significantly increase the number of RBCs, Hb levels, and virus replication in tumors as well as to enhance tumor regression in the A549 tumor xenograft model. Moreover, locally expressed hEPO did not promote tumor angiogenesis, tumor growth, and immune infiltration but was shown to causing enlarged tumoral microvessels which facilitated virus spreading. It is conceivable that in a possible clinical application, anemic cancer patients could benefit from the EPO-VACVs, where they could serve as “wellness pills” to decrease anemic symptoms, while simultaneously destroying tumors.
In this dissertation, I examine the relationship between specialisation and stability of plant-pollinator networks, with a focus on two issues: Diversity maintenance in animal-pollinated plant communities and robustness of plant-pollinator systems against disturbances such as those caused by anthropogenic climate change. Chapter 1 of this thesis provides a general introduction to the concepts of ecological stability and specialisation with a focus on plant-pollinator systems, and a brief outline of the following chapters. Chapters 2-5 each consist of a research article addressing a specific question. While chapters 2 and 3 deal with different aspects of diversity maintenance in animal-pollinated plant communities, chapters 4 and 5 are concerned with the consequences of climate change in the form of temporary disturbances caused by extreme climatic events (chapter 4) and shifts in phenology of plants and pollinators (chapter 5). From a methodological perspective, the first three articles (chapter 2-4) can be grouped together as they all employ mathematical models of plant-pollinator systems, whereas chapter 5 describes an empirical study of plant-pollinator interactions along an altitudinal gradient in the Alps. The final chapter (6) provides a review of current knowledge on each of the two main themes of this thesis and places the findings of the four research articles in the context of related studies.
Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells.
Fanconi anemia (FA) is an autosomal recessive or X-chromosomal inherited disorder, which is not only phenotypically but also genotypically very heterogeneous. While its hallmark feature is progressive bone marrow failure, many yet not all patients suffer additionally from typical congenital malformations like radial ray defects and growth retardation. In young adulthood the cumulative risk for developing hematological or other malignancies is compared to the general population several hundred-fold increased. The underlying molecular defect is the deficiency of DNA interstrand crosslink (ICL) repair. ICLs are deleterious lesions, which interfere with crucial cellular processes like transcription and replication and thereby can lead to malignant transformation, premature senescence or cell death. To overcome this threat evolution developed a highly complex network of interacting DNA repair pathways, which is conserved completely only in vertebrates. The so called FA/BRCA DNA damage response pathway is able to recognize ICLs on stalled replication forks and promotes their repair through homologous recombination (HR). Today we know 15 FA genes (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O and -P) whose products are involved in this pathway. Although more than 80% of FA patients carry biallelic mutations in either FANCA, FANCC or FANCG, there are still some who cannot be assigned to any of the known complementation groups. This work aimed to indentify the di¬sease causing mutations in a cohort of those unassigned patients. Initial screens of the candidate genes FAN1, MHF1 and MHF2 did not reveal any pathogenic alterations. Moreover, FAN1 could be excluded as FA candidate gene because patients carrying a homozygous microdeletion including the FAN1 locus did not show a phenotype comparable to FA patients. In the case of MHF1 and MHF2 the reason for the negative screening result is not clear. Mutation carriers might be rare or, regarding the diverse and also FA pathway independent protein functions, phenotypically not comparable to FA patients. Nevertheless, this study contri¬buted to the identification and characterization of the most recent members of the FA pathway - RAD51C (FANCO), SLX4 (FANCP) and XPF (FANCQ). FANCO is one of the RAD51 paralogs and is involved in crucial steps of HR. But since the only reported FA-O patient has so far not developed any hematological anomalies, FANCO is tentatively designated as gene underlying an FA-like disorder. In contrast, patients carrying biallelic mutations in FANCP do not only show hematological anomalies, but as well congenital malformations typical for FA. The distinct role of FANCP in the FA pathway could not be determined, but it is most likely the coordination of structure-specific nucleases during ICL excision. One of these nucleases is the heterodimer XPF/ERCC1. XPF is probably disease causing in the complementation group FA-Q and is the first FA gene, which was identified by Next Generation Sequencing (NGS). Extraordinarily is that mutations in this gene had previously been reported to cause two other disorders, xeroderma pigmentosum and segmental progeria. Despite some overlaps, it was shown that the divergent phenotypes could clearly be distinguished and are caused by distinct functional defects of XPF. Additionally, this work aimed to improve and accelerate the genotyping process of FA patients in general. Therefore, classical approaches should be complemented or fully replaced by approa¬ches using NGS. Massively parallel sequencing of the whole exome proved to be most appro¬priate and the establishment of an FA-specific analysis pipeline facilitated improved molecular diagnostics by combining complementation group assignment and mutation analysis in one step. Consequently two NGS studies revealed the pathogenic defect in several previously unassigned FA patients and thereby added another patient to one of the most recent subtypes, FA-P. In summary, this work contributed not only to further completion of the FA/BRCA DNA repair network by adding three novel genes, it also showed that classical molecular approaches for re¬search as well as for diagnostics could be replaced by NGS.
Bei einer Vielzahl neuromuskulärer und neurodegenerativer Erkrankungen spielen Fehlfunktionen der Mitochondrien eine wichtige Rolle. Da die Proteine der Atmungsketten-komplexe sowohl durch die mitochondriale DNA als auch durch das Kerngenom codiert werden, können Mutationen in beiden Genomen die Auslöser dieser Erkrankungen darstellen. Veränderungen der mitochondrialen DNA lassen sich - im Gegensatz zum Kerngenom - bisher nicht korrigieren, weshalb bei einem großen Teil der Erkrankungen nur die Symptome und nicht die Auslöser behandelt werden können. Das grundlegende Problem stellt dabei der Transport der DNA in die Mitochondrien dar. Ziel dieser Arbeit war es, mit Hilfe von physikalischen Transfektionsmethoden exogene DNA in die Mitochondrien menschlicher Kulturzellen einzubringen. Dazu wurden unterschiedliche Vektoren hergestellt, die in Mitochondrien das an die Mitochondrien angepasste grün fluoreszierende mtEGFP exprimieren sollen. Die Expressionsfähigkeit und Prozessierung dieser Konstrukte konnte in in-vitro-Assays mit einem Mitochondrienextrakt nachgewiesen werden. Bei Transfektionsversuchen mit der Gene Gun gelang es erstmals, exogene Plasmid-DNA in die Mitochondrien menschlicher Zellen einzubringen. Das durch die transfizierten Vektoren exprimierte mtEGFP konnte am Fluoreszenzmikroskop eindeutig in den Mitochondrien der Zellen lokalisiert werden. Eine Transfektion mit Hilfe magnetischer Partikel erwies sich jedoch nicht als zielführend, da die die Partikel eine Eigenfluoreszenz aufwiesen, die eine Detektion der mtEGFP-Expression verhinderten. Eine wichtige Voraussetzung für die Transfektion von Mitochondrien durch mechanische Methoden wie die Mikroinjektion ist die reversible Induktion von Megamitochondrien, da sie erst in diesem Zustand penetriert werden können. Durch eine Ansäuerung des Kulturmediums mit Natriumacetat bzw. Essigsäure konnten Mitochondrien erzeugt werden, die beinahe die Größe des Zellkerns aufwiesen und somit ideale Bedingungen für die Mikroinjektion darstellen. Bei den anschließenden Mikroinjektionsversuchen mit den hergestellten mitochondrialen Expressionsvektoren wurden wiederum Zellen mit eindeutig grün fluoreszierenden Mitochondrien gefunden. Zusammenfassend wurden im Rahmen dieser Arbeit erstmalig menschliche Mitochondrien mit exogener DNA transfiziert. Dies stellt einen grundlegenden Schritt für die Entwicklung neuer Therapieformen bei mitochondrialen Myopathien dar. Zuvor müssen die Transfektionsmethoden jedoch noch weiter optimiert werden, um eine höhere Transfektionseffizienz zu erreichen.
The DREAM complex plays an important role in regulation of gene expression during the cell cycle. It was previously shown that the DREAM subunits LIN9 and B-MYB are required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this work the effect of LIN9 or B-MYB depletion on embryonic stem cells (ESC) was examined. It demonstrates that LIN9 and B-MYB knock down changes the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. By using genome-wide expression studies it was revealed that the depletion of LIN9 leads to downregulation of mitotic genes and to upregulation of differentiation-specific genes. ChIP-on chip experiments determined that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of the pluripotency markers Sox2 and Oct4 and LIN9 depleted ESCs retain alkaline phosphatase activity. I conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis. The exact molecular mechanisms behind this gene activation are still unclear as no DREAM subunit features a catalytically active domain. It is assumed that DREAM interacts with other proteins or co-factors for transcriptional activation. This study discovered potential binding proteins by combining in vivo isotope labeling of proteins with mass spectrometry
(MS) and further analysed the identified interaction of the tight junction protein ZO-2 with DREAM which is cell cycle dependent and strongest in S-phase. ZO-2 depletion results in reduced cell proliferation and decreased G1 gene expression. As no G2/M genes, typical DREAM targets, are affected upon ZO-2 knock down, it is unlikely that ZO-2 binding is needed for a functional DREAM complex. However, this work demonstrates that with (MS)-based quantitative proteomics, DREAM interacting proteins can be identified which might help to elucidate the mechanisms underlying DREAM mediated gene activation.
Cancer cells frequently escape from immune surveillance by down-regulating two important components of the immune defence: antigen-presenting MHC and costimulatory molecules. Therefore several novel anti-tumour compounds that aim to assist the immune system in recognising and fighting cancer are currently under development. Recombinant bispecific antibodies represent one group of such novel therapeutics. They target two different antigens and recruit cytotoxic effector cells to tumour cells. For cancer immunotherapy, bispecific T cell-engaging antibodies are already well characterised. These antibodies target a tumour-associated antigen and CD3ε, the constant molecule of the T cell receptor complex.
On the one hand, this study presents the development of a bispecific antibody targeting CD3ε and the rhabdomyosarcoma-associated fetal acetylcholine receptor. On the other hand, it describes a novel two-part trispecific antibody format for the treatment of leukaemia and other haematological malignancies in the context of haematopoietic stem cell transplantation (HSCT).
For HSCT, an HLA-identical donor is preferred, but very rarely available. In an HLA-mismatched setting, the HLA disparity could be exploited for targeted cancer treatment. In the present study, a two-part trispecific HLA-A2 × CD45 × CD3 antibody was developed for potential cases in which the patient is HLA-A2-positive, but the donor is not. This holds true for about half the cases in Germany, since HLA-A2 is the most common HLA molecule found here. Combinatorial targeting of HLA-A2 and the leucocyte-common antigen CD45 allows for highly specific dual-antigen restricted tumour targeting.
More precisely, two single-chain antibody constructs were developed: i) a single-chain variable fragment (scFv) specific for HLA-A2, and ii) a scFv against CD45, both linked to the VL and the VH domain of a CD3ε-specific antibody, respectively. It turned out that, after the concomitant binding of these constructs to the same HLA-A2- and CD45-expressing cell, the unpaired variable domains of a CD3ε-specific antibody assembled to a functional scFv. In a therapeutic situation, this assembly should exclusively occur on the recipient’s blood cancer cells, leading to T cell-mediated cancer cell destruction. In this way, a relapse of disease might be prevented, and standard therapy (radiation and chemotherapy) might be omitted.
For both approaches, the antibody constructs were periplasmically expressed in E. coli, purified via His tag, and biochemically characterised. Their binding to the respective targets was proven by flow cytometry. The stimulatory properties of the antibodies were assayed by measuring IL-2 release after incubation with T cells and antigen-expressing target cells. Both the bispecific antibody against rhabdomyosarcoma and the assembled trispecific antibody against blood cancer mediated T-cell activation in a concentration-dependent manner at nanomolar concentrations. For the trispecific antibody, this effect indeed proved to be dual antigen-restricted, as it could be blocked by prior incubation of either HLA-A2- or CD45-specific scFv and did not occur on single-positive (CD45+) or double-negative (HLA-A2- CD45-) target cells. Furthermore, antibodies from both approaches recruited T cells for tumour cell destruction in vitro.
Mutationen in Genen, die für Kernhüllproteine codieren sind mit einer stetig zunehmenden Anzahl menschlicher Erkrankungen verbunden, die als Envelopathien bezeichnet werden. Erstaunlicherweise betrifft die Pathologie dieser Krankheiten spezifische Gewebe und Organe, obwohl entsprechende Proteine meist ubiquitär exprimiert werden. So führen beispielsweise Defekte in Emerin, einem Protein der inneren Kernhülle, zur X-chromosomalen Emery-
Dreifuss Muskeldystrophie (EDMD). Diese Krankheit ist durch Muskelschwäche oder –
schwund gekennzeichnet. Defekte im Kernhüllprotein MAN1 sind dagegen mit Krankheiten verbunden, die Knochen- und Hautgewebe betreffen. Interessanterweise besitzen beide
Proteine eine evolutionär hoch konservierte Domäne, die sog. LEM-Domäne. LEM-Domänen Proteine können mit der Kernlamina interagieren, ebenso mit dem sog. Barrier-to-
Autointegration Factor (BAF) sowie mit zahlreichen Transkriptionsfaktoren. Dennoch ist die
funktionelle Rolle der LEM-Domänen Proteine bis dato nicht vollständig aufgeklärt. In der vorliegenden Studie sollten daher die Funktionen von MAN1 und Emerin während der Frühentwicklung von Xenopus laevis untersucht werden.
Vorangehende Untersuchungen zeigten, dass Mikroinjektionen von XMAN1-
Antikörpern in Zwei-Zell-Stadien befruchteter Eizellen zu einem Arrest der Zellteilung in der injizierten Blastomere führten. Da dabei eine Störung der Kernhüllbildung spekuliert wurde, sollte durch Antikörper-vermittelter Inhibition von XMAN1 die Bildung von in vitro Kernen im Xenopus Eiextrakt untersucht werden. Dabei wurden Kerne beobachtet, die dekondensiertes
Chromatin zeigten, bei denen jedoch eine Fusion von Membranvesikeln zu einer durchgehenden Kernhülle nicht stattgefunden hatte. Frühere Charakterisierungen von MAN1 und Emerin zeigten unterschiedliche Expressionsmuster während der Entwicklung von X. laevis. Da XMAN1 ubiquitär exprimiert und Xemerin jedoch erstmals ab Stadium 41 nachweisbar ist, war es mittels Mikroinjektion von
Xemerin möglich zu zeigen, dass es in der Lage ist den Arrest der Zellteilung zu verhindern. Es wurde daher die These aufgestellt, dass MAN1 und Emerin während der Frühentwicklung von Xenopus überlappende Funktionen besitzen. Um diese These zu prüfen, wurde zunächst
unter Verwendung des Proximity Ligation Assays untersucht, ob beide Proteine miteinander interagieren können. Mit Hilfe dieser Methode konnte gezeigt werden, dass Interaktionen beider Proteine innerhalb der Kernhülle lokalisieren. Die Interaktionen blieben während der Mitose bestehen und waren erst wieder zum Ende der Mitose in der Kernhülle nachweisbar. Diese Resultate deuten daher darauf hin, dass XMAN1/Xemerin-Interaktionen während der ...
Learning and memory is considered to require synaptic plasticity at presynaptic specializations of neurons. Kenyon cells are the intrinsic neurons of the primary olfactory learning center in the brain of arthropods – the mushroom body neuropils. An olfactory mushroom body memory trace is supposed to be located at the presynapses of Kenyon cells. In the calyx, a sub-compartment of the mushroom bodies, Kenyon cell dendrites receive olfactory input provided via projection neurons. Their output synapses, however, were thought to reside exclusively along their axonal projections outside the calyx, in the mushroom body lobes. By means of high-resolution imaging and with novel transgenic tools, we showed that the calyx of the fruit fly Drosophila melanogaster also comprised Kenyon cell presynapses. At these presynapses, synaptic vesicles were present, which were capable of neurotransmitter release upon stimulation. In addition, the newly identified Kenyon cell presynapses shared similarities with most other presynapses: their active zones, the sites of vesicle fusion, contained the proteins Bruchpilot and Syd-1. These proteins are part of the cytomatrix at the active zone, a scaffold controlling synaptic vesicle endo- and exocytosis. Kenyon cell presynapses were present in γ- and α/β-type KCs but not in α/β-type Kenyon cells.
The newly identified Kenyon cell derived presynapses in the calyx are candidate sites for an olfactory associative memory trace. We hypothesize that, as in mammals, recurrent neuronal activity might operate for memory retrieval in the fly olfactory system.
Moreover, we present evidence for structural synaptic plasticity in the mushroom body calyx. This is the first demonstration of synaptic plasticity in the central nervous system of Drosophila melanogaster. The volume of the mushroom body calyx can change according to changes in the environment. Also size and numbers of microglomeruli - sub-structures of the calyx, at which projection neurons contact Kenyon cells – can change. We investigated the synapses within the microglomeruli in detail by using new transgenic tools for visualizing presynaptic active zones and postsynaptic densities. Here, we could show, by disruption of the projection neuron - Kenyon cell circuit, that synapses of microglomeruli were subject to activity-dependent synaptic plasticity. Projection neurons that could not generate action potentials compensated their functional limitation by increasing the number of active zones per microglomerulus. Moreover, they built more and enlarged microglomeruli. Our data provide clear evidence for an activity-induced, structural synaptic plasticity as well as for the activity-induced reorganization of the olfactory circuitry in the mushroom body calyx.
In the last decades, both the incidence and the severity of asthma have steadily increased. Furthermore, available therapies only treat the symptoms but do not cure the disease. Immune modulation induced by TLR agonists may be a promising novel approach to effectively treat asthma as it targets the underlying immunopathology directly rather than one mediator alone. The aim of this thesis was to investigate if the immunostimulatory properties of Toll-like receptor (TLR) agonists can be utilized to develop novel therapeutic intervention strategies for the treatment of asthma using murine models of allergic inflammation. For this purpose five different TLR agonists were tested in preclinical mouse models of acute and chronic asthma, both in preventive and therapeutic settings. Firstly, TLR-2, 3, 4, 7/8 and 9 agonists were delivered intratracheally at different doses before pulmonary allergen exposure in the asthma model of acute inflammation. TLR9 agonist CpG-containing oligodeoxynucleotides (CpG) > TLR7 agonist Resiquimod (R848) > TLR3 agonists poly(I:C) strongly reduced allergen induced airway eosinophilia and IL-4 levels in a dose-dependent manner. All TLR agonists increased neutrophil numbers, TLR4 agonist lipopolysaccharide (LPS) > TLR2 agonist lipoteichonic acid (LTA) > poly(I:C) > CpG > R848 and, with the exception of R848, the amount of pro-inflammatory cytokines in the airways. Suppressive effects were not dependent upon IFN-γ and IL-10 or associated with increased numbers of regulatory T cells in the airways. All TLR agonists, except LTA, similarly reduced airway eosinophilia and IL-4 levels when applied therapeutically after allergen challenge. These results show that the TLR agonists have different suppressive effects on TH2 responses in the airways which further depend on the dose and the experimental setup in which they were tested. Interestingly, all agonists induced airway neutrophilia, albeit to different degrees, raising the question if TLR ligands are safe for human use when applied directly into the lung. Different TLR agonists are also being developed for human use as adjuvants combined with allergen in specific immunotherapy. Recent clinical data suggest that this may be achieved by induction of allergen-specific TH1 responses. For this reason, the ability of different TLR agonists to induce allergen-specific TH1 and suppress allergen-specific TH2 responses in a preclinical setting was investigated in this thesis. Different doses of the TLR agonists were applied together with allergen, then mice were exposed to allergen aerosol. CpG > LPS >LTA dose-dependently strongly suppressed the development of airway eosinophilia with poly(I:C) and R848 having no effect. The decrease in eosinophilic numbers was associated withincreased neutrophils present in the airways. IL-4 and IL-5 levels in the bronchoalveolar lavage fluid were also decreased when poly(I:C), LPS, and CpG were used. All TLR agonists increased allergen-specific IgG2a, and with the exception of poly(I:C), reduced allergen-specific IgE levels in the serum. Cutaneous anaphylaxis to allergen was completely prevented when LPS or CpG were given as adjuvant. The strongest TH1 responses were induced by CpG and poly(I:C), characterized by the presence of IFN-γ in the bronchoalveolar lavage and the highest allergen-specific IgG2a levels in the serum. This data supports approaches to use TLR9 or TLR4 agonists for human therapy as adjuvant in combination with allergen in novel specific immunotherapy formulations. In the last part of the thesis, it was investigated if TLR activation can also affect the pathology of severe chronic asthma. Therapeutic administration of R848 or CpG reduced features of inflammation and remodeling. Both agonists showed superior effects to dexamethasone, with CpG being more efficient than R848. This result again supports a TLR9-based therapy as a viable option for the treatment of severe chronic asthma which may present a potential alternative for anti-inflammatory therapy with steroids. Taken together, the results of this thesis support the use of TLR agonists to treat asthma. The most favorable efficacy/safety ratio is to be expected from TLR-based therapies combining TLR4 or TLR9 agonists with allergen in specific immunotherapy. In regard to TLR agonist monotherapy, R848 and CpG showed the most promising profiles, CpG particularly in a model of severe chronic asthma. However, since all TLR agonists used in this study also showed pro-inflammatory potential, the safety aspect of such an approach needs to be taken into account.
Precise control of progression through mitosis is essential to maintain genomic stability and to prevent aneuploidy. The DREAM complex is an important regulator of mitotic gene expression. Depletion of Lin9, one core-subunit of DREAM, leads to reduced expression of G2/M genes and impaired proliferation. In conditional mouse knockout cells (MEFs) Lin9 deletion causes defects in mitosis and cytokinesis and cells undergo premature senescence in order to prevent further proliferation. In this work it could be shown that the senescence phenotype in Lin9 knockout MEFs is independently mediated by the two tumor suppressor pathways p53-p21 and p16-pRB. Studies using the conditional Lin9 knockout mouse model demonstrated an important function of Lin9 in the regulation of mitotic gene expression and proliferation in vivo. Deletion of Lin9 caused reduced proliferation in the intestinal crypts resulting in atrophy of the intestinal epithelium and in rapid death of the animals. In the second part of this work, the pathways leading to p53 mediated G1 arrest after failed cytokinesis were analyzed by using a chemical inhibitor of the mitotic kinase Aurora B. In a high throughput siRNA screen the MAP kinase MAP3K4 was identified as an upstream activator of p53. It could be shown that MAP3K4 activates the downstream stress kinase p38b to induce the p53 mediated cell cycle arrest of tetraploid cells. p38b was required for the transcriptional activation of the p53 target gene p21 in response to Aurora B inhibition. In contrast, phosphorylation, stabilization and recruitment of p53 to the p21 promoter occured independently of p38 signaling. Partial inhibition of Aurora B demonstrated that chromosome missegregation also activates the MAP3K4-p38-p53 pathway, suggesting that subtle defects in mitosis are sufficient for inducing this stress signaling pathway. Although p38 was required for the G1 cell cycle arrest after mitotic failures, long-term co-inhibition of p38 and Aurora B resulted in reduced proliferation probably due to increased apoptosis. Presumably, MAP3K4-p38-p53 signaling is a common pathway that is activated after errors in mitosis or cytokinesis to arrest cells in G1 and to prevent chromosomal instability.
A major goal of the main topics of ecology is to answer the question of how species can co-exist and maintain biodiversity. To understand how community dynamics operate in different spatio-temporal dimensions to govern biodiversity patterns requires a process-based knowledge. Thus, this study focused primarily on biodiversity patterns and ecological processes at both spatial and temporal scales. Spatially, the diversity and similarity of spider communities in high, intermediate, and low strata of beech trees represented a set of age-related effects: Old-growth trees provided unique and distinct resources to spiders and in turn possessed discrete spider compositions. Intra-annually, spider communities in different seasons showed a repeated, predictable temporal dynamics. Inter-annually, comparison revealed that neutral and niche models can operate in tandem, and that both are needed to fully explain the dynamics of arboreal spider assemblages among different canopy strata in this beech forest.
Bone Morphogenetic Proteins (BMPs) are key regulators for a lot of diverse cellular processes. During embryonic development these proteins act as morphogens and play a crucial role particularly in organogenesis. BMPs have a direct impact on distinct cellular fates by means of concentration-gradients in the developing embryos. Using the diverse signaling input information within the embryo due to the gradient, the cells transduce the varying extracellular information into distinct gene expression profiles and cell fate decisions. Furthermore, BMP proteins bear important functions in adult organisms like tissue homeostasis or regeneration. In contrast to TGF-ß signaling, currently only little is known about how cells decode and quantify incoming BMP signals. There is poor knowledge about the quantitative relationships between signal input, transducing molecules, their states and location, and finally their ability to incorporate graded systemic inputs and produce qualitative responses. A key requirement for efficient pathway modulation is the complete comprehension of this signaling network on a quantitative level as the BMP signaling pathway, just like many other signaling pathways, is a major target for medicative interference. I therefore at first studied the subcellular distribution of Smad1, which is the main signal transducing protein of the BMP signaling pathway, in a quantitative manner and in response to various types and levels of stimuli in murine c2c12 cells. Results indicate that the subcellular localization of Smad1 is not dependent on the initial BMP input. Surprisingly, only the phospho-Smad1 level is proportionally associated to ligand concentration. Furthermore, the activated transducer proteins were entirely located in the nucleus. Besides the subcellular localization of Smad1, I have analyzed the gene expression profile induced by BMP signaling. Therefore, I examined two endogenous immediate early BMP targets as well as the expression of the stably transgenic Gaussia Luciferase. Interestingly, the results of these independent experimental setups and read-outs suggest oscillating target gene expression. The amplitudes of the oscillations showed a precise concentration-dependence for continuous and transient stimulation. Additionally, even short-time stimulation of 15’ activates oscillating gene-expression pulses that are detectable for at least 30h post-stimulation. Only treatment with a BMP type I receptor kinase inhibitor leads to the complete abolishment of the target gene expression. This indicated that target gene expression oscillations depend directly on BMP type I receptor kinase activity.
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A chromatographic procedure 1 is described by means of which cytochrome oxidase has been purified from a variety of organisms including the fungus N eurospora crassa,2,3 the unicellular alga Po/ytoma mirum, 4 the insect Locusta migratoria ,5 the frog Xenopus muel/eri,4 and the mammal Rattus norwegicus. 4 This procedure can be used to equal effect for large-scale preparations, starting from grams of mitochondrial protein, or for small-scale preparations starting from milligrams. The cytochrome oxidase preparations from the different organisms are enzymically active. They show similar subunit compositions.
The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewhere
The A TPase eomplex has been isolated from mitoehondria of N eurospora crassa by immunologieal teehniques. The protein ean be obtained rapidly and qua ntitatively in high purity by miero- or large-seale immunopreeipitation. Immunopreeipitation has been applied to labeled and doubly labeled mitoehondrial proteins in order to investigate the number and moleeular weights of subunit polypeptides , the site of synthesis of subunit polypeptides, and the dieycIohexyIcarbodiimide-binding protein . The A TPase complex obtained by large-seale immunopreeipitation has been used as starting ma terial for the isolation of hydrophobie polypeptides.
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Millionen Menschen weltweit leiden an den verschiedensten Autoimmunerkrankungen. Diese Krankheiten entstehen, wenn das Immunsystem gesundes körpereigenes Gewebe angreift und zerstört. An der Pathogenese sind sowohl Komponenten des angeborenen Immunsystems als auch Bestandteile des adaptiven Immunsystems, wie Lymphozyten und Antikörper, beteiligt. Da die Ursachen und molekularen Mechanismen der Pathogenese dieser Erkrankungen bis heute weitgehend unbekannt sind, wurden in dieser Arbeit autoaggressive Lymphozyten bei den humanen Autoimmunerkrankungen Polymyositis und Multiple Sklerose näher untersucht. Die Polymyositis ist eine chronisch entzündliche Erkrankung der Skelettmuskulatur. Die Muskelfasern werden dabei von zytotoxischen CD8+ gd-T-Lymphozyten infiltriert, attackiert und schließlich zerstört. In einem seltenen Fall der Polymyositis wurden die Muskelzellen hingegen in ähnlicher Weise von CD8- gd-T-Lymphozyten angegriffen. Die gd-T-Lymphozyten waren monoklonal expandiert und ihr Rezeptor, im Folgenden als M88 bezeichnet, wurde als Vg1.3+Vd2+ identifiziert. Frühere Untersuchungen der Antigenspezifität dieser Zellen zeigten, dass M88 mehrere funktionell und strukturell verschiedene Proteine aus unterschiedlichen Spezies erkennt. Die Bindung erfolgt spezifisch durch die Antigenerkennungsregionen beider Rezeptorketten von M88. In dieser Arbeit wurden verschiedene bakterielle und humane Proteine des Translationsapparates als Antigene von M88 identifiziert. Weitere ausführliche Untersuchungen eines paradigmatischen bakteriellen Antigens, dem Translationsinitiationsfaktor EcIF1, zeigten, dass M88 an Oberflächen-exponierte Konformationsepitope von Proteinen bindet. Interessanterweise erkennt M88 mehrere humane Aminoacyl-tRNA-Synthetasen, Antigene, die in anderen Formen der Myositis von Autoantikörpern angegriffen werden. Diese Beobachtung ergibt eine bemerkenswerte Verbindung zwischen T-Zell- und Antikörper-vermittelten B-Zell-Antworten bei der autoimmunen Myositis. Bei der Multiplen Sklerose ist das zentrale Nervensystem betroffen. Autoaggressive Lymphozyten greifen die Myelinschicht der Nervenzellen im Gehirn und Rückenmark an und zerstören sie. Im Liquor cerebrospinalis von Patienten lassen sich klonal expandierte und affinitätsgereifte B-Zellen sowie „oligoklonale Banden“ (OKB) Antikörper nachweisen. Obwohl diese Merkmale auf eine Antigen-induzierte Immunantwort hindeuten, sind die zugrundeliegenden Antigene und die Rolle der OKB bei der Pathogenese bis heute unbekannt. In dieser Arbeit wurde die Antigenspezifität von fünf IgG OKB-Antikörpern aus drei Patienten untersucht. Durch verschiedene proteinbiochemische Methoden konnten intrazelluläre Kandidatenantigene identifiziert werden. Interessanterweise sind darunter mehrere nukleäre Proteine, die an der Transkriptionsregulation oder der RNA-Prozessierung beteiligt sind. Reaktivitäten gegen intrazelluläre Antigene treten auch bei anderen Autoimmunerkrankungen, wie beispielsweise dem systemischen Lupus erythematodes, auf. Diese Ergebnisse könnten auf einen allgemeinen Mechanismus der Entstehung und Funktion von Autoantikörpern bei diesen humanen Autoimmunerkrankungen hindeuten.
Professor Harold Gamet Callan, honorary member of the German Society for Cell Biology, died on the 3rd November 1993, at the age of 76. His name is inseparably connected with lampbrush chromosomes, the most spectacular and aesthetically ailuring form of chromosomes, which occupied the major part of his scientific career. " Mick" Callan's pioneering studies led to fruitful new concepts, served as a building block for many subsequent studies by others, and contributed enormously to our current understanding of chromosome organization and activity ...
The Xiphophorus tumor system has provided the opportunity to reduce the enormous complexity of cancer etiology to a few biological elements basically involved in neoplasia. The development of a tumor requires an oncogene which, after impairment, deletion, or elimination of its regulatory genes is permitted to mediate neoplastic transformation. Emphasis is being placed today in cancer research on the actual oncogenes themselves, but, in our opinion, the most important genes involved in neoplasia are these regulatory genes. However, although detected by c1assical genetics in the Xiphophorus system, th ese genes are not at present open to a more fin ely detailed molecular biological analysis. Their actual mode of action is therefore still far from being understood.
The livebearing all-female fish Poecilia formosa reproduces by gynogenesis, a modified form of parthenogenesis. P. formosa forms at least two breeding complexes: in its northern range it exists sympatrically with Poecilia latipinna and in its southern range with Poecilia mexicana. Differences between these complexes and their possible origin are discussed. Embryogenesis is triggered by sperm of males of these closely related sympatric species. Because inheritance is stricdy maternal, from the male point of view energy and time invested are totally lost. In this study we wanted to elucidate whether males are able to distinguish between conspecific and parasitic females. It could be shown that males are able to distinguish females optically, but that this ability was obscured as soon as chemical and/or tactile contact was possible. Furthermore, we found that females in an attractive phase of their sexual cycle are always preferred, regardless of species. This is possibly the mechanism by which parasitic females obtain the matings they need to reproduce.
In one of the simplest metazoan organisms, the sponge Spongilla lacustris, at least four different src-related kin ase genes (srkl-4) are expressed, aD of which show a high degree of similarity to the c-src genes of vertebrates. Whereas srk2 and srk3 are c1early unrelated at the nucleic acid level, srkl and srk4 share identical sequences in the 5' parts of their cDNAs. The cloning of several primer extension clones and genomic polymerase chain re action experiments confirmed the hypo thesis of an alternative splicing of tandemly arranged carboxyterminal parts of srkl and srk4. The genomic sequence encoding both proteins was found to be interrupted at the splice point by an intron which is located in the same position as one of the introns in the chicken src gene, which is the only gene conserved in invertebrates and vertebrates. All four srk genes are expressed in adult sponges as mRNA transcripts of about 2.2 kb. Tyrosine kin ase activity of a src-related kin ase could be detected in adult sponges but not in their resting form (gemmulae), and may reflect the activity of the srk protein products. Spongilla lacustris is the simplest organism from which a pro tein tyrosine kinase gene has been isolated. The presence of at least four such genes in the evolutionary ancient and primitive phylum Porifera suggests that tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms and that their activity may be involved in aggregation and cell-cell recognition.
Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order “cortical” brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.
A large number of metabolic waste products accumulate in the blood of patients with renal failure. Since these solutes have deleterious effects on the biological functions, they are called uremic toxins and have been classified in three groups: 1) small water soluble solutes (MW < 500 Da), 2) small solutes with known protein binding (MW < 500 Da), and 3) middle molecules (500 Da < MW < 60 kDa). Protein bound uremic toxins are poorly removed by conventional hemodialysis treatments because of their high protein binding and high distribution volume. The prototypical protein bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (pCS) are associated with the progression of chronic kidney disease, cardiovascular outcomes, and mortality of patients on maintenance hemodialysis. Furthermore, these two compounds are bound to albumin, the main plasma protein, via electrostatic and/or Van-der-Waals forces. The aim of the present thesis was to develop a dialysis strategy, based on the reversible modification of the ionic strength in the blood stream by increasing the sodium chloride (NaCl) concentration, in order to enhance the removal of protein bound substances, such as IS and pCS, with the ultimate goal to improve clinical patient outcomes. Enhancing the NaCl concentration ([NaCl]) in both human normal and uremic plasma was efficient to reduce the protein bound fraction of both IS and pCS by reducing their binding affinity to albumin. Increasing the ionic strength was feasible during modified pre-dilution hemodiafiltration (HDF) by increasing the [NaCl] in the substitution fluid. The NaCl excess was adequately removed within the hemodialyzer. This method was effective to increase the removal rate of both protein bound uremic toxins. Its ex vivo hemocompatibility, however, was limited by the osmotic shock induced by the high [NaCl] in the substituate. Therefore, modified pre-dilution HDF was further iterated by introducing a second serial cartridge, named the serial dialyzers (SDial) setup. This setting was validated for feasibility, hemocompatibility, and toxin removal efficiency. A better hemocompatibility at similar efficacy was obtained with the SDial setup compared with the modified pre-dilution HDF. Both methods were finally tested in an animal sheep model of dialysis to verify biocompatibility. Low hemolysis and no activation of both the complement and the coagulation systems were observed when increasing the [NaCl] in blood up to 0.45 and 0.60 M with the modified pre-dilution HDF and the SDial setup, respectively. In conclusion, the two dialysis methods developed to transitory enhance the ionic strength in blood demonstrated adequate biocompatibility and improved the removal of protein bound uremic toxins by decreasing their protein bound fraction. The concepts require follow-on clinical trials to assess their in vivo efficacy and their impact on long-term clinical outcomes.
The role of meiotic nuclear envelope components in chromosome dynamics and meiotic progression
(2013)
Meiosis is the specialised cell division which produces haploid germ cells, capable of developing into fertile gametes, from diploid progenitor cells. During meiosis, chromosomes undergo strictly regulated and strongly conserved dynamic processes, at the beginning of which the telomeres are actively tethered and intimately attached to the nuclear envelope (NE). The attached telomeres are then moved within the NE through cytoskeletal forces to cluster within a restricted region, forming the highly conserved bouquet stage. Subsequently, the bouquet is released simultaneously to the completion of the synaptonemal complex assembly tightly linking homologous chromosome pairs together. In combination these processes are essential for the successful completion of meiosis. Because the meiotic NE serves as a platform for telomere attachment and movement it can be assumed to be critically involved in these events crucial for fertility. However, the precise roles of many meiotic NE proteins in the attachment and movement of telomeres still remain elusive. Therefore, it was the aim of this thesis to investigate the functions of two mammalian meiotic NE components in telomere attachment and dynamics. The first part of this thesis is concerned with the meiosis-specific lamin C2. Lamin C2 is the only A-type lamin expressed during meiosis and has in previous studies shown to feature altered meiosis-specific properties, clearly distinguishing it from somatic lamins. Because lamin C2 is enriched at sites of telomere attachment, exhibits a high mobility within the nuclear lamina and influences NE integrity, it has been postulated that it may locally increase NE flexibility to allow efficient meiotic telomere movement. Therefore, possible functions of lamin C2 in the movement of attached telomeres were investigated in this thesis by studying the bouquet formation and release of pubertal mice specifically lacking lamin C2. This revealed that lamin C2 deficient mice show a delayed bouquet release, leading to severe defects in the synaptic pairing of homologous chromosomes, which in turn results in infertility of the males. Therefore, the efficient repositioning of attached meiotic telomeres, facilitated by lamin C2, seems essential for completing meiosis. The second part of this thesis focuses on the protein complex responsible for the attachment of meiotic telomeres to the NE and their coupling to the cytoskeleton. The so-called LINC complex is composed of SUN domain proteins in the inner nuclear membrane interacting with KASH domain proteins of the outer nuclear membrane. In previous studies it had been shown that SUN1, SUN2 and KASH5 localise to the attached meiotic telomeres. Regarding the meiotic role of SUN2, however, contradicting results have recently been discussed, showing the need for further investigations. Using an available SUN1 deficient mouse strain, this thesis was able to show that SUN2 is sufficient for telomere attachment per se although telomere attachment is impaired in SUN1 deficient mice leading to infertility. It is also demonstrated that SUN2 forms a functional LINC complex together with KASH5 to mediate this telomere attachment. This LINC complex in the absence of SUN1 is able to move attached telomeres into a bouquet-like cluster formation. Therefore, this demonstrates that SUN2 is involved in the functional attachment and movement of meiotic telomeres. In summary, this thesis has shown SUN2 and the meiotic nuclear lamina to be directly involved in or essential for the highly conserved attachment and movement of telomeres, making them critical for a successful meiosis. The meiotic NE is therefore in this thesis demonstrated to be a determinant of mammalian fertility.
Für Patienten mit malignem Melanom im Stadium der Fernmetastasierung gibt es bis heute lediglich Therapieoptionen mit sehr eingeschränkten Erfolgsaussichten. Diese Tatsache bestätigt die Notwendigkeit von Biomarkern zur Vorhersage des Erfolgs verschiedener Therapien. Der ATP-basierende ex vivo Chemosensitivitätsassay hat sich als erfolgreiche Methode zur individuellen Vorhersage eines Chemotherapieerfolgs herausgestellt. Tatsächlich zeigte der Assay ein heterogenes Sensitivitätsprofil gegen verschiedene Chemotherapeutika und ließ in getesteten Patienten ein ex vivo wirksames Chemotherapieregime identifizieren, das anschließend auch klinische Therapieerfolge bei Verwendung der Therapie mit dem besten individuellen Chemosensitivitätsindex(BICSI) zeigte. Um diesen sehr aufwendigen Assay zukünftig zu umgehen, sollten in der vorliegenden Arbeit prädiktive molekulare Biomarker der Chemosensitivität identifiziert werden. Hierfür wurden im Voraus durch einen Microarray die Kandidaten Secernin 1 (SCRN1), Lysyl oxidaselike 1 (LOXL1), Thymosin beta 4 X-linked (TMSB4X), Vesicle-associated membrane protein 5 (VAMP5) und Serine protease inhibitor B1 (SERPINB1) als differentiell exprimierte Gene in chemosensitivem gegenüber chemoresistentem Gewebe identifiziert. Die relative Expression dieser Kandidatengene wurde daraufhin in bis zu 128 verschiedenen Melanomgeweben mit dem Chemosensitivitätsindex verschiedener Chemotherapeutika korreliert. Hierbei konnte eine signifikante Korrelation zwischen SerpinB1 mit der Chemosensitivität gegenüber der Therapiekombination mit Paclitaxel und Cisplatin auf Gen- aber nicht auf Proteinebene identifiziert werden. Weiterhin konnte eine differentielle Expression ebenfalls in chemosensitiven und -resistenten Melanomzelllinien nachgewiesen werden, die allerdings im Vergleich mit dem analysierten Gewebe in gegensätzlicher Richtung verlief. Zusammenfassend lässt sich sagen, dass SerpinB1 ein vielversprechender Marker für die Chemosensitivität gegenüber Paclitaxel und Cisplatin ist, dessen funktionelle Bedeutung aber unklar bleibt. Das Merkelzellkarzinom (MCC) ist ein seltener und hoch aggressiver Tumor der mit dem Merkelzellpolyomavirus (MCV) in Zusammenhang steht. Da MCC Zelllinien zur Aufrechterhaltung ihrer Viabilität die MCV T-Antigene benötigen, könnte der Einsatz von Interferonen (IFN) ein möglicher therapeutischer Ansatz zur Behandlung dieser Krebserkrankung sein. In der vorliegenden Arbeit haben wir daher die Effekte von IFNs auf MCC Zelllinien, mit besonderer Berücksichtigung der MCV+ Linien, untersucht. IFNs vom Typ I (hier Multiferon, ein Mix verschiedener IFN α Subtypen, und IFN β) wirkten stark inhibierend auf die zelluläre Viabilität. Die Zellzyklusanalyse zeigte eine Erhöhung des sub-G Anteils der Zellen nach Behandlung mit IFN, was auf Apoptose als ausschlagebenden Grund schließen ließ. Diese Effekte waren für die Behandlung mit IFN β weniger stark ausgeprägt. Der inhibitorische Effekt von Typ I IFNs auf MCV+ MCC Zelllinien war assoziiert mit einer verringerten Expression des viralen großen T-Antigens (LTA) und einer Erhöhung in der Expression von promyelocytic leukemia protein (PML), das dafür bekannt ist, die Funktion des LTA störend zu beeinflussen. Zusätzlich führte die intratumorale Anwendung von Multiferon in vivo zu einer Regression im Wachstum von MCV+, aber nicht MCV- MCC Xenotransplantaten. Die Ergebnisse zeigen das Typ I IFNs einen starken antitumoralen Effekt haben, der zum Teil durch die Regulierung des LTA herbeigeführt wird. Neben diesen direkten Effekten der IFNs auf die Zellproliferation induzieren diese auch die Expression von MHC Klasse I Molekülen in MCC Zelllinien. Die Durchflusszytometrie zeigte eine Induktion der MHC Klasse I Expression in drei MHC I negativen MCC Zelllinien und eine Erhöhung der Expression, die vor der Behandlung eine geringe Menge an MHC I aufwiesen. Diese Effekte konnten auch in den in vivo Xenotransplantaten beobachtet werden. Die Ergebnisse zeigen, dass die Behandlung mit IFN sowohl direkte als auch indirekte Effekte auf das MCC hat und eine breite Anwendung in Patienten mit MCV+ und MCV- Tumoren finden kann. Neben IFNs sind auch Artemisinin und seine Derivate bekannt für ihre antitumoralen und antiviralen Eigenschaften. Daher haben wir den Effekt des Artemisininderivats Artesunate auf MCV+ und MCV- MCC Zelllinien getestet. Tatsächlich konnten wir auch hier einen antiproliferativen Effekt des Stoffes nachweisen, der stärker auf MCV+ als auf MCV- Zelllinien wirkte und bei ersteren wiederum mit einer reduzierten LTA Expression einherging. Im Vergleich dazu blieben Fibroblasten von der Behandlung unbeeinflusst. Das verringerte Tumorwachstum konnte ebenfalls für in vivo Xenotransplantationsmodelle gezeigt werden. Auf Grundlage dieser Erkenntnis sollte eine genauere Untersuchung dieses alten Naturheilstoffes für die Behandlung von MCC Patienten in Betracht gezogen werden.
Is behaviour response or action? In this Thesis I study this question regarding a rather simple organism, the larva of the fruit fly Drosophila melanogaster. Despite its numerically simple brain and limited behavioural repertoire, it is nevertheless capable to accomplish surprisingly complex tasks. After association of an odour and a rewarding or punishing reinforcement signal, the learnt odour is able to retrieve the formed memory trace. However, the activated memory trace is not automatically turned into learned behaviour: Appetitive memory traces are behaviourally expressed only in absence of the rewarding tastant whereas aversive memory traces are behaviourally expressed in the presence of the punishing tastant. The ‘decision’ whether to behaviourally express a memory trace or not relies on a quantitive comparison between memory trace and current situation: only if the memory trace (after odour-sugar training) predicts a stronger sugar reward than currently present, animals show appetitive conditioned behaviour. Learned appetitive behaviour is best seen as active search for food – being pointless in the presence of (enough) food. Learned aversive behaviour, in turn, can be seen as escape from a punishment – being pointless in absence of punishment. Importantly, appetitive and aversive memory traces can be formed and retrieved independent from each other but also can, under appriate circumstances, summate to jointly organise conditioned behaviour. In contrast to learned behaviour, innate olfactory behaviour is not influenced by gustatory processing and vice versa. Thus, innate olfactory and gustatory behaviour is rather rigid and reflexive in nature, being executed almost regardless of other environmental cues. I suggest a behavioural circuit-model of chemosensory behaviour and the ‘decision’ process whether to behaviourally express a memory trace or not. This model reflects known components of the larval chemobehavioural circuit and provides clear hypotheses about the kinds of architecture to look for in the currently unknown parts of this circuit. The second chapter deals with gustatory perception and processing (especially of bitter substances). Quinine, the bitter tastant in tonic water and bitter lemon, is aversive for larvae, suppresses feeding behaviour and can act as aversive reinforcer in learning experiments. However, all three examined behaviours differ in their dose-effect dynamics, suggesting different molecular and cellular processing streams at some level. Innate choice behaviour, thought to be relatively reflexive and hard-wired, nevertheless can be influenced by the gustatory context. That is, attraction toward sweet tastants is decreased in presence of bitter tastants. The extent of this inhibitory effect depends on the concentration of both sweet and bitter tastant. Importantly, sweet tastants differ in their sensitivity to bitter interference, indicating a stimulus-specific mechanism. The molecular and cellular processes underlying the inhibitory effect of bitter tastants are unknown, but the behavioural results presented here provide a framework to further investigate interactions of gustatory processing streams.
The Xiphophorus melanoma system is a useful animal model for the study of the genetic basis of tumor formation. The development of hereditary melanomas in interspecific hybrids of Xiphophorus is connected to pigment cell specific overexpression of the mutationally activated receptor tyrosine kinase Xmrk. In purebred fish the oncogenic function of xmrk is suppressed by the molecularly still unidentified locus R. The xmrk oncogene was generated by a gene duplication event from the Xiphophorus egfrb gene and thereby has acquired a new 5’ regulatory sequence, which has probably altered the transcriptional control of the oncogene. So far, the xmrk promoter region was still poorly characterized and the molecular mechanism by which R controls xmrk-induced melanoma formation in Xiphophorus still remained to be elucidated. To test the hypothesis that R controls melanoma development in Xiphophorus on the transcriptional level, the first aim of the thesis was to gain a deeper insight into the transcriptional regulation of the xmrk oncogene. To this end, a quantitative analysis of xmrk transcript levels in different Xiphophorus genotypes carrying either the highly tumorigenic xmrkB or the non-tumorigenic xmrkA allele was performed. I was able to demonstrate that expression of the tumorigenic xmrkB allele is strongly increased in malignant melanomas of R-free backcross hybrids compared to benign lesions, macromelanophore spots, and healthy skin. The expression level of the non-tumorigenic xmrkA allele, in contrast, is not influenced by the presence or absence of R. These findings strongly indicate that differential transcriptional regulation of the xmrk promoter triggers the tumorigenic potential of these xmrk alleles. To functionally characterize the xmrk promoter region, I established a luciferase assay using BAC clones containing the genomic regions where xmrk and egfrb are located for generation of reporter constructs. This approach showed for the first time a melanoma cell specific transcriptional activation of xmrkB by its flanking regions, thereby providing the first functional evidence that the xmrk oncogene is controlled by a pigment cell specific promoter region. Subsequent analysis of different deletion constructs of the xmrkB BAC reporter construct strongly indicated that the regulatory elements responsible for the tumor-inducing overexpression of xmrkB in melanoma cells are located within 67 kb upstream of the xmrk oncogene. Taken together, these data indicate that melanoma formation in Xiphophorus is regulated by a tight transcriptional control of the xmrk oncogene and that the R locus acts through this mechanism. As the identification of the R-encoded gene(s) is necessary to fully understand how melanoma formation in Xiphophorus is regulated, I furthermore searched for alternative R candidate genes in this study. To this end, three genes, which are located in the genomic region where R has been mapped, were evaluated for their potential to be a crucial constituent of the regulator locus R. Among these genes, I identified pdcd4a, the ortholog of the human tumor suppressor gene PDCD4, as promising new candidate, because this gene showed the expression pattern expected from the crucial tumor suppressor gene encoded at the R locus.
Durch das Auftreten neuer Stämme resistenter Krankheitserreger ist die Suche nach neuartigen Wirkstoffen gegen diese, sich ständig weiter ausbreitende Bedrohung, dringend notwendig. Der interdisziplinäre Sonderforschungsbereich 630 der Universität Würzburg stellt sich dieser Aufgabe, indem hier neuartige Xenobiotika synthetisiert und auf ihre Wirksamkeit getestet werden. Die hier vorgelegte Dissertation fügt sich hierbei nahtlos in die verschiedenen Fachbereiche des SFB630 ein: Sie stellt eine Schnittstelle zwischen Synthese und Analyse der Effekte der im Rahmen des SFB630 synthetisierten Isochinolinalkaloid-Derivaten. Mit den hier angewandten bioinformatischen Methoden wurden zunächst die wichtigsten Stoffwechselwege von S. epidermidis R62A, S. aureus USA300 und menschlicher Zellen in sogenannten metabolischen Netzwerkmodellen nachgestellt. Basierend auf diesen Modellen konnten Enzymaktivitäten für verschiedene Szenarien an zugesetzten Xenobiotika berechnet werden. Die hierfür benötigten Daten wurden direkt aus Genexpressionsanalysen gewonnen. Die Validierung dieser Methode erfolgte durch Metabolommessungen. Hierfür wurde S. aureus USA300 mit verschiedenen Konzentrationen von IQ-143 behandelt und gemäß dem in dieser Dissertation vorgelegten Ernteprotokoll aufgearbeitet. Die Ergebnisse hieraus lassen darauf schließen, dass IQ-143 starke Effekte auf den Komplex 1 der Atmungskette ausübt – diese Resultate decken sich mit denen der metabolischen Netzwerkanalyse. Für den Wirkstoff IQ-238 ergaben sich trotz der strukturellen Ähnlichkeiten zu IQ-143 deutlich verschiedene Wirkeffekte: Dieser Stoff verursacht einen direkten Abfall der Enzymaktivitäten in der Glykolyse. Dadurch konnte eine unspezifische Toxizität dieser Stoffe basierend auf ihrer chemischen Struktur ausgeschlossen werden. Weiterhin konnten die bereits für IQ-143 und IQ-238 auf Bakterien angewandten Methoden erfolgreich zur Modellierung der Effekte von Methylenblau auf verschiedene resistente Stämme von P. falciparum 3D7 angewandt werden. Dadurch konnte gezeigt werden, dass Methylenblau in einer Kombination mit anderen Präparaten gegen diesen Parasiten zum einen die Wirkung des Primärpräparates verstärkt, zum anderen aber auch in gewissem Maße vorhandene Resistenzen gegen das Primärpräparat zu verringern vermag. Somit konnte durch die vorgelegte Arbeit eine Pipeline zur Identifizierung der metabolischen Effekte verschiedener Wirkstoffe auf unterschiedliche Krankheitserreger erstellt werden. Diese Pipeline kann jederzeit auf andere Organismen ausgeweitet werden und stellt somit einen wichtigen Ansatz um Netzwerkeffekte verschiedener, potentieller Medikamente aufzuklären.
Many ant species excavate underground nests. One of the most impressive examples is the Chaco leaf-cutting ant Atta vollenweideri from the Gran Chaco region in South America. The nests excavated by the workers of that species are among the largest insect-built structures on the planet. They are ecavated over years possibly involving millions of working individuals. However, the mechanisms underlying the organisation of collective nest digging in ants remain largely unknown. Considering the sheer dimensions of the nest in comparison to the size and presumably limited perceptual and cognitive abilities of the single worker, the assumption can be made that organising mechanisms are mostly based on responses of individuals to local stimuli within their perceptual range. Among these local stimuli that guide nest digging we can expect environmental variables, stimuli that relate to the requirements of the colony, and stimuli related to the spatial coordination of collective effort. The present thesis investigates the role of local stimuli from these three categories in the organisation of collective digging behaviour in the Chaco leaf-cutting ant. It describes experiments on (1) how workers respond in the context of digging to differences in soil moisture, which comprises an important environmental variable; (2) how available nest space influences nest enlargement; (3) and how the spatial coordination of excavating workers is implemented by responding to stimuli arising from nest mates while engaged in digging behaviour. The experiments on soil water content show that workers prefer to dig in moist materials that allow for fast excavation and transport rates. Accordingly, an unequal distribution of water in the soil around a nest can influence how the nest shape develops. On the other hand, results also indicate that workers strongly avoid excavating in extremely moist materials. Regarding the abundant occurrence of flooding events in the Gran Chaco region, the latter can be interpreted as an adaptation to avoid water inflow into the nest. In the experiments on the effect of nest space, the ants excavated less when presented with larger nests. When a large amount of space was suddenly added to the nest during the digging process, excavation rates decreased according to the new volume. These observations confirm the hypothesis that digging activity is regulated according to space requirements, possibly because crowding conditions inside the nest influence excavation behaviour. However, observations also indicate an intrinsic decrease of digging motivation with time. Moreover, excavation rates correlate with nest size only when comparing nests of similar shape. Distributing a similar nest volume to three smaller chambers, instead of one, resulted in drastically decreased digging rates. A possible explanation for that observation lies in the distribution of workers inside the nest that may vary according to nest geometry: a different distribution of individuals can lead to in different local crowding conditions in similar nest volumes. Furthermore, two different stimuli are described that are used in the spatial coordination of collective digging effort. First, fresh soil pellets deposited close to the digging site on their way from the surface increase the probability that arriving workers join excavation efforts at the same site. The deposition of pellets on the way is a consequence of sequential task partitioning during soil transport. The pellets are carried in transport chains that closely resemble the modalities of leaf transport observed at the surface. Second, workers stridulate while digging. The short-ranged vibrational signals produced thereby also attract nest mates to excavate at the same location. Accordingly, two mutually complementing mechanisms are described that allow to concentrate excavators at one location. In both cases, a local stimulus that is generated by current close-by excavation activity increases the probability of the stimulus receiver to dig close to other excavators. In an environment otherwise poor in digging stimuli, these mechanisms can be especially important to give collective digging efforts a common direction. As a consequence it can be argued that the spatial organisation of collective digging is based on choice copying. Individuals copy nest mate decisions on where to excavate by responding to local stimuli provided by nest mate digging activity. Taken together, responses to local stimuli can determine the direction of nest growth, aid in preventing the inflow of surface water into the nest, guide the adjustment of nest size to colony requirements and spatially coordinate collective digging efforts. Even though it cannot be ruled out that digging responses based e.g. on spatial memory or long-term experience exist, the results presented here clearly demonstrate that responses to local information account for many important aspects of nest development.
Trotz beträchtlicher Anstrengung Malaria zu kontrollieren bzw. zu eradizieren, stellt die Krankheit weiterhin eines der gravierendsten Gesundheitsprobleme unseres Jahrtausends dar. Malaria fordert jährlich zwischen 0,7 und 2,7 Millionen Menschenleben, beeinträchtigt schulische und soziale Entwicklung und hemmt erheblich das Wirtschaftswachstum der betroffenen Länder. In Burkina Faso, einem der ärmsten Länder der Welt, ist Malaria eines der größten Gesundheitsprobleme und ca. ein Drittel aller Todesfälle werden hier Malaria angelastet. Die sich weiter ausbreiteten Resistenzen gegen die gängigen Malariamedikamente machen die Bekämpfung der Malaria zunehmend schwierig. Artemisinin basierende Kombinationstherapien sind aktuell, trotz relativ hoher Therapiekosten und erster Resistenzen, die Erstlinien Behandlung. Effektive und billige neue Kombinationstherapien werden dringend benötigt. In dieser Doktorarbeit wurde das Resistenzpotential von Artemisinin modelliert. Die Homologiemodellierungen unterstützen die These von Krishna und Kollegen von SERCA als einzige Zielstruktur von Artemisinin. Des Weiteren wurde Methylenblau als neues altes Malariamittel evaluiert. Methylenblau ist das erste gegen Malaria eingesetzte Medikament, agiert als ein prooxidatives Agens und inhibiert selektiv und nicht-kompetitiv die P. falciparum Glutathion Reduktase. Die additiven und multiplen Zielprotein Effekte von Methylenblau wurden experimentell untersucht und hier in einem bioinformatischem Modell getestet. Unter dem Einfluss von Methylenblau werden einige Schlüsselenzyme des Redoxstoffwechsels in ihrer Aktivität beeinträchtigt und der Parasit wird verstärkt oxidativem Stress ausgesetzt. Des Weiteren konnte in dieser Dr. Arbeit eine starke Kooperationsbereitschaft der urbanen und ländlichen Bevölkerung an zukünftigen Malaria Projekten gezeigt werden.
The neuronal ceroid lipofuscinoses (NCLs) are fatal neurodegenerative disorders in which the visual system is affected in early stages of disease. A typical accompanying feature is neuroinflammation, the pathogenic impact of which is presently unknown. In this study, the role of inflammatory cells in the pathogenesis was investigated in Palmitoyl-protein thioesterase 1-deficient (Ppt1-/-) and Ceroidlipofuscinosis, neuronal 3-deficient (Cln3-/-) mice, models of the infantile and juvenile forms of NCL, respectively. Focusing predominantly on the visual system, an infiltration of CD8+ cytotoxic Tlymphocytes and an activation of microglia/macrophage-like cells was observed early in disease. To analyze the pathogenic impact of lymphocytes, Ppt1-/- mice were crossbred with mice lacking lymphocytes (Rag1-/-) and axonal transport, perturbation and neuronal survival were scored. Lack of lymphocytes led to a significant amelioration of neuronal disease and reconstitution experiments revealed a crucial role of CD8+ cytotoxic T-lymphocytes. Lack of lymphocytes also caused an improved clinical phenotype and extended longevity. To investigate the impact of microglia/macrophage-like cells, Ppt1-/- and Cln3-/- mice were crossbred with mice lacking sialoadhesin (Sn-/-), a monocyte lineage-restricted cell adhesion molecule important for interactions between macrophage-like cells and lymphocytes. Similar to the lack of lymphocytes, absence of sialoadhesin significantly ameliorated the disease in Ppt1-/- and Cln3-/- mice. Taken together, both T-lymphocytes and microglia/macrophage-like cells were identified as pathogenic mediators in two distinct forms of fatal inherited neurodegenerative storage disorders. These studies expand the concept of secondary inflammation as a common pathomechanistic feature in some neurological diseases and provide novel insights that may be crucial for developing treatment strategies for different forms of NCL.
Zurzeit sterben jährlich ca. 11.000 Männer in Deutschland am Prostatakarzinom. Damit stellt dies die zweithäufigste Krebstodesursache von Männern dar. Da das Prostatakarzinom häufig asymptomatisch verläuft, wird die Erkrankung oftmals erst so spät erkannt, dass zum Zeitpunkt der Diagnose bereits eine Metastasierung stattgefunden hat. Durch metastasierende Prostatakarzinomzellen werden Lymphknoten, Knochen und Lungen befallen. Es sind zwei unterschiedliche Verbreitungsarten von metastasierenden Tumorzellen beschrieben. Zum einen kann eine Migration über Lymphgefäße erfolgen, ein Prozess der als lymphatische Metastasierung bezeichnet wird. Zum anderen können Tumorzellen über das Blutsystem im Körper zirkulieren: die hämatogene Metastasierung. In dieser Arbeit wurde die lymphatische Metastasierung der humanen Prostatakarzinomzellline PC-3 im Detail analysiert und Teilaspekte der hämatogenen Verteilung untersucht. Ausgangspunkt der Untersuchungen bildete die Vergrößerung lumbaler und renaler Lymphknoten in PC-3-Tumor-tragenden Mäusen 60 Tage nach der Implantation von PC-3-Zellen. Es wurde daraufhin der zeitliche Verlauf der Vergrößerung untersucht und festgestellt, dass sowohl das Volumen als auch die Anzahl vergrößerter Lymphknoten von Woche zu Woche nach Implantation der PC-3-Tumore zunehmen. Anschließend wurden alle vergrößerten Lymphknoten bezüglich des Vorhandenseins von metastasierenden humanen PC-3-Zellen in den Mäusen untersucht. Dies geschah mit Hilfe einer RT-PCR unter Verwendung von Primern für humanes β-Aktin. Sechs Wochen nach Implantation konnten in 90 % der vergrößerten Lymphknoten PC-3-Zellen nachgewiesen werden. Weiterhin wurde durch lentivirale Transduktion das Gen für das rot fluoreszierende Protein (RFP) in die PC-3-Zellen inseriert, wodurch eine Visualisierung dieser Zellen in der Maus ermöglicht wurde. Es konnten metastasierende PC-3-RFP-Zellen in lumbalen und renalen Lymphknoten PC-3-RFP-Tumor-tragender Mäuse nachgewiesen werden. Ebenso konnte mittels RFP gezeigt werden, dass die Lymphknotenmetastasierung in Abhängigkeit von der Lokalisation des PC-3-RFP-Tumors erfolgt. Es kam zur Metastasierung jener Lymphknoten, in deren Einzugsgebiet sich der PC-3-Tumor befand. Es wurde eine PC-3-RFP-Zellmigration zwischen lumbalen und renalen Lymphknotenmetastasen nachgewiesen und bei immunhistologischen Untersuchungen stellte sich heraus, dass PC-3-RFP-Zellen tatsächlich in lymphatischen Bahnen zwischen lumbalen und renalen Lymphknotenmetastasen migrieren. Außerdem wurde gezeigt, dass es von Woche zu Woche nach Implantation von PC-3-Zellen zu einer Zunahme der Anzahl von Lymphgefäßen in PC-3-Tumoren kommt. Die Zunahme der Lymphgefäßdichte korrelierte hierbei positiv mit der Bildung von Lymphknotenmetastasen. Es konnten weiterhin neben Lymphknotenmetastasen hämatogene Mikrometastasen in den Lungen PC-3-RFP-Tumor-tragender Mäuse beobachtet werden. Da die Haupttodesursache von Prostatakarzinompatienten in der Bildung von Metastasen liegt, ist es von herausragender Bedeutung eine effektive Therapie gegen lymphatische und hämatogene Metastasen zu entwickeln. Aus diesem Grund erlangt die onkolytische Virustherapie große Bedeutung. Deshalb wurde als zweiter Aspekt in dieser Arbeit der Einfluss des onkolytischen Vaccinia-Virus GLV-1h68 auf den Prozess der PC-3-Zellmetastasierung untersucht. Dabei konnte zunächst gezeigt werden, dass GLV-1h68 in der Lage ist, erfolgreich sowohl migrierende PC-3-Zellen als auch metastasierende PC-3-Zellen in Lymphknoten zu kolonisieren. In der Folge wurde deshalb ein möglicher Metastasen-inhibierender Effekt von GLV-1h68 untersucht. Hierbei stellte sich heraus, dass GLV-1h68 drei Wochen nach intravenöser Injektion eine signifikante Reduktion der Anzahl der für PC-3-Zellen positiven Lymphknoten bewirkt. Des Weiteren konnte ein inhibierender Effekt von GLV-1h68 auf die im Blut zirkulierenden PC-3-Zellen und auf hämatogene Metastasen in den Lungen beobachtet werden. Durch intravenöse Injektion von GLV-1h68 in PC-3-RFP-Tumor-tragenden Mäusen konnte gezeigt werden, dass es zu einer präferentiellen Virus-Kolonisierung der Lymphknotenmetastasen im Vergleich zu den Tumoren kommt. Auch nach intraperitonealer und intratumoraler Injektion von GLV-1h68 konnte eine präferentielle Virus-Kolonisierung der Lymphknotenmetastasen gezeigt werden. Darüber hinaus wurden die Lymph- und Blutgefäße von PC-3-Tumoren und Lymphknotenmetastasen analysiert. Hierbei wurde gezeigt, dass es sieben Tage nach intravenöser Injektion von GLV-1h68 zu einer signifikanten Abnahme von beiden Gefäßarten kam. Es wurde in dieser Arbeit somit gezeigt, dass GLV-1h68 in der Lage ist, sowohl lymphatische als auch hämatogene Metastasen der Prostatakarzinomzelllinie PC-3 erfolgreich zu eliminieren. Folglich dürften onkolytische Vaccinia-Viren ein vielversprechendes Therapeutikum für die Behandlung des fortgeschrittenen Prostatakarzinoms darstellen.
Thrombozyten (Blutplättchen) sind die Vermittler der zellulären Hämostase. Ihre Fähigkeit zu Aggregieren und sich an das umgebende Gewebe verletzter Blutgefässe anzulagern, wird durch ein komplexes intrazelluläres Signaltransduktionsnetzwerk bestimmt, das sowohl aktivierende, als auch inhibierende Subnetzwerke beinhaltet. Das Verständnis dieser Prozesse ist von hoher medizinischer Bedeutung. Im Rahmen dieser Arbeit wurde die thrombozytäre Signaltransduktion sowohl mittels eines Boole'schen, als auch verschiedener dynamischer Modelle analysiert. Die Boole'sche Modellierung führte zu interessanten Erkenntnissen über das Zusammenwirken einzelner Subnetzwerke bei der Vermittlung irreversibler Plättchenaktivierung und zeigte Mechanismen der Interaktion mit dem hemmenden Prostaglandinsystem auf. Das Modell beinhaltet unter Anderem wichtige Systemkomponenten wie Calciumsignalgebung, Aktivierung von Schlüsselkinasen wie Src und PKC, Integrin-vermitteltes outside-in sowie inside-out Signalgebung und autokrine ADP- und Thromboxan-Produktion. Unter Verwendung dieses Boole'schen Ansatzes wurde weiterhin das System-eigene Schwellenwertverhalten analysiert. Dabei stellte sich eine umgekehrt proportionale Abhängigkeit des relativen aktivierenden Reizes, der notwendig ist um den Schwellenwert zu überschreiten, vom absoluten hemmenden Input heraus. Das System adaptiert demnach an höhere Prostaglandinkonzentrationen durch eine Erhöhung der Sensitivität für Aktivatoren wie dem van-Willebrandt-Faktor und Kollagen, und ermöglicht somit auch unter lokal hemmenden Bedingungen eine Plättchen-vermittelte Hämostase. Der nächste Schritt bestand in der Implementierung eines Differentialgleichungs-basierten Modells der thrombozytären Prostaglandin-Signaltransduktion, um einen detaillierten Überblick über die Dynamik des inhibierenden Netzwerkteils zu erhalten. Die kinetischen Parameter dieses Modells wurden teilweise der Literatur entnommen. Der andere Teil wurde anhand einer umfassenden Kombination dosis- und zeitabhängiger cAMP und phospho-VASP Messdaten geschätzt. Der Prozess beinhaltete mehrere Iterationen aus Modellvorhersagen einerseits und experimentellem Design andererseits. Das Modell liefert die quantitativen Effekte der Prostaglandinrezeptoren IP, DP1, EP3 und EP4 und des ADP-Rezeptors P2Y12 auf die zugrunde liegende Signalkaskade. EP4 zeigt den stärksten Effekt in der aktivierenden Fraktion, wohingegen EP3 einen stärkeren inhibitorischen Effekt ausübt, als der durch Clopidogrel hemmbare ADP-Rezeptor P2Y12. Weiterhin wurden die Eigenschaften des negativen feedback-loops der PKA auf den cAMP-Spiegel untersucht, und eine direkte Beeinflussung der Adenylatzyklase durch die PKA festgestellt, in Form einer Reduzierung der maximalen katalytischen Geschwindigkeit. Die Identifizierbarkeit der geschätzten Parameter wurde mittels profile-Likelihood-Schätzung untersucht. In einem dritten Schritt wurde ein sowohl die aktivierenden, als auch die hemmenden Netzwerkteile umfassendes dynamisches Modell implementiert. Die Topologie dieses Modells wurde in Anlehnung an die des Boole'schen Modells auf der Basis von a priori Wissen festgelegt. Die Modellparameter wurden anhand von Western-Blot, Calcium- und Aggregationsmessungen geschätzt. Auch hier wurde die Identifizierbarkeit der Modellparameter durch profile-likelihood-Schätzung überprüft. Die bei niedrigen Ligandenkonzentrationen auftretende Reversibilität der Plättchen-Aggregation konnte mittels dieses Modells reproduziert werden. Jedoch zeigte sich bei mittleren ADP-Konzentrationen ein Fließgleichgewicht in einem teilweise aktivierten Zustand, und damit kein bistabiles Schwellenwertverhalten. Inwiefern dieses Verhalten durch einen Umgebungs-basierteren Mechanismus des Alles-Oder-Nichts-Verhaltens begründet wird, bei dem der Übergang von reversibler zu irreversibler Aggregation mehr durch parakrine Effekte des gesammten Thrombus bestimmt wird, als durch spezifische Signaltransduktionseigenschaften der einzelnen Zelle, müssen zukünftige Experimente zeigen. Insgesamt geben die erstellten Modelle interessante Einblicke in die Funktionsweise der Thrombozyten und ermöglichen die Simulation von pharmakologischen und genetischen Einflüssen, wie Rezeptormodulationen und knock-outs. Sie geben damit Implikationen zur Entstehung und Behandlung pathophysiologischer Zustände, und wertvolle Denkanstöße für die weitere Forschung.
Bees have had an intimate relationship with humans for millennia, as pollinators of fruit, vegetable and other crops and suppliers of honey, wax and other products. This relationship has led to an extensive understanding of their ecology and behavior. One of the most comprehensively understood species is the Western honeybee, Apis mellifera. Our understanding of sex-specific investment in other bees, however, has remained superficial. Signals and cues employed in bee foraging and mating behavior are reasonably well understood in only a handful of species and functional adaptations are described in some species. I explored the variety of sensory adaptations in three model systems within the bees. Females share a similar ecology and similar functional morphologies are to be expected. Males, engage mainly in mating behavior. A variety of male mating strategies has been described which differ in their spatiotemporal features and in the signals and cues involved, and thus selection pressures. As a consequence, males’ sensory systems are more diverse than those of females. In the first part I studied adaptations of the visual system in honeybees. I compared sex and caste-specific eye morphology among 5 species (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera). I found a strong correlation between body size and eye size in both female castes. Queens have a relatively reduced visual system which is in line with the reduced role of visual perception in their life history. Workers differed in eye size and functional morphology, which corresponds to known foraging differences among species. In males, the eyes are conspicuously enlarged in all species, but a disproportionate enlargement was found in two species (A. dorsata, A. florea). I further demonstrate a correlation between male visual parameters and mating flight time, and propose that light intensities play an important role in the species-specific timing of mating flights. In the second study I investigated eye morphology differences among two phenotypes of drones in the Western honeybee. Besides normal-sized drones, smaller drones are reared in the colony, and suffer from reduced reproductive success. My results suggest that the smaller phenotype does not differ in spatial resolution of its visual system, but suffers from reduced light and contrast sensitivity which may exacerbate the reduction in reproductive success caused by other factors. In the third study I investigated the morphology of the visual system in bumblebees. I explored the association between male eye size and mating behavior and investigated the diversity of compound eye morphology among workers, queens and males in 11 species. I identified adaptations of workers that correlate with distinct foraging differences among species. Bumblebee queens must, in contrast to honeybees, fulfill similar tasks as workers in the first part of their life, and correspondingly visual parameters are similar among both female castes. Enlarged male eyes are found in several subgenera and have evolved several times independently within the genus, which I demonstrate using phylogenetic informed statistics. Males of these species engage in visually guided mating behavior. I find similarities in the functional eye morphology among large-eyed males in four subgenera, suggesting convergent evolution as adaptation to similar visual tasks. In the remaining species, males do not differ significantly from workers in their eye morphology. In the fourth study I investigated the sexual dimorphism of the visual system in a solitary bee species. Males of Eucera berlandi patrol nesting sites and compete for first access to virgin females. Males have enlarged eyes and better spatial resolution in their frontal eye region. In a behavioral study, I tested the effect of target size and speed on male mate catching success. 3-D reconstructions of the chasing flights revealed that angular target size is an important parameter in male chasing behavior. I discuss similarities to other insects that face similar problems in visual target detection. In the fifth study I examined the olfactory system of E. berlandi. Males have extremely long antennae. To investigate the anatomical grounds of this elongation I studied antennal morphology in detail in the periphery and follow the sexual dimorphism into the brain. Functional adaptations were found in males (e.g. longer antennae, a multiplication of olfactory sensilla and receptor neurons, hypertrophied macroglomeruli, a numerical reduction of glomeruli in males and sexually dimorphic investment in higher order processing regions in the brain), which were similar to those observed in honeybee drones. The similarities and differences are discussed in the context of solitary vs. eusocial lifestyle and the corresponding consequences for selection acting on males.
Trypanosome Motion Represents an Adaptation to the Crowded Environment ofthe Vertebrate Bloodstream
(2012)
Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy
Ofall amphibians living in arid habitats, reed frogs (belonging to the super species Hyperolius viridiflavus) are the most peculiar. Froglets are able to tolerate dry periods of up to 35 days or longer immediately after metamorphosis, in climatically exposed positions. They face similar problems to estivating juveniles, i.e. enduranee of long periods of high temperature and low RH with rather limited energy and water reserves. In addition, they must have had to develop meehanisms to prevent poisoning by nitrogenous wastes that rapidly accumulate during dry periods as a metabolie consequenee of maintaining a non-torpid state. During dry periods, plasma osmolarity of H. v. taeniatus froglets strongly increased, mainly through urea accumulation. Urea accumulation was also observed during metamorphic climax. During postmetamorphic growth, chromatophores develop with the density and morphology typical of the adult pigmentary pattern. The dermal iridophore layer, which is still incomplete at this time, is fully developed within 4-8 days after metamorphosis, irrespective of maintenance conditions. These iridophores mainly contain the purines guanine and hypoxanthine. The ability of these purines to reflect light provides an excellent basis for the role of iridophores in temperature regulation. In individuals experiencing dehydration stress, the initial rate of purine synthesis is doubled in eomparison to specimens continuously maintained under wet season conditions. This increase in synthesis rate leads to a rapid increase in the thiekness of the iridophore layer, thereby effectively reducing radiation absorption. Thus, the danger of overheating is diminished during periods of water shortage when evaporative cooling must be avoided. After the development of an iridophore layer of sufficient thickness for effective radiation reflectance, synthesis of iridophore pigments does not cease. Rather, this pathway is further used during the remaining dry season for solving osmotic problems eaused by accumulation of nitrogenous wastes. During prolonged water deprivation, in spite of reduced metabolic rates, purine pigments are produced at the same rate as in wet season conditions. This leads to a higher relative proportion of nitrogen end products being stored in skin pigments under dry season conditions. At the end of an experimental dry season lasting 35 days, up to 38% of the accrued nitrogen is stored in the form of osmotically inactive purines in thc skin. Thus the osmotic problems caused by evaporative water loss and urea production are greatly reduced.
Reed frogs of the superspecies Hyperolius viridiflavus occur throughout the seasonally very dry and hot African savannas. Despite their small size (300-700 mg), estivating reed frogs do not avoid stressful conditions above ground by burrowing into the soil, but endure the inhospitable climate relatively unprotected, clinging to mostly dry grass sterns. They must have emcient mechanisms to enable them to survive e.g. very high temperatures, low relative hurnidities, and high solar radiation loads. Mechanisms must also have developed to prevent poisoning by the nitrogenous wastes that inevitably result from protein and nucleotide turnover. In contrast to fossorial amphibians, estivating reed frogs do not become torpid. Reduction in metabolism is therefore rather Iimited so that nitrogenous wastes accumulate faster in these frogs than in fossorial amphibians. This severely aggravates the osmotic problems caused by dehydration. During dry periods total plasma osmolarity greatly increases, mainly due to urea accumulation. Of the total urea accumulated over 42 days of experimental water deprivation, 30% was produced during the first 7 days. In the next 7 days rise in plasma urea content was negligible. This strong initial increase of urea is seen as a byproduct of elevated amino acid catabolism following the onset of dry conditions. Tbe rise in total plasma osmolarity due to urea accumulation, however, is not totally disadvantageous, but enables fast rehydration when water is available for very short periods only. Voiding of urine and feces eeases once evaporative water loss exceeds 10% of body weight. Tberefore, during continuous water deprivation, nitrogenous end products are not excreted. After 42 days of water deprivation, bladder fluid was substantially depleted, and urea coneentration in the remaining urine (up to 447 mM) was never greater than in plasma fluid. Feces voided at the end of the dry period after water uptake contained only small amounts of nitrogenous end products. DSF (dry season frogs) seemed not to be uricotelic. Instead, up to 35% of the total nitrogenous wastes produced over 42 days of water deprivation were deposited in an osmotically inert and nontoxic form in iridophore crystals. The increase in skin purine content averaged 150 µg/mg dry weight. If urea had been the only nitrogenous waste product during an estivation period of 42 days, lethal limits of total osmolarity (about 700 mOsm) would have been reached 10-14 days earlier. Thus iridophores are not only involved in colour change and in reducing heat load by radiation remission, but are also important in osmoregulation during dry periods. The seIective advantages of deposition of guanine rather than uric acid are discussed.
Scorpions, living in North African semideserts are - in spite of disrupting experimental interferences - able to maintain a certain direction in their natural environment in the dark on a plane surface. Under comparable laboratory conditions, excluding the possibility of light or gravity orientation, they can orient themselves if a directed air current passes over the "arena." In most cases the scorpions do not run necessarily with or against the wind, but rather maintain constant angles to the air current for anywhere from minutes to many hours. They are running anemomenotactically (ref. 1). Under identical conditions many species of beetles also orient themselves to air currents (refs. 2 to 4). The main problems to be solved in the study of anemomenotactic orientation are: (1) Which physical qualities of the air current have an influence on the anemomenotaxis? (2) With which sense organs do beetles and scorpions perceive wind directions? (3) Which physiological mechanism is the basis of anemomenotactic orientation? (4) What is the biological significance of anemomenotaxis in beetles and scorpions? With respect to these problems, more study has been done on beetles than on scorpions. Therefore, due to lack of space, I shall discuss mainly some of the results obtained in experiments with dung beetles (Geotrupes silvaticus, G. ,Stercorarius, G. armifrons, G. niger, Scarabaeus variolosus) and tenebrionid beetles (Tenebrio molitor, Pimelia grossa, P. tenuicomis, Scaurus dubius).