@phdthesis{Winkler2015,
  author    = {Winkler, Ann-Cathrin Nicole},
  title     = {Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Staphylococcus aureus is both a human commensal and a pathogen. 20\%-30\% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options. In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen. In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death. In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger. All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys. Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Schneider2015,
  author    = {Schneider, Gudrun},
  title     = {Effects of adjacent habitats and landscape composition on biodiversity in semi-natural grasslands and biological pest control in oilseed rape fields},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113549},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {1) Modern European agricultural landscapes form a patchy mosaic of highly fragmented natural and semi-natural habitat remnants embedded in a matrix of intensively managed agricultural land. In those landscapes many organism frequently cross habitat borders including the crop - non-crop boundary, hereby connecting the biotic interactions of multiple habitat types. Therefore biodiversity and ecosystem functions within habitats are expected to depend on adjacent habitat types and the surrounding landscape matrix. In this thesis the biodiversity of non-crop habitats, and ecosystem services and disservices in crop habitats were studied in the human-dominated agricultural landscape in the district Lower Franconia, Bavaria, Germany. First we examined the effect of adjacent habitat type on species composition, diversity and ecosystem functions in semi-natural calcareous grasslands, a biodiversity-rich habitat of high conservation value (chapter 2 and 3). Second we studied the effect of habitat composition in the landscape on herbivory, biological pest control and yield in oilseed rape fields (chapter 4). 2) We examined the effect of adjacent habitat type on the diversity of carabid beetles in 20 calcareous grasslands using pitfall traps. Half of the grasslands were adjacent to a coniferous forest and half to a cereal crop field. We found different species compositions of carabid beetles depending on adjacent habitat type. In addition calcareous grasslands adjacent to crop fields harboured a higher species richness and activity density but a lower evenness of carabid beetles than calcareous grasslands adjacent to forests. These differences can be explained by the spillover of carabid beetles from the adjacent habitats. After crop harvest carabid beetle activity density in crop fields decreased while in parallel the activity density in the calcareous grasslands adjacent to the crop fields increased, indicating an unidirectional carabid beetle spillover. Our results underline that type and management of adjacent habitats affect community composition and diversity in calcareous grasslands. Therefore nature conservation measures, which focused on the improvement of local habitat quality so far, additionally need to consider adjacent habitat type. 3) In addition to carabid beetle communities we also surveyed predation rates of ground-dwelling predators on the same calcareous grasslands in two study periods (June and late August). As ground-dwelling predators of forests or crop fields can move into adjacent calcareous grasslands we expected different predation rates depending on adjacent habitat type. We exposed in total 32.000 lady bird eggs as prey items on the calcareous grasslands in distances of 5 and 20m from the habitat border. We found higher predation rates on calcareous grasslands adjacent to forests than on calcareous grasslands adjacent to crop fields, but only on cool days. On warm days a very high extent (often 100\%) of the exposed prey items were consumed adjacent to both habitat types, which did not allow the detection of possible differences between the adjacent habitat types. Predation rates differed not between the two study periods or the two distances to the habitat edge. The higher predation rates adjacent to forests can be explained by the spillover of ground-dwelling predators from forests into calcareous grasslands. Our results show, that spillover into semi-natural habitats affects ecosystem functioning in addition to species composition and diversity. 4) In chapter 4 of this thesis we examined the effect of spatiotemporal changes in crop cover on pest - natural enemy interactions and crop yields. During two study years we surveyed the abundance of adult and larval pollen beetles, parasitism of pollen beetle larvae by a hymenopteran parasitoid and oilseed rape yields of 36 oilseed rape fields. The surrounding landscape of the fields (1 km radius) differed in the oilseed rape proportion and in the inter-annual change in the oilseed rape proportion since the previous year. We found a dilution effect, i.e. a decreasing abundance with increasing oilseed rape proportions, for pollen beetle larvae and parasitoids in both study years and for adult pollen beetles in one study year. Oilseed rape yields increased with increasing oilseed rape proportions. Inter-annual changes in oilseed rape proportions led to inter-annual crowding and dilution effects for pollen beetles, but had no effect on parasitism or yield. Our results indicate the potential to reduce pest loads and increase yields in intensively managed oilseed rape fields by a coordinated management of the spatiotemporal oilseed rape cover in the landscape. 5) In summary, we showed in this thesis that the biodiversity and functioning of crop and non-crop habitats within agricultural landscapes is affected by the spillover of organisms and thus by the habitat composition in the close surrounding and in the broader landscape context. Spillover affects also ecosystem services and disservices and therefore crop productivity. Thereby the spatial and temporal variation of specific crop types in the landscape can be of particular importance for crop yields. Thus a coordinated landscape wide management can help to optimize both biodiversity conservation and the delivery of ecosystem services and thus crop yields. Future studies integrating landscape effects across several ecosystem functions, multiple taxonomic groups and different crop types are necessary to develop definite landscape management schemes.},
  subject      = {Landschafts{\"o}kologie},
  language  = {en}
}
@phdthesis{MeirgebRother2015,
  author    = {Meir [geb. Rother], Juliane},
  title     = {Influence of oncolytic vaccinia viruses on metastases of human and murine tumors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118530},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Cancer is one of the leading causes of death. 90\% of all deaths are caused by the effects of metastases. It is of major importance to successfully treat the primary tumor and metastases. Tumors and metastases often differ in their properties and therefore, treatment is not always successful. In contrast, those therapeutic agents can even promote formation and growth of metastases. Hence, it is indispensable to find treatment options for metastatic disease. One promising candidate represents the oncolytic virus therapy with vaccinia viruses. The aim of this work was to analyze two cell lines regarding their metastatic abilities and to investigate whether oncolytic vaccinia viruses are useful therapy options. The cell lines used were the human cervical cancer cell line C33A implanted into immune-compromised mice and the murine melanoma cell line B16F10, implanted into immune-competent mice. The initial point of the investigations was the observation of enlarged lumbar und renal lymph nodes in C33A tumor-bearing mice 35 days post implantation of C33A cells subcutaneously into immune-compromised nude mice. Subsequently, the presence of human cells in enlarged lymph nodes was demonstrated by RT-PCR. To facilitate the monitoring of cancer cell spreading, the gene encoding for RFP was inserted into the genome of C33A cells. In cell culture experiments, it was possible to demonstrate that this insertion did not negatively affect the susceptibility of the cells to virus infection, replication and virus-mediated cell lysis. The analysis of the metastatic process in a xenografted mouse model revealed the continuous progression of lumbar (LN) and renal (RN) lymph node metastasis after C33A-RFP tumor cell implantation. The lymph node volume and the amount of RFP-positive LNs and RNs was increasing from week to week in accordance with the gain of the primary tumor volume. Moreover, the metastatic spread of cancer cells in lymph vessels between lumbar and renal lymph nodes was visualized. Additionally, the haematogenous way of cancer cell migration was demonstrated by RFP positive cancer cells in blood vessels. The haematogenous route of spreading was confirmed by detecting micrometastases in lungs of tumor bearing mice. The next step was to investigate whether the recombinant oncolytic vaccinia virus GLV-1h68 is a suitable candidate to cure the primary tumor and metastases. Therefore, GLV-1h68 was systemically injected into C33A-RFP tumor bearing mice 21 days after tumor cell implantation. It was demonstrated that the volume of the primary tumor was drastically reduced, and the volume and the amount of RFP positive lumbar and renal lymph nodes were significantly decreasing compared to the untreated control group. Subsequently, this process was analyzed further by investigating the colonization pattern in the C33A-RFP model. It was shown that first the primary tumor was colonized with highest detectable virus levels, followed by LN and RN lymph nodes. Histological analyses revealed the proliferative status of tumor cells in the tumor and lymph nodes, the amount of different immune cell populations and the vascular permeability in primary tumors and lymph nodes having an influence on the colonization pattern of the virus. Whereby, the vascular permeability seems to have a crucial impact on the preferential colonization of tumors compared to lymph node metastases in this tumor model. C33A turned out to be a useful model to study the formation and therapy of metastases. However, a metastatic model in which the influence of the immune system on tumors and especially on tumor therapy can be analyzed would be preferable. Therefore, the aim of the second part was to establish a syngeneic metastatic mouse model. Accordingly, the murine melanoma cell line B16F10 was analyzed in immunocompetent mice. First, the highly attenuated GLV 1h68 virus was compared to its parental strain LIVP 1.1.1 concerning infection, replication and cell lysis efficacy in cell culture. LIVP 1.1.1 was more efficient than GLV-1h68 and was subsequently used for following mouse studies. Comparative studies were performed, comparing two different implantation sites of the tumor cells, subcutaneously and footpad, and two different mouse strains, FoxN1 nude and C57BL/6 mice. Implantation into the footpad led to a higher metastatic burden in lymph nodes compared to the subcutaneous implantation site. Finally, the model of choice was the implantation of B16F10 into the footpad of immune-competent C57BL/6 mice. Furthermore, it was inevitable to deliver the virus as efficient as possible to the tumor and metastases. Comparison of two different injection routes, intravenously and intratumorally, revealed, that the optimal injection route was intratumorally. In summary, the murine B16F10 model is a promising model to study the effects of the immune system on vaccinia virus mediated therapy of primary tumors and metastases.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Kober2015,
  author    = {Kober, Christina},
  title     = {Characterization of Murine GL261 Glioma Models for Oncolytic Vaccinia Virus Therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118556},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Glioblastoma multiforme (GBM) is one of the most frequent and malignant forms of brain cancer in adults. The prognosis is poor with a median survival time of 12-15 months. There is a broad range of alternative treatment options studied in preclinical and clinical trials for GBM. One alternative treatment option is oncolytic virotherapy, defined as the use of replication-competent viruses that selectively infect and destroy cancer cells while leaving, non-transformed cells unharmed. Vaccinia virus (VACV) is one favorable candidate. Although oncolytic viruses can kill tumor cells grown in vitro with high efficiency, they often exhibit reduced replication capacity in vivo suggesting that physiological aspects of the tumor microenvironment decrease the virus' therapeutic potential. The percentage and composition of immune cells varies between cancer types and patients and is investigated as a biomarker in several studies. Making oncolytic virotherapy successful for GBM, it is necessary to understand the individual tumor biology, the interaction with the microenvironment and immune system. It was demonstrated that the attenuated VACV wild-type (wt) isolate LIVP 1.1.1 replicate and lyse the murine GL261 glioma cell line in vitro. In the following, the replication efficacy was characterized in a comparative approach in vivo. Immunocompetent C57BL/6 (wt) mice and immunodeficient mouse strains of different genetic background C57BL/6 athymic and Balb/c athymic mice were used. In addition, subcutaneous and intracranial locations were compared. The results revealed viral replication exclusively in Balb/c athymic mice with subcutaneous tumors but in none of the other models. In the following, the tumor microenvironment of the subcutaneous tumor models at the time of infection was performed. The study showed that implantation of the same tumor cells in different mouse strains resulted in a different tumor microenvironment with a distinct composition of immune cells. Highest differences were detected between immunodeficient and immunocompetent mice. The study showed major differences in the expression of MHCII with strongest expression in C57BL/6 wt and weakest in Balb/c athymic tumors. In the following, the influence of the phenotypic change associated with the upregulation of MHCII on GL261 tumor cells on viral replication was analyzed. Comparison of C57BL/6 wt and C57BL/6 IFN-γ knockout mice revealed endogenous IFN-γ levels to upregulate MHCII on GL261 tumor cells and to reduce viral replication in C57BL/6 wt mice. Analysis of single cell suspensions of tumor homogenates of C57BL/6 and Balb/c athymic mice showed that the IFN-γ-mediated anti-tumor effect was a reversible effect. Furthermore, reasons for inhibition of virus replication in orthotopic glioma models were elucidated. By immunohistochemical analysis it was shown that intratumoral amounts of Iba1+ microglia and GFAP+ astrocytes in Gl261 gliomas was independent from intratumoral VACV injection. Based on these findings virus infection in glioma, microglia and astrocytes was compared and analyzed in cell culture. In contrast to the GL261 glioma cells, replication was barely detectable in BV-2 microglia and IMA2.1 astrocytic cells. Co-culture experiments revealed that microglia compete for virus uptake in cell culture. It was further shown that BV-2 cells showed apoptotic characteristics after VACV infection while GL261 cells showed signs of necrotic cell death. Additionally, in BV-2 cells with M1-phenotype a further reduction of viral replication and inhibition of cell lysis was detected. Infection of IMA 2.1 cells was independent of the M1/M2-phenotype. Application of BV-2 microglia with M1-phenotype onto organotypic slice cultures with implanted GL261 tumors resulted in reduced infection of BV-2 cells with LIVP 1.1.1, whereas GL261 cells were significantly infected. Taken together, the analyzed GL261 tumors were imprinted by the immunologic and genetic background in which they grow. The experimental approach applied in this thesis can be used as suitable model which reflects the principles of personalized medicine In an additional project, based on gene expression data and bioinformatic analyses, the biological role and function of the anti-apoptotic factor AVEN was analyzed with regard to oncolytic VACV therapy. Besides a comparison of the replication efficacy of GLV-1h68 and VACV-mediated cell killing of four human tumor cell lines, it was shown that AVEN was expressed in all analyzed cells. Further, shown for HT-29 and 1936-MEL, the knockdown of AVEN by siRNA in cell culture resulted in an increase of apoptotic characteristics and a decrease of VACV infection. These findings provide essential insights for future virus development.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Graver2015,
  author    = {Graver, Shannon},
  title     = {Molecular and cellular cross talk between angiogenic, immune and DNA mismatch repair pathways},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108302},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {VEGF is a main driver of tumor angiogenesis, playing an important role not only in the formation of new blood vessels, but also acts as a factor for cell migration, proliferation, survival and apoptosis. Angiogenesis is a universal function shared by most solid tumors and its inhibition was thought to have the potential to work across a broad patient population. Clinical evidence has shown that inhibiting pathological angiogenesis only works in a subset of patients and the identification of those patients is an important step towards personalized cancer care. The first approved antiangiogenic therapy was bevacizumab (Avastin®), a monoclonal antibody targeting VEGF in solid tumors including CRC, BC, NSCLC, RCC and others. In addition to endothelial cells, VEGF receptors are present on a number of different cell types including tumor cells, monocytes and macrophages. The work presented in this thesis looked at the in vitro cellular changes in tumor cells and leukocytes in response to the inhibition of VEGF signaling with the use of bevacizumab. In the initial experiments, VEGF was induced by hypoxia in tumor cells to evaluate changes in survival, proliferation, migration and changes in gene or protein expression. There was a minimal direct response of VEGF inhibition in tumor cells that could be attributed to bevacizumab treatment, with minor variations in some of the cell lines screened but no uniform or specific response noted. MMR deficiency often results in microsatellite instability (MSI) in tumors, as opposed to microsatellite stable (MSS) tumors, and accounts for up to 15\% of colorectal carcinomas (CRCs). It has been suggested in clinical data that MMR deficient tumors responded better to bevacizumab regimens, therefore further research used isogenic paired CRC tumor cell lines (MMR deficient and proficient). Furthermore, a DNA damaging agent was added to the treatment regimen, the topoisomerase inhibitor SN-38 (the active metabolite of irinotecan). Inhibiting VEGF using bevacizumab significantly inhibited the ability of MMR deficient tumor cells to form anchor dependent colonies, however conversely, bevacizumab treatment before damaging cells with SN-38, showed a significant increase in colony numbers. Moreover, VEGF inhibition by bevacizumab pretreatment also significantly increased the mutation fraction in MMR deficient cells as measured by transiently transfecting a dinucleotide repeat construct, suggesting VEGF signaling may have an intrinsic role in MMR deficient cells. A number of pathways were analyzed in addition to changes in gene expression profiles resulting in the identification of JNK as a possible VEGF targeted pathway. JUN expression was also reduced in these conditions reinforcing this hypothesis, however the intricate molecular mechanisms remain to be elucidated. In order to remain focused on the clinical application of the findings, it was noted that some cytokines were differentially regulated by bevacizumab between MMR proficient and deficient cells. Treatment regimens employed in vitro attempted to mimic the clinical setting by inducing DNA damage, then allowing cells to recover with or without VEGF using bevacizumab treatment. Inflammatory cytokines, CCL7 and CCL8, were found to have higher expression in the MMR deficient cell line with bevacizumab after DNA damage, therefore the cross talk via tumor derived factors to myeloid cells was analyzed. Gene expression changes in monocytes induced by tumor conditioned media showed CCL18 to be a bevacizumab regulated gene by MMR deficient cells and less so in MMR proficient cells. CCL18 has been described as a prognostic marker in gastric, colorectal and ovarian cancers, however the significance is dependent on tumor type. CCL18 primarily exerts its function on the adaptive immune system to trigger a TH2 response in T cells, but is also described to increase non-specific phagocytosis. The results of this study did show an increase in the phagocytic activity of macrophages in the presence of bevacizumab that was significantly more apparent in MMR deficient cells. Furthermore, after DNA damage MMR deficient cells treated with bevacizumab released a cytokine mix that induced monocyte migration in a bevacizumab dependent manner, showing a functional response with the combination of MMR deficiency and bevacizumab. In summary, the work in this thesis has shown evidence of immune cell modulation that is specific to MMR deficient tumor cells that may translate into a marker for the administration of bevacizumab in a clinical setting. VEGF ist ein zentraler Regulator der Tumor-Angiogenese, und spielt eine wichtige Rolle nicht nur in der Bildung von neuen Blutgef{\"a}ßen, sondern ist auch f{\"u}r die Migration, Proliferation, das {\"U}berleben und Apoptose von Tumorzellen essentiell. Angiogenese ist eine der universellen Funktionen, welche das Wachstum der meisten soliden Tumoren charakterisiert. Eine der klassischen therapeutischen Ideen wurde auf der Basis entwickelt, dass die spezifische Hemmung der Angiogenese das Potenzial hat in einer breiten Patientenpopulation einen klinischen Effekt zu zeigen. Die klinische Erfahrung und Anwendung hat jedoch gezeigt, dass die Hemmung der pathologischen Angiogenese nur in einem Teil der Patienten einen therapeutischen Nutzen aufweist. Somit stellt die Identifikation derjenigen Patienten, welche von der anti-angiogenen Therapie profitieren, einen wichtiger Schritt zur personalisierten Krebsbehandlung dar. Die erste zugelassene antiangiogene Therapie war Bevacizumab (Avastin®), ein monoklonaler Antik{\"o}rper gegen VEGF, welcher unter anderem in soliden Tumoren wie CRC, BC, nicht-kleinzelligem Lungenkrebs (NSCLC) und dem Nierenzellkarzinom angewandt wird. VEGF-Rezeptoren befinden sich nicht nur auf Endothelzellen, sondern sind auch auf einer Anzahl von verschiedenen Zelltypen, einschließlich Tumorzellen, Monozyten und Makrophagen nachweisbar. Die in dieser Arbeit vorgestellten Ergebnisse befassen sich mit den zellul{\"a}ren Ver{\"a}nderungen an Tumorzellen und Leukozyten als Reaktion auf die Hemmung der VEGF-Signalkaskade durch Bevacizumab in-vitro. In den Initialen Experimenten wurde VEGF durch Hypoxie in Tumorzellen induziert und Ver{\"a}nderungen der {\"U}berlebensrate, der Proliferation, Migration als auch in der Gen- oder Protein-Expression gemessen. Es konnte eine minimale direkte Reaktion der VEGF-Hemmung auf Tumorzellen beobachtet werden, welche auf die Bevacizumab Behandlung zur{\"u}ckgef{\"u}hrt werden k{\"o}nnte. Es zeigten sich aber auch geringf{\"u}gige Abweichungen in einigen der verwendeten Zellinien, die keine einheitliche Interpretation erlauben oder auf eine uniformelle Reaktion hinweisen w{\"u}rden. Das ph{\"a}notypische Korrelat einer „Mismatch" Reparatur (MMR)-Defizienz ist die Mikrosatelliteninstabilit{\"a}t im Gegensatz zu mikrosatellitenstabilen Tumoren und findet sich bei bis zu 15\% der kolorektalen Karzinomen (CRC) wieder. Klinischen Daten deuten daraufhin, dass Bevacizumab besser in MMR-defizienten Tumoren wirkt. Daher wurden die weiteren Untersuchungen in gepaarten MMR stabilen und MMR instabilen CRC-Tumorzelllinien (MMR defizient und kompetent) durchgef{\"u}hrt. Weiterhin wurde ein DNA-sch{\"a}digendes Agens, SN-38, ein Topoisomerase-Inhibitor (der aktive Metabolit von Irinotecan) dem Behandlungsschema zugef{\"u}gt. Es zeigte sich, dass die Hemmung von VEGF mittels Bevacizumab die F{\"a}higkeit der MMR defizienten Tumorzellen Kolonien zu bilden signifikant inhibiert. Im Gegensatz dazu, hatte die Behandlung von Bevacizumab vor der Zugabe des DNA sch{\"a}digenden Agens zu einer vermehrten Kolonienzahl gef{\"u}hrt. Außerdem erh{\"o}hte die Vorbehandlung mit Bevacizumab deutlich die Mutationsrate in MMR-defizienten Zellen, was durch die transiente Transfektion eines Dinukleotid-Repeat-Konstrukts nachgewiesen werden konnte. Dies deutete darauf hin, dass VEGF eine intrinsische Rolle in der Signalkaskade des MMR-Systems haben k{\"o}nnte. Deshalb wurde eine Anzahl von Signalalkaskaden zus{\"a}tzlich zu Ver{\"a}nderungen von Genexpressionsprofilen untersucht und JNK als m{\"o}gliche Verbindungsstelle der beiden Signalkaskaden, VEGF und MMR, identifiziert. Diese Hypothese wurde zus{\"a}tzlich unterst{\"u}tzt durch die Tatsache, dass die JUN Expression unter diesen experimentellen Bedingungen reduziert war. Die Aufkl{\"a}rung der komplexen molekularen Mechanismen der potentiellen Interaktion bleibt zuk{\"u}nftigen Untersuchungen vorbehalten. In Hinblick auf die klinische Konsequenz der erhaltenen Ergebnisse war es auff{\"a}llig, dass einige Zytokine durch Bevacizumab in den MMR defizienten Zellen im Gegensatz zu den MMR kompetenten Zellen unterschiedlich reguliert wurden. Die in-vitro verwendeten Behandlungsschemata waren den klinisch zur Anwendung kommenden Protokollen nachempfunden. Zuerst wurde ein DNA-Schaden gesetzt, und den Zellen erm{\"o}glicht, sich mit oder ohne Bevacizumab zu erholen. Es konnte gezeigt werden, dass die inflammatorischen Zytokine CCL7 und CCL8 eine h{\"o}here Expression in der MMR-defiziente Zelllinie in Kombination mit Bevacizumab aufweisen. Daher wurde ein m{\"o}glicher Crosstalk zwischen von Tumorzellen sezernierten Faktoren und myeloischen Zellen weiter verfolgt. Ver{\"a}nderungen der Genexpression in Monozyten durch Tumorzell- konditionierte Medien zeigte CCL18 als ein Bevacizumab reguliertes Gen in MMR-defizienten Zellen, aber nicht in MMR kompetenten Zellen. CCL18 {\"u}bt seine Funktion prim{\"a}r im adaptiven Immunsystems aus um eine TH2-Antwort in T-Zellen auszul{\"o}sen Ausserdem wird eine Erh{\"o}hung der nicht-spezifische Phagozytose als weitere Funktion beschrieben. CCL18 wurde bereits als prognostischer Marker in Magen-, Dickdarm- und Eierstockkrebsarten beschrieben; die klinische Bedeutung ist jedoch abh{\"a}ngig von Tumortyp. Die Ergebnisse dieser Arbeit zeigen, dass eine Erh{\"o}hung der phagozytischen Aktivit{\"a}t von Makrophagen in Gegenwart von Bevacizumab wesentlich deutlicher in MMR-defizienten Zellen ausgepr{\"a}gt war. Weiterhin wurde gefunden, dass nach DNA-Sch{\"a}digung in Bevacizumab behandelten MMR-defizienten Zellen Zytokine freigesetzt werden, welche eine Monozytenmigration in einer Bevacizumab-abh{\"a}ngigen Weise induzieren. Dies weist auf eine funktionelle Interaktion von MMR-Defizienz und Bevacizumab hin. Zus{\"a}tzlich zeigen die Ergebnisse dieser Arbeit eine Immunzellmodulation, die spezifisch f{\"u}r Mismatch-Reparatur defiziente Tumorzellen ist und in der klinischen Praxis als Marker f{\"u}r die Verabreichung von Bevacizumab verwendet werden k{\"o}nnte.},
  subject      = {Vascular endothelial Growth Factor},
  language  = {en}
}
@phdthesis{Kuhtz2015,
  author    = {Kuhtz, Juliane},
  title     = {Epimutationen humaner Keimzellen und Infertilit{\"a}t},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108248},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Infertilit{\"a}t stellt in unserer heutigen Gesellschaft ein zunehmendes Problem dar. Bei der Suche nach den der Infertilit{\"a}t zugrunde liegenden Ursachen ger{\"a}t immer mehr die Epigenetik in den Fokus. Epigenetische Prozesse sind nicht nur in die Embryo-nalentwicklung und Wachstumsprozesse des Kindes involviert, sondern auch in korrekte Funktionsweisen von Gameten. St{\"o}rungen k{\"o}nnen die Fertilit{\"a}t beeintr{\"a}ch-tigen. Eine besondere Rolle spielen gepr{\"a}gte Gene, die auf einem ihrer Allele, je nach parentaler Herkunft, ein Imprint in Form von DNA-Methylierung tragen. Fehl-regulationen solcher gepr{\"a}gter Gene k{\"o}nnen zu Imprinting-Erkrankungen f{\"u}hren. Seit Einf{\"u}hrung der In-vitro-Fertilisation (IVF) wurden verschiedene assistierte Re-produktionstechniken (ART) entwickelt, um infertilen Paaren zu helfen. Die Sicher-heit dieser Techniken ist nicht abschließend gekl{\"a}rt. Immer wieder wird von nach ART-Behandlung geh{\"a}uft auftretenden Imprinting-Erkrankungen berichtet. Diese Erkrankungen stehen jedoch eher in Zusammenhang mit der zugrunde liegenden Infertilit{\"a}t, als mit ART selbst. Dennoch ist es notwendig zu untersuchen inwieweit sich ART eventuell auf den Gesundheitszustand dieser Kinder auswirken k{\"o}nnte. In der hier vorgelegten Arbeit wurde der Zusammenhang von Epigenetik, Infertilit{\"a}t und ART von verschiedenen Standpunkten aus beleuchtet. In humanen Spermien wurde die DNA-Methylierung verschiedener gepr{\"a}gter Gene hinsichtlich Epimutationen untersucht. ICSI (intracytoplasmatic sperm injection) und IMSI (intracytoplasmic morphologically selected sperm injection) sind verschiedene Techniken zur Selektion von Spermien f{\"u}r eine ART-Behandlung. Hier wurde un-tersucht, ob IMSI epigenetisch bessere Spermien selektiert als die konventionelle ICSI-Methode. Außerdem ist bekannt, dass in Spermienk{\"o}pfen fertiler und infertiler M{\"a}nner Vakuolen vorkommen k{\"o}nnen, deren epigenetische Bedeutung jedoch un-bekannt ist. Ob diese Vakuolen in Zusammenhang mit Epimutationen stehen k{\"o}nn-ten, wurde ebenfalls {\"u}berpr{\"u}ft. Dazu wurde bisulfitkonvertierte DNA weniger Sper-mien (11 je Probe) mithilfe der Limiting Dilution (LD)-Technik und Pyrosequenzie-rung analysiert. Insgesamt standen 880 Spermien f{\"u}r diese Untersuchung zur Ver-f{\"u}gung. Es konnte kein Unterschied zwischen IMSI- und ICSI-selektierten Spermien gefunden werden. Vorhandene Vakuolen im Spermienkopf beeintr{\"a}chtigten nicht die DNA-Methylierung der Gene hGTL2, hLIT1 und hPEG3. Ein weiteres Projekt befasste sich mit der Frage, inwieweit die Technik der In-vitro-Maturation (IVM) DNA-Methylierung in humanen Oocyten beeinflussen k{\"o}nnte. Bisulfitkonvertierte DNA einzelner humaner Oocyten wurde mit LD und Pyrose-quenzierung analysiert. Verglichen wurden IVM und in vivo gereifte Oocyten. Hier-f{\"u}r standen 139 Oocyten zur Verf{\"u}gung, wovon 90 mittels IVM und 49 in vivo gereift waren. Untersucht wurden vier gepr{\"a}gte Gene (hGTL2, hLIT1, hPEG3 und hSNRPN) und drei nicht gepr{\"a}gte Gene (hDNMT3Lo, hNANOG und hOCT4). Es konnten keine IVM-bedingten Epimutationen gefunden werden. Im dritten Projekt wurde untersucht, ob sich die DNA-Methylierung normaler Sper-mien von Spermien aus Oligozoospermie-Asthenozoospermie-Teratozoospermie (OAT)-Syndrom-Patienten unterscheidet. Eine weitere Frage war, ob Epimutationen einen Einfluss auf den ART-Ausgang haben. Untersucht wurden 54 Spermienpro-ben von Paaren in ART-Behandlung. Zur Untersuchung der DNA-Methylierungsmuster der gepr{\"a}gten Gene hGTL2 und hPEG3 sowie der beiden nicht gepr{\"a}gten Pluripotenzgene hNANOG und hOCT4 wurde die Methode Deep Bisulfite Sequencing (DBS) verwendet. Dies ist eine Next Generation Sequencing (NGS)-Technik, angewandt an bisulfitkonvertierter DNA. Diese Technik erm{\"o}glicht es mehrere Proben sowie Gene gleichzeitig zu analysieren. Es zeigte sich, dass OAT-Spermien, die zu einer Lebendgeburt gef{\"u}hrt hatten, sich epigenetisch nicht von normalen Spermien unterschieden. Besonders viele Epimutationen konnten hingegen in OAT-Spermien gefunden werden, die zu keiner Schwangerschaft ge-f{\"u}hrt hatten. Zwischen Spermien die zu einer Lebendgeburt oder keiner Schwan-gerschaft gef{\"u}hrt hatten, zeigten sich Unterschiede in der H{\"a}ufigkeit von hGTL2-Epimutationen. {\"U}ber die H{\"a}ufigkeit von Epimutationen konnte eine pr{\"a}diktive Aus-sage zum ART-Ausgang getroffen werden. Zusammenfassend konnte in dieser Arbeit festgestellt werden, dass sich eine H{\"a}u-fung von Epimutationen darauf auswirkt, ob eine Schwangerschaft erreicht werden kann oder nicht. Diese Epimutationen liegen bereits im parentalen Genom vor. Sie werden nicht durch ART verursacht. Allerdings muss man Techniken finden, mit denen man Gameten mit m{\"o}glichst wenig Epimutationen selektiert, um eine {\"U}ber-tragung solcher auf das Kind zu verhindern.},
  subject      = {Epigenetik},
  language  = {de}
}
@phdthesis{Stangler2015,
  author    = {Stangler, Eva},
  title     = {Effects of habitat fragmentation on trap-nesting bees, wasps and their natural enemies in small secondary rainforest fragments in Costa Rica},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108254},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Summary (English) I. Human induced global change threatens biodiversity and trophic interactions. Fragmentation is considered as one of the major threats to biodiversity and can cause reduced species richness, population declines, loss of genetic diversity and disruption of trophic interactions such as predation and parasitism. However forest fragmentation effects can be eclectic due to species specific traits. Specialist species with narrower niches or at higher trophic levels may be in danger of extinction whereas generalist species with less specific habitat requirements may even profit from fragmentation. In the tropics, known as "the" terrestrial biodiversity hotspots, even biodiversity inventories are often lacking, especially in forest canopies. Ongoing deforestation and resulting fragmentation in tropical regions are expected to heavily affect ecosystem functions by changes in biodiversity, community compositions and disruption of trophic interactions. It is even less unknown in what extent different global change drivers for example climate change and fragmentation interact. It is unlikely that deforestation will end, so that small secondary forest fragments will be important habitat elements that must be investigated to optimize their potential contribution to biodiversity conservation. This dissertation aimed to disentangle the effects of forest fragmentation on trap-nesting bee and wasp communities in small secondary forest fragments addressing the following main questions: 1) Are there interactive effects between microclimate and fragmentation on the abundance of bees and wasps, their mortality - and parasitism rates (Chapter II)? 2) How does fragmentation affect bee biodiversity from canopy to the understory with considerations of single species patterns (Chapter III)? 3) How is fragmentation affecting diversity and community composition of different trophic levels between understory and canopy with emphasis on the host-antagonist relation? (Chapter IV). II. A variety of global change drivers affect biodiversity and trophic interactions. The combined effects of habitat fragmentation and climate change are poorly understood and with ongoing deforestation and agricultural intensification secondary rainforest fragments might contribute to biodiversity conservation and mitigation of climate warming. This chapter investigated the interactive effects of habitat fragmentation and microclimate on the abundance and biotic interactions of trap-nesting bees and wasps in secondary forest fragments in the Northeastern lowlands of Costa Rica. Habitat area did not affect hymenopteran abundance, parasitism and mortality rates, but tree location- from the forest border to the forest center- influenced all variables. Interactive effects were found such as in the higher mortality rates at interior locations in larger fragments. Mean temperature at edge and interior locations led to significant effects on all tested variables and interactive effects between temperature and tree locations were found. Abundances at interior locations were significantly higher with increasing temperatures. Mortality rates at interior location increased at lower mean temperatures, whereas higher temperatures at edges marginally increased mortality rates. Our results indicate, that edge effects, mediated by altered microclimatic conditions, significantly change biotic interactions of trap-nesting hymenopterans in small secondary fragments. III. This chapter focusses on the vertical distribution of bees, their parasitism and mortality rates as well as single species patterns in relation to fragment size and edge effects in secondary rainforest remnants. No size effects on bee abundance, bee diversity and on parasitism- and mortality rates were found. Bees were least abundant at the intermediate height and were most abundant in the understory; whereas the highest diversity was found in the canopy. Tree location had no effect on bee abundance, but on bee diversity since most species were found in the forest interior. The cuckoo bees Aglaomelissa duckei and Coelioxys sp. 1 only partly followed the patterns of their hosts, two Centris species. Edge effects greatly influenced the bee community, so that the amount of edge habitat in secondary forest fragments will influence the conservation value for bees. IV. In this section the effects of habitat fragmentation on biodiversity, on community structure of hosts and natural enemies as well as the relation of hosts and antagonists were investigated from the understory to the canopy. The results stress the importance to monitor biodiversity, community composition and trophic interactions from the understory to the canopy. The higher trophic level of the antagonists was found to be more sensitive to fragment size compared to their hosts. Again edge effects were found to be the dominant driver since both host and antagonist richness, as well as community compositions were strongly affected. Ongoing fragmentation and increased amount of edge habitat could favor few abundant disturbance-adapted species over the rare and more diverse forest-adapted species. A positive-density dependent parasitism rate was demonstrated, as well as an increase of the parasitism rate not only with antagonist abundance but also diversity. Small secondary forest fragments surely can contribute to the conservation of biodiversity and trophic interactions, but increase of edge habitat will have negative consequences on above-ground nesting Hymenoptera, so that important interactions such as pollination, predation and parasitism could be disrupted. Therefore small forest fragments could contribute to biodiversity conservation but will not be able to compensate for the loss of large areas of primary forests. V. This dissertation contributes to the understanding of habitat area - and edge effects as well as the interaction of those with microclimatic conditions in small secondary rainforest fragments. As study system trap nests inhabited by solitary above-ground nesting bees, wasps and their natural enemies were chosen because they allow to study trophic interactions along their whole vertical distribution from the understory to the canopy. The effect of fragment size was rather weak, however, larger sizes affected the diversity of natural enemies positively, proofing the hypothesis that higher trophic levels react more sensitive to habitat loss. Edge effects heavily affected the abundance, diversity and community composition of hosts and their natural enemies as well as parasitism and mortality rates. Increased edge conditions resulting from ongoing fragmentation and deforestation will therefore negatively affect bees, wasps and their trophic interactions with natural enemies. Those changes affect important processes such as pollination, predation and parasitism, which could result in changes of ecosystem functioning. This study showed the importance to include all strata in biodiversity monitoring since height did matter for the trap-nesting communities. Diversity was shown to be higher in the canopy and community composition did change significantly. To conclude we could show that secondary forest fragments can sustain a trap-nesting bee and wasp community, but the amount of interior habitat is highly important for the conservation of forest-adapted species. Probably the conservation of large primary forest in combination with a high habitat connectivity, for example with small secondary forest fragments, will help to sustain biodiversity and ecosystem functioning better than the mere presence of small forest fragments.},
  subject      = {Costa Rica},
  language  = {en}
}