@phdthesis{Roemer2014, author = {R{\"o}mer, Daniela}, title = {Where and how to build? Influence of social and environmental cues on nest building behavior in leaf-cutting ants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109409}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {This thesis explores the influence of social and environmental cues on the nest building behavior of leaf-cutting ants. Especially, the investigations are aimed at evaluating the mechanisms of nest building and how the nest environment can spatially guide building responses that lead to an adaptive nest architecture. The emergence of nest chambers in the nest of the leaf-cutting ant Acromyrmex lundi were evaluated. Rather than excavating nest chambers in advance, at places where workers encounter suitable environmental conditions for brood and fungus rearing, these items have to be present at a site. When presented in the laboratory with a choice between two otherwise identical digging sites, offering suitable environmental conditions, but one containing brood, the workers displayed a higher excavation activity at the site where they encountered the putative content of a chamber. The shape of the excavated cavity was also more round and chamber-like. It is concluded that leaf-cutting ants respond to social cues during nest building. Excavation is a costly process and colonies have to spend a part of their energy stores on nest building, so that regulatory responses for the control of nest excavation are expected to occur. Worker density at the beginning of the digging process influenced digging activity while the presence of in-nest stores did not. Stored brood and fungus did however influence the architecture of the excavated nest, leading to the excavation of larger chambers and smaller tunnels. While self-organized mechanisms appear to be involved in the nest building process, the social cues of the ants' environment during building clearly influence the nest architecture and lead to an adjustment of the nest size to the current space needs of the colony. Workers secondarily regulated nest size by the opportunistic refilling of unused space with excavated soil pellets. As the ants should provide suitable conditions for brood and fungus rearing, they should show a behavioral response to CO2 concentrations, as the gas is known to hinder fungus respiration. Workers of A. lundi did indeed avoid high CO2-levels for fungus rearing but actually preferred CO2-values in the range encountered close to the soil surface, where this species excavates their nests. However, different CO2-levels did not affect their excavation behavior. While fungus chambers make up part of a leaf-cutting ant nest, most leaf-cutting ants of the genus Atta also spent part of the colony's energy on excavating large, voluminous chambers for waste disposal, rather than scattering the material aboveground. It is expected that leaf-cutting ants also show environmental preferences for waste management. In experiments Atta laevigata workers preferred deposition in a warm and dry environment and showed no preference for specific CO2-levels. The continued accumulation of waste particles in a waste chamber seems to be based on the use of volatiles. These originate from the waste itself, and seem to be used as an orientation cue by workers relocating the material. The ensuing large accumulation of waste at one site should result in the emergence of more voluminous chambers for waste disposal.}, subject = {Nestbau}, language = {en} } @phdthesis{Huang2013, author = {Huang, Ting}, title = {Vaccinia Virus-mediated Therapy of Solid Tumor Xenografts: Intra-tumoral Delivery of Therapeutic Antibodies}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91327}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Over the past 30 years, much effort and financial support have been invested in the fight against cancer, yet cancer still represents the leading cause of death in the world. Conventional therapies for treatment of cancer are predominantly directed against tumor cells. Recently however, new treatments options have paid more attention to exploiting the advantage of targeting the tumor stroma instead. Vaccinia virus (VACV) has played an important role in human medicine since the 18th century as a vaccination against smallpox. In our laboratory, the recombinant, replication-competent vaccinia virus, GLV-1h68, was shown to enter, colonize and destroy cancer cells both in cell culture, and in vivo, in xenograft models (Zhang, Yu et al. 2007). In addition, combined therapy of GLV-1h68 and anti-VEGF immunotherapy significantly enhanced antitumor therapy in vivo (Frentzen, Yu et al. 2009). In this study, we constructed several new recombinant VACVs carrying genes encoding different antibodies against fibroblast activation protein (FAP) in stroma (GLV-1h282), nanobody against the extracellular domain of epidermal growth factor receptor (EGFR, GLV-1h442) or antibodies targeting both vascular endothelial growth factor (VEGF) and EGFR (GLV-1h444) or targeting both VEGF and FAP (GLV-1h446). The expression of the recombinant proteins was first verified using protein analytical methods, SDS-gel electrophoresis, Western blot analysis, immunoprecipitation (IP) assays and ELISA assays. The proteins were detected after infection of the cells with the different VACVs and the recombinant proteins purified by affinity adsorption. The purified antibodies were shown to specifically bind to their respective antigens. Secondly, the infection and replication capability of all the virus strains was analyzed in cell culture using several human tumor cell lines (A549, FaDu or DU145), revealing that all the new recombinant VACVs were able to infect cancer cells with comparable efficiency to the parental viruses from which they were derived. Thirdly, the antitumor efficacy of the new recombinant VACVs was evaluated in vivo using several human cancer xenograft models in mice. In A549 and DU145 xenografts, the new recombinant VACVs exhibited an enhanced therapeutic efficacy compared to GLV-1h68 with no change in toxicity in mice. In the FaDu xenograft, treatment with GLV-1h282 (anti-FAP) significantly slowed down the speed of tumor growth compared to GLV-1h68. Additionally, treatment with the recombinant VACVs expressed the various antibodies achieved comparable or superior therapeutic effects compared to treatment with a combination of GLV-1h68 and the commercial therapeutic antibodies, Avastin, Erbitux or both. Next, the virus distribution in tumors and organs of treated mice was evaluated. For most of the viruses, the virus titer in tumors was not signficantly diffferent than GLV-1h68. However, for animals treated with GLV-1h282, the virus titer in tumors was significantly higher than with GLV-1h68. This may be the reason for enhanced antitumor efficacy of GLV-1h282 in vivo. Lastly, the underlying mechanisms of therapeutic antibody-enhanced antitumor effects were investigated by immunohistochemistry. Blood vessels density and cell proliferation in tumors were suppressed after treatment with the antibody-encoded VACVs. The results indicated that the suppression of angiogenesis or cell proliferation in tumors may cause the observed therapeutic effect. In conclusion, the results of the studies presented here support the hypothesis that the treatment of solid tumors with a combination of oncolytic virotherapy and immunotherapy has an additive effect over each treatment alone. Moreover, expression of the immunotherapeutic antibody by the oncolytic VACV locally in the tumor enhances the antitumor effect over systemic treatment with the same antibody. Combined, these results indicate that therapy with oncolytic VACVs expressing-therapeutic antibodies may be a promising approach for the treatment of cancer.}, subject = {Vaccinia-Virus}, language = {en} } @phdthesis{Geissinger2010, author = {Geissinger, Ulrike}, title = {Vaccinia Virus-mediated MR Imaging of Tumors in Mice: Overexpression of Iron-binding Proteins in Colonized Xenografts}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48099}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Vaccinia virus plays an important role in human medicine and molecular biology ever since the 18th century after E. Jenner discovered its value as a vaccination virus against smallpox. After the successful eradication of smallpox, vaccinia virus, apart from its use as a vaccine carrier, is today mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes encoding therapeutic or diagnostic proteins to be expressed within the tumor. The emphasis in this study was the diagnosis of tumors using different vaccinia virus strains. Viruses with metal-accumulating capabilities for tumor detection via MRI technology were generated and tested for their usefulness in cell culture and in vivo. The virus strains GLV-1h131, GLV-1h132, and GLV-1h133 carry the gene encoding the two subunits of the iron storage protein ferritin under the control of three different promoters. GLV-1h110, GLV-1h111, and GLV-1h112 encode the bacterial iron storage protein bacterioferritin, whereas GLV-1h113 encodes the codon-optimized version of bacterioferritin for more efficient expression in human cells. GLV-1h22 contains the transferrin receptor gene, which plays an important role in iron uptake, and GLV-1h114 and GLV-1h115 contain the murine transferrin receptor gene. For possibly better iron uptake the virus strains GLV-1h154, GLV-1h155, GLV-1h156, and GLV-1h157 were generated, each with a version of a ferritin gene and a transferrin receptor gene. GLV-1h154 carries the genes that encode bacterioferritin and human transferrin receptor, GLV-1h155 the human ferritin H-chain gene and the human transferrin receptor gene. GLV-1h156 and GLV-1h157 infected cells both express the mouse transferrin receptor and bacterioferritin or human ferritin H-chain, respectively. The virus strains GLV-1h186 and GLV-1h187 were generated to contain a mutated form of the ferritin light chain, which was shown to result in iron overload and the wildtype light chain gene, respectively. The gene encoding the Divalent Metal Transporter 1, which is a major protein in the uptake of iron, was inserted in the virus strain GLV-1h102. The virus strain GLV-1h184 contains the magA gene of the magnetotactic bacterium Magnetospirillum magnetotacticum, which produces magnetic nanoparticles for orientation in the earth's magnetic field. Initially the infection and replication capability of all the virus strains were analyzed and compared to that of the parental virus strain GLV-1h68, revealing that all the viruses were able to infect cells of the human cancer cell lines A549 and GI-101A. All constructs exhibited a course of infection comparable to that of GLV-1h68. Next, to investigate the expression of the foreign proteins in GI-101A and A549 cells with protein analytical methods, SDS-gelelectrophoresis, Western blots and ELISAs were performed. The proteins, which were expressed under the control of the strong promoters, could be detected using these methods. To be able to successfully detect the protein expression of MagA and DMT1, which were expressed under the control of the weak promoter, the more sensitive method RT-PCR was used to at least confirm the transcription of the inserted genes. The determination of the iron content in infected GI-101A and A549 cells showed that infection with all used virus strains led to iron accumulation in comparison to uninfected cells, even infection with the parental virus strain GLV-1h68. The synthetic phytochelatin EC20 was also shown to enhance the accumulation of different heavy metals in bacterial cultures. In vivo experiments with A549 tumor-bearing athymic nude mice revealed that 24 days post infection virus particles were found mainly in the tumor. The virus-mediated expression of recombinant proteins in the tumors was detected successfully by Western blot. Iron accumulation in tumor lysates was investigated by using the ferrozine assay and led to the result that GLV-1h68-infected tumors had the highest iron content. Histological stainings confirmed the finding that iron accumulation was not a direct result of the insertion of genes encoding iron-accumulating proteins in the virus genome. Furthermore virus-injected tumorous mice were analyzed using MRI technology. Two different measurements were performed, the first scan being done with a seven Tesla small animal scanner seven days post infection whereas the second scan was performed using a three Tesla human scanner 21 days after virus injection. Tumors of mice injected with the virus strains GLV-1h113 and GLV-1h184 were shown to exhibit shortened T2 and T2* relaxation times, which indicates enhanced iron accumulation. In conclusion, the experiments in this study suggest that the bacterioferritin-encoding virus strain GLV-1h113 and the magA-encoding virus strain GLV-1h184 are promising candidates to be used for cancer imaging after further analyzation and optimization.}, subject = {Vaccinia-Virus}, language = {en} } @phdthesis{Hess2013, author = {Heß, Michael}, title = {Vaccinia virus-encoded bacterial beta-glucuronidase as a diagnostic biomarker for oncolytic virotherapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86789}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Oncolytic virotherapy represents a promising approach to revolutionize cancer therapy. Several preclinical and clinical trials display the safety of oncolytic viruses as wells as their efficiency against solid tumors. The development of complementary diagnosis and monitoring concepts as well as the optimization of anti-tumor activity are key points of current virotherapy research. Within the framework of this thesis, the diagnostic and therapeutic prospects of beta-glucuronidase expressed by the oncolytic vaccinia virus strain GLV-1h68 were evaluated. In this regard, a beta-glucuronidase-based, therapy-accompanying biomarker test was established which is currently under clinical validation. By using fluorescent substrates, the activity of virally expressed beta-glucuronidase could be detected and quantified. Thereby conclusions about the replication kinetics of oncolytic viruses in animal models and virus-induced cancer cell lysis could be drawn. These findings finally led to the elaboration and establishment of a versatile biomarker assay which allows statements regarding the replication of oncolytic viruses in mice based on serum samples. Besides the analysis of retrospective conditions, this test is able to serve as therapy-accompanying monitoring tool for virotherapy approaches with beta-glucuronidase-expressing viruses. The newly developed assay also served as complement to routinely used plaque assays as well as reference for virally expressed anti-angiogenic antibodies in additional preclinical studies. Further validation of this biomarker test is currently taking place in the context of clinical trials with GL-ONC1 (clinical grade GLV-1h68) and has already shown promising preliminary results. It was furthermore demonstrated that fluorogenic substrates in combination with beta-glucuronidase expressed by oncolytic viruses facilitated the optical detection of solid tumors in preclinical models. In addition to diagnostic purposes, virus-encoded enzymes could also be combined with prodrugs resulting in an improved therapeutic outcome of oncolytic virotherapy. In further studies, the visualization of virus-induced immune reactions as well as the establishment of innovative concepts to improve the therapeutic outcome of oncolytic virotherapy could be accomplished. In conclusion, the results of this thesis provide crucial findings about the influence of virally expressed beta-glucuronidase on various diagnostic concepts in the context of oncolytic virotherapy. In addition, innovative monitoring and therapeutic strategies could be established. Our preclinical findings have important clinical influence, particularly by the development of a therapy-associated biomarker assay which is currently used in different clinical trials.}, subject = {Vaccinia-Virus}, language = {en} } @phdthesis{Nguyen2012, author = {Nguyen, Hoang Duong}, title = {Vaccinia virus mediated expression of human erythropoietin in colonized human tumor xenografts results in faster tumor regression and increased red blood cell biogenesis in mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85383}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {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.}, subject = {Erythropoietin}, language = {en} } @phdthesis{PrietoGarcia2022, author = {Prieto Garc{\´i}a, Cristian}, title = {USP28 regulates Squamous cell oncogenesis and DNA repair via ΔNp63 deubiquitination}, doi = {10.25972/OPUS-27033}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270332}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer resistance toward therapies inducing DNA damage. SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited, and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities, the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a potential alternative to improve the therapeutic response and clinical outcomes of SCC patients. However, ∆Np63 is considered "undruggable." As is commonly observed in transcription factors, ∆Np63 does not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of different pathways and cellular processes, making it difficult to counteract its function by targeting downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin-proteasome system (UPS), the development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target ∆Np63 in SCC treatment. This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability, and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time, ∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in preclinical mouse models. Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance, thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28 inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such as Cisplatin.}, language = {en} } @phdthesis{Gnamlin2015, author = {Gnamlin, Prisca}, title = {Use of Tumor Vasculature for Successful Treatment of Carcinomas by Oncolytic Vaccinia Virus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119019}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Tumor-induced angiogenesis is of major interest for oncology research. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor characterized so far. VEGF blockade was shown to be sufficient for angiogenesis inhibition and subsequent tumor regression in several preclinical tumor models. Bevacizumab was the first treatment targeting specifically tumor-induced angiogenesis through VEGF blockade to be approved by the Food and Drugs Administration (FDA) for cancer treatment. However, after very promising results in preclinical evaluations, VEGF blockade did not show the expected success in patients. Some tumors became resistant to VEGF blockade. Several factors have been accounted responsible, the over-expression of other angiogenic factors, the noxious influence of VEFG blockade on normal tissues, the selection of hypoxia resistant neoplastic cells, the recruitment of hematopoietic progenitor cells and finally the transient nature of angiogenesis inhibition by VEGF blockade. The development of blocking agents against other angiogenic factors like placental growth factor (PlGF) and Angiopoietin-2 (Ang-2) allows the development of an anti-angiogenesis strategy adapted to the profile of the tumor. Oncolytic virotherapy uses the natural propensity of viruses to colonize tumors to treat cancer. The recombinant vaccinia virus GLV-1h68 was shown to infect, colonize and lyse several tumor types. Its descendant GLV-1h108, expressing an anti-VEGF antibody, was proved in previous studies to inhibit efficiently tumor induced angiogenesis. Additional VACVs expressing single chain antibodies (scAb) antibodies against PlGF and Ang-2 alone or in combination with anti VEGF scAb were designed. In this study, VACV-mediated anti-angiogenesis treatments have been evaluated in several preclinical tumor models. The efficiency of PlGF blockade, alone or in combination with VEGF, mediated by VACV has been established and confirmed. PlGF inhibition alone or with VEGF reduced tumor burden 5- and 2-folds more efficiently than the control virus, respectively. Ang-2 blockade efficiency for cancer treatment gave controversial results when tested in different laboratories. Here we demonstrated that unlike VEGF, the success of Ang-2 blockade is not only correlated to the strength of the blockade. A particular balance between Ang-2, VEGF and Ang-1 needs to be induced by the treatment to see a regression of the tumor and an improved survival. We saw that Ang-2 inhibition delayed tumor growth up to 3-folds compared to the control virus. These same viruses induced statistically significant tumor growth delays. This study unveiled the need to establish an angiogenic profile of the tumor to be treated as well as the necessity to better understand the synergic effects of VEGF and Ang-2. In addition angiogenesis inhibition by VACV-mediated PlGF and Ang-2 blockade was able to reduce the number of metastases and migrating tumor cells (even more efficiently than VEGF blockade). VACV colonization of tumor cells, in vitro, was limited by VEGF, when the use of the anti-VEGF VACV GLV-1h108 drastically improved the colonization efficiency up to 2-fold, 72 hours post-infection. These in vitro data were confirmed by in vivo analysis of tumors. Fourteen days post-treatment, the anti-VEGF virus GLV-1h108 was colonizing 78.8\% of the tumors when GLV-1h68 colonization rate was 49.6\%. These data confirmed the synergistic effect of VEGF blockade and VACV replication for tumor regression. Three of the tumor cell lines used to assess VACV-mediated angiogenesis inhibition were found, in certain conditions, to mimic either endothelial cell or pericyte functions, and participate directly to the vascular structure. The expression by these tumor cells of e-selectin, p-selectin, ICAM-1 and VCAM-1, normally expressed on activated endothelial cells, corroborates our findings. These proteins play an important role in immune cell recruitment, and there amount vary in presence of VEGF, PlGF and Ang-2, confirming the involvement of angiogenic factors in the immuno-modulatory abilities of tumors. In this study VACV-mediated angiogenesis blockade proved its potential as a therapeutic agent able to treat different tumor types and prevent resistance observed during bevacizumab treatment by acting on different factors. First, the expression of several antibodies by VACV would prevent another angiogenic factor to take over VEGF and stimulate angiogenesis. Then, the ability of VACV to infect tumor cells would prevent them to form blood vessel-like structures to sustain tumor growth, and the localized delivery of the antibody would decrease the risk of adverse effects. Next, the blockade of angiogenic factors would improve VACV replication and decrease the immune-modulatory effect of tumors. Finally the fact that angiogenesis blockade lasts until total regression of the tumor would prevent the recovery of the tumor-associated vasculature and the relapse of patients.}, subject = {Vaccinia-Virus}, language = {en} } @phdthesis{Regneri2013, author = {Regneri, Janine}, title = {Transcriptional regulation of cancer genes in the Xiphophorus melanoma system}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-82319}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {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.}, subject = {Melanom}, language = {en} } @phdthesis{Eidel2020, author = {Eidel, Matthias T. A. M.}, title = {Training Effects of a Tactile Brain-Computer Interface System During Prolonged Use by Healthy And Motor-Impaired People}, doi = {10.25972/OPUS-20851}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208511}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Background - Brain-Computer Interfaces (BCI) enable their users to interact and communicate with the environment without requiring intact muscle control. To this end, brain activity is directly measured, digitized and interpreted by the computer. Thus, BCIs may be a valuable tool to assist severely or even completely paralysed patients. Many BCIs, however, rely on neurophysiological potentials evoked by visual stimulation, which can result in usability issues among patients with impaired vision or gaze control. Because of this, several non-visual BCI paradigms have been developed. Most notably, a recent study revealed promising results from a tactile BCI for wheelchair control. In this multi-session approach, healthy participants used the BCI to navigate a simulated wheelchair through a virtual apartment, which revealed not only that the BCI could be operated highly efficiently, but also that it could be trained over five sessions. The present thesis continues the research on this paradigm in order to - confirm its previously reported high performance levels and trainability - reveal the underlying factors responsible for observed performance increases - establish its feasibility among potential impaired end-users Methods - To approach these goals, three studies were conducted with both healthy participants and patients with amyotrophic lateral sclerosis (ALS). Brain activity during BCI operation was recorded via electroencephalography (EEG) and interpreted using a machine learning-based linear classifier. Wheelchair navigation was executed according to the classification results and visualized on a monitor. For offline statistical analysis, neurophysiological features were extracted from EEG data. Subjective data on usability were collected from all participants. Two specialized experiments were conducted to identify factors for training. Results and Discussion - Healthy participants: Results revealed positive effects of training on BCI performances and their underlying neurophysiological potentials. The paradigm was confirmed to be feasible and (for a non-visual BCI) highly efficient for most participants. However, some had to be excluded from analysis of the training effects because they could not achieve meaningful BCI control. Increased somatosensory sensitivity was identified as a possible mediator for training-related performance improvements. Participants with ALS: Out of seven patients with various stages of ALS, five could operate the BCI with accuracies significantly above chance level. Another ALS patient in a state of near-complete paralysis trained with the BCI for several months. Although no effects of training were observed, he was consistently able to operate the system above chance level. Subjective data regarding workload, satisfaction and other parameters were reported. Significance - The tactile BCI was evaluated on the example of wheelchair control. In the future, it could help impaired patients to regain some lost mobility and self-sufficiency. Further, it has the potential to be adapted to other purposes, including communication. Once visual BCIs and other assistive technologies fail for patients with (progressive) motor impairments, vision-independent paradigms such as the tactile BCI may be among the last remaining alternatives to interact with the environment. The present thesis has strongly confirmed the general feasibility of the tactile paradigm for healthy participants and provides first clues about the underlying factors of training. More importantly, the BCI was established among potential end-users with ALS, providing essential external validity.}, subject = {Myatrophische Lateralsklerose}, language = {en} } @phdthesis{Fink2008, author = {Fink, Kristin}, title = {Toxins in Renal Disease and Dialysis Therapy : Genotoxic Potential and Mechanisms}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31082}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {In patients suffering from end-stage renal disease who are treated by hemodialysis genomic damage as well as cancer incidence is elevated. One possible cause for the increased genomic damage could be the accumulation of genotoxic substances in the blood of patients. Two possible sources for those toxins have to be considered. The first possibility is that substances from dialysers, the blood tubing system or even contaminated dialysis solutions may leach into the blood of the patients during dialysis. Secondly, the loss of renal filtration leads to an accumulation of substances which are normally excreted by the kidney. If those substances possess toxic potential, they are called uremic toxins. Several of these uremic toxins are potentially genotoxic. Within this thesis several exemplary uremic toxins have been tested for genotoxic effects (homocysteine, homocysteine-thiolactone,leptine, advanced glycated end-products). Additionally, it was analysed whether substances are leaching from dialysers or blood tubing and whether they cause effects in in vitrotoxicity testing. The focus of chemical analytisis was on bisphenol A (BPA), the main component of plastics used in dialysers and dialyser membranes.}, subject = {Bisphenol A}, language = {en} } @phdthesis{Michels2008, author = {Michels, Birgit}, title = {Towards localizing the Synapsin-dependent olfactory memory trace in the brain of larval Drosophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-36338}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Animals need to adapt and modify their behaviour according to a changing environment. In particular, the ability to learn about rewarding or punishing events is crucial for survival. One key process that underlies such learning are modifications of the synaptic connection between nerve cells. This Thesis is concerned with the genetic determinants of such plasticity, and with the site of these modifications along the sensory-to-motor loops in Drosophila olfactory learning. I contributed to the development and detailed parametric description of an olfactory associative learning paradigm in larval fruit flies (Chapter I.1.). The robustness of this learning assay, together with a set of transgenic Drosophila strains established during this Thesis, enabled me to study the role for Synapsin, a presynaptic phosphoprotein likely involved in synaptic plasticity, in this form of learning (Chapter I.2.), and to investigate the cellular site of the corresponding Synapsin-dependent memory trace (Chapter I.3.). These data provide the first comprehensive account to-date of the neurogenetic bases of learning in larval Drosophila. The role for Synapsin was also analyzed with regard to pain-relief learning in adult fruit flies (Chapter II.1.); that is, if an odour precedes an electric shock during training, flies subsequently avoid that odour ('punishment learning'), whereas presentation of the odour upon the cessation of shock subsequently leads to approach towards the odour ('relief larning'). Such pain-relief learning was also the central topic of a study concerning the white gene (Chapter II.2.), which as we report does affect pain-relief as well as punishment learning in adult flies, but leaves larval odour-food learning unaffected. These studies regarding pain-relief learning provide the very first hints, in any experimental system, concerning the genetic determinants of this form of learning.}, subject = {Taufliege}, language = {en} } @phdthesis{Griffoni2019, author = {Griffoni, Chiara}, title = {Towards advanced immunocompetent skin wound models for in vitro drug evaluation}, doi = {10.25972/OPUS-19212}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192125}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models.}, subject = {Haut}, language = {en} } @phdthesis{Liang2009, author = {Liang, Chunguang}, title = {Tools for functional genomics applied to Staphylococci, Listeriae, Vaccinia virus and other organisms}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48051}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Genome sequence analysis A combination of genome analysis application has been established here during this project. This offers an efficient platform to interactively compare similar genome regions and reveal loci differences. The genes and operons can be rapidly analyzed and local collinear blocks (LCBs) categorized according to their function. The features of interests are parsed, recognized, and clustered into reports. Phylogenetic relationships can be readily examined such as the evolution of critical factors or a certain highly-conserved region. The resulting platform-independent software packages (GENOVA and inGeno), have been proven to be efficient and easy to handle in a number of projects. The capabilities of the software allowed the investigation of virulence factors, e.g., rsbU, strains' biological design, and in particular pathogenicity feature storage and management. We have successfully investigated the genomes of Staphylococcus aureus strains (COL, N315, 8325, RN1HG, Newman), Listeria spp. (welshimeri, innocua and monocytogenes), E.coli strains (O157:H7 and MG1655) and Vaccinia strains (WR, Copenhagen, Lister, LIVP, GLV-1h68 and parental strains). Metabolic network analysis Our YANAsquare package offers a workbench to rapidly establish the metabolic network of such as Staphylococcous aureus bacteria in genome-scale size as well as metabolic networks of interest such as the murine phagosome lipid signalling network. YANAsquare recruits reactions from online databases using an integrated KEGG browser. This reduces the efforts in building large metabolic networks. The involved calculation routines (METATOOL-derived wrapper or native Java implementation) readily obtain all possible flux modes (EM/EP) for metabolite fluxes within the network. Advanced layout algorithms visualize the topological structure of the network. In addition, the generated structure can be dynamically modified in the graphic interface. The generated network as well as the manipulated layout can be validated and stored (XML file: scheme of SBML level-2). This format can be further parsed and analyzed by other systems biology software, such as CellDesigner. Moreover, the integrated robustness-evaluation routine is able to examine the synthesis rates affected by each single mutation throughout the whole network. We have successfully applied the method to simulate single and multiple gene knockouts, and the affected fluxes are comprehensively revealed. Recently we applied the method to proteomic data and extra-cellular metabolite data of Staphylococci, the physiological changes regarding the flux distribution are studied. Calculations at different time points, including different conditions such as hypoxia or stress, show a good fit to experimental data. Moreover, using the proteomic data (enzyme amounts) calculated from 2D-Gel-EP experiments our study provides a way to compare the fluxome and the enzyme expression. Oncolytic vaccinia virus (VACV) We investigated the genetic differences between the de novo sequence of the recombinant oncolytic GLV-1h68 and other related VACVs, including function predictions for all found genome differences. Our phylogenetic analysis indicates that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. Functions of viral genes were either strain-specific, tissue-specific or host-specific comparing viral genes in the Lister, WR and COP strains. This helps to rationally design more optimized oncolytic virus strains to benefit cancer therapy in human patients. Identified differences from the comparison in open reading frames (ORFs) include genes for host-range selection, virulence and immune modulation proteins, e.g. ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. The contribution of foreign gene expression cassettes in the therapeutic and oncolytic virus GLV-1h68 was studied, including the F14.5L, J2R and A56R loci. The contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence data of GLV-1h68 with its F14.5L-null and revertant viruses. The comparison suggests that insertion of a foreign gene expression cassette in a nonessential locus in the viral genome is a practical way to attenuate VACVs, especially if the nonessential locus itself contains a virulence gene. This reduces the virulence of the virus without compromising too much the replication competency of the virus, the key to its oncolytic activity. The reduced pathogenicity of GLV-1h68 was confirmed by our experimental collaboration partners in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. In conclusion, bioinformatics and experimental data show that GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.}, subject = {Genanalyse}, language = {en} } @phdthesis{SchenkneeWolf2018, author = {Schenk [n{\´e}e Wolf], Mariela}, title = {Timing of wild bee emergence: mechanisms and fitness consequences}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161565}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Solitary bees in seasonal environments have to align their life-cycles with favorable environmental conditions and resources. Therefore, a proper timing of their seasonal activity is highly fitness relevant. Most species in temperate environments use temperature as a trigger for the timing of their seasonal activity. Hence, global warming can disrupt mutualistic interactions between solitary bees and plants if increasing temperatures differently change the timing of interaction partners. The objective of this dissertation was to investigate the mechanisms of timing in spring-emerging solitary bees as well as the resulting fitness consequences if temporal mismatches with their host plants should occur. In my experiments, I focused on spring-emerging solitary bees of the genus Osmia and thereby mainly on O. cornuta and O. bicornis (in one study which is presented in Chapter IV, I additionally investigated a third species: O. brevicornis). Chapter II presents a study in which I investigated different triggers solitary bees are using to time their emergence in spring. In a climate chamber experiment I investigated the relationship between overwintering temperature, body size, body weight and emergence date. In addition, I developed a simple mechanistic model that allowed me to unite my different observations in a consistent framework. In combination with the empirical data, the model strongly suggests that solitary bees follow a strategic approach and emerge at a date that is most profitable for their individual fitness expectations. I have shown that this date is on the one hand temperature dependent as warmer overwintering temperatures increase the weight loss of bees during hibernation, which then advances their optimal emergence date to an earlier time point (due to an earlier benefit from the emergence event). On the other hand I have also shown that the optimal emergence date depends on the individual body size (or body weight) as bees adjust their emergence date accordingly. My data show that it is not enough to solely investigate temperature effects on the timing of bee emergence, but that we should also consider individual body conditions of solitary bees to understand the timing of bee emergence. In Chapter III, I present a study in which I investigated how exactly temperature determines the emergence date of solitary bees. Therefore, I tested several variants degree-day models to relate temperature time series to emergence data. The basic functioning of such degree-day models is that bees are said to finally emerge when a critical amount of degree-days is accumulated. I showed that bees accumulate degree-days only above a critical temperature value (~4°C in O. cornuta and ~7°C in O. bicornis) and only after the exceedance of a critical calendar date (~10th of March in O. cornuta and ~28th of March in O. bicornis). Such a critical calendar date, before which degree-days are not accumulated irrespective of the actual temperature, is in general less commonly used and, so far, it has only been included twice in a phenology model predicting bee emergence. Furthermore, I used this model to retrospectively predict the emergence dates of bees by applying the model to long-term temperature data which have been recorded by the regional climate station in W{\"u}rzburg. By doing so, the model estimated that over the last 63 years, bees emerged approximately 4 days earlier. In Chapter IV, I present a study in which I investigated how temporal mismatches in bee-plant interactions affect the fitness of solitary bees. Therefore, I performed an experiment with large flight cages serving as mesocosms. Inside these mesocosms, I manipulated the supply of blossoms to synchronize or desynchronize bee-plant interactions. In sum, I showed that even short temporal mismatches of three and six days in bee-plant interactions (with solitary bee emergence before flower occurrence) can cause severe fitness losses in solitary bees. Nonetheless, I detected different strategies by solitary bees to counteract impacts on their fitness after temporal mismatches. However, since these strategies may result in secondary fitness costs by a changed sex ratio or increased parasitism, I concluded that compensation strategies do not fully mitigate fitness losses of bees after short temporal mismatches with their food plants. In the event of further climate warming, fitness losses after temporal mismatches may not only exacerbate bee declines but may also reduce pollination services for later-flowering species and affect populations of animal-pollinated plants. In conclusion, I showed that spring-emerging solitary bees are susceptible to climate change as in response to warmer temperatures bees advance their phenology and show a decreased fitness state. As spring-emerging solitary bees not only consider overwintering temperature but also their individual body condition for adjusting emergence dates, this may explain differing responses to climate warming within and among bee populations which may also have consequences for bee-plant interactions and the persistence of bee populations under further climate warming. If in response to climate warming plants do not shift their phenologies according to the bees, bees may experience temporal mismatches with their host plants. As bees failed to show a single compensation strategy that was entirely successful in mitigating fitness consequences after temporal mismatches with their food plants, the resulting fitness consequences for spring-emerging solitary bees would be severe. Furthermore, I showed that spring-emerging solitary bees use a critical calendar date before which they generally do not commence the summation of degree-days irrespective of the actual temperature. I therefore suggest that further studies should also include the parameter of a critical calendar date into degree-day model predictions to increase the accuracy of model predictions for emergence dates in solitary bees. Although our retrospective prediction about the advance in bee emergence corresponds to the results of several studies on phenological trends of different plant species, we suggest that more research has to be done to assess the impacts of climate warming on the synchronization in bee-plant interactions more accurately.}, subject = {wild bees}, language = {en} } @phdthesis{Nuernberger2018, author = {N{\"u}rnberger, Fabian}, title = {Timing of colony phenology and foraging activity in honey bees}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155105}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {I. Timing is a crucial feature in organisms that live within a variable and changing environment. Complex mechanisms to measure time are wide-spread and were shown to exist in many taxa. These mechanisms are expected to provide fitness benefits by enabling organisms to anticipate environmental changes and adapt accordingly. However, very few studies have addressed the adaptive value of proper timing. The objective of this PhD-project was to investigate mechanisms and fitness consequences of timing decisions concerning colony phenology and foraging activity in the honey bee (Apis mellifera), a social insect species with a high degree of social organization and one of the most important pollinators of wild plants and crops. In chapter II, a study is presented that aimed to identify the consequences of disrupted synchrony between colony phenology and the local environment by manipulating the timing of brood onset after hibernation. In a follow-up experiment, the importance of environmental factors for the timing of brood onset was investigated to assess the potential of climate change to disrupt synchronization of colony phenology (Chapter III). Chapter IV aimed to prove for the first time that honey bees can use interval time-place learning to improve foraging activity in a variable environment. Chapter V investigates the fitness benefits of information exchange between nest mates via waggle dance communication about a resource environment that is heterogeneous in space and time. II. In the study presented in chapter II, the importance of the timing of brood onset after hibernation as critical point in honey bee colony phenology in temperate zones was investigated. Honey bee colonies were overwintered at two climatically different sites. By translocating colonies from each site to the other in late winter, timing of brood onset was manipulated and consequently colony phenology was desynchronized with the local environment. Delaying colony phenology in respect to the local environment decreased the capability of colonies to exploit the abundant spring bloom. Early brood onset, on the other hand, increased the loads of the brood parasite Varroa destructor later in the season with negative impact on colony worker population size. This indicates a timing related trade-off and illustrates the importance of investigating effects of climate change on complex multi-trophic systems. It can be concluded that timing of brood onset in honey bees is an important fitness relevant step for colony phenology that is highly sensitive to climatic conditions in late winter. Further, phenology shifts and mismatches driven by climate change can have severe fitness consequences. III. In chapter III, I assess the importance of the environmental factors ambient temperature and photoperiod as well as elapsed time on the timing of brood onset. Twenty-four hibernating honey bee colonies were placed into environmental chambers and allocated to different combinations of two temperature regimes and three different light regimes. Brood onset was identified non-invasively by tracking comb temperature within the winter cluster. The experiment revealed that ambient temperature plays a major role in the timing of brood onset, but the response of honey bee colonies to temperature increases is modified by photoperiod. Further, the data indicate the involvement of an internal clock. I conclude that the timing of brood onset is complex but probably highly susceptible to climate change and especially spells of warm weather in winter. IV. In chapter IV, it was examined if honey bees are capable of interval time-place learning and if this ability improves foraging efficiency in a dynamic resource environment. In a field experiment with artificial feeders, foragers were able to learn time intervals and use this ability to anticipate time periods during which feeders were active. Further, interval time-place learning enabled foragers to increase nectar uptake rates. It was concluded that interval time-place learning can help honey bee foragers to adapt to the complex and variable temporal patterns of floral resource environments. V. The study presented in chapter V identified the importance of the honey bee waggle dance communication for the spatiotemporal coordination of honey bee foraging activity in resource environments that can vary from day to day. Consequences of disrupting the instructional component of honey bee dance communication were investigated in eight temperate zone landscapes with different levels of spatiotemporal complexity. While nectar uptake of colonies was not affected, waggle dance communication significantly benefitted pollen harvest irrespective of landscape complexity. I suggest that this is explained by the fact that honey bees prefer to forage pollen in semi-natural habitats, which provide diverse resource species but are sparse and presumably hard to find in intensively managed agricultural landscapes. I conclude that waggle dance communication helps to ensure a sufficient and diverse pollen diet which is crucial for honey bee colony health. VI. In my PhD-project, I could show that honey bee colonies are able to adapt their activities to a seasonally and daily changing environment, which affects resource uptake, colony development, colony health and ultimately colony fitness. Ongoing global change, however, puts timing in honey bee colonies at risk. Climate change has the potential to cause mismatches with the local resource environment. Intensivation of agricultural management with decreased resource diversity and short resource peaks in spring followed by distinctive gaps increases the probability of mismatches. Even the highly efficient foraging system of honey bees might not ensure a sufficiently diverse and healthy diet in such an environment. The global introduction of the parasitic mite V. destructor and the increased exposure to pesticides in intensively managed landscapes further degrades honey bee colony health. This might lead to reduced cognitive capabilities in workers and impact the communication and social organization in colonies, thereby undermining the ability of honey bee colonies to adapt to their environment.}, subject = {Biene}, language = {en} } @phdthesis{Basile2009, author = {Basile, Rebecca}, title = {Thermoregulation and Resource Management in the Honeybee (Apis mellifera)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39793}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Ein grundlegender Faktor, der f{\"u}r das {\"U}berleben einer Kolonie sozialer Insekten ausschlaggebend ist, liegt in der F{\"a}higkeit Nahrung durch sogenannte „Trophallaxis" auszutauschen. Diese F{\"u}tterungskontakte sorgen f{\"u}r die gleichm{\"a}ßige Verteilung der Nahrung innerhalb der Kolonie und werden als einer der Grundpfeiler der Sozialit{\"a}t der Staatenbildenden Insekten erachtet. Im Fall der Honigbienen finden diese Kontakte in vollkommener Dunkelheit statt. Damit es in dieser Situation {\"u}berhaupt zum Nahrungsaustausch kommen kann, sind die Antennen von großer Wichtigkeit. Ein erster Schritt in den Verhaltensweisen, die der Rezipient eines trophallaktischen Kontaktes zeigt, ist der Kontakt einer Antennenspitze mit den Mundwerkzeugen des Donoren, da sich dort die regurgitierte Nahrung befindet. Diese Ber{\"u}hrung hat aufgrund der gustatorischen Sensibilit{\"a}t der Antenne den Zweck, das angebotene Futter zu „erschmecken". Die rechte Antenne wird vom Rezipienten eines trophallaktischen Kontakts signifikant h{\"a}ufiger eingesetzt als die linke Antenne. Die Pr{\"a}ferenz f{\"u}r die rechte Antenne bleibt dabei auch erhalten, wenn ein Teil der Antennengeisel abgetrennt wurde, also die sensorischen F{\"a}higkeiten der rechten Antenne stark beeintr{\"a}chtigt wurden. Der Grund f{\"u}r die Pr{\"a}ferenz der rechten Antenne k{\"o}nnte ihrer erh{\"o}hten Sensibilit{\"a}t gegen{\"u}ber Zuckerwasser zugrunde liegen, da die rechte Antenne im Laborversuch signifikant st{\"a}rker auf Stimulationen mit Zuckerwasser verschiedener Konzentrationen reagierte als die linke. Trophallaktische Kontakte sichern Individuen innerhalb einer Kolonie den Zugang zur lebenswichtigen Nahrung. Im Beispiel der Honigbienen ist st{\"a}ndige Zugriff auf Nahrung besonders wichtig, da es sich um ein heterothermes Tier handelt, das die F{\"a}higkeit besitzt, aktiv seine K{\"o}rpertemperatur zu regulieren. Obgleich jedes Individuum in der Lage ist, seine K{\"o}rpertemperatur den eigenen Bed{\"u}rfnissen anzupassen, ist diese F{\"a}higkeit streng durch den in der Nahrung aufgenommenen Zucker reguliert. Im Gegensatz zu den S{\"a}ugetieren oder V{\"o}geln, die f{\"u}r eine Erh{\"o}hung des Blutzuckerspiegels auch auf Fett- oder Eiweißressourcen zur{\"u}ckgreifen k{\"o}nnen, ist die Honigbiene auf die Glucose aus der aufgenommenen Nahrung angewiesen. Die Ergebnisse dieser Untersuchung zeigen, dass der Zuckergehalt der aufgenommenen Nahrung positiv mit der Thoraxtemperatur der Bienen korreliert. Dieser Zusammenhang tritt auf, selbst wenn keine W{\"a}rmeerzeugung f{\"u}r die Brutpflege oder f{\"u}r das Erw{\"a}rmen der Wintertraube notwendig ist und die Tiere außerhalb des Stockes ohne eigentliche Notwendigkeit f{\"u}r die W{\"a}rmeerzeugung in einem K{\"a}fig gehalten werden. Die Ergebnisse der Untersuchung zeigen, dass die Rezipienten beim Nahrungsaustausch eine signifikant h{\"o}here Thoraxtemperatur haben als die Donoren. Außerdem zeigen die Rezipienten nach der F{\"u}tterung signifikant h{\"a}ufiger Brutw{\"a}rmeverhalten als die Donoren. Letztere haben eine signifikant niedrigere Thoraxtemperatur als die Rezipienten und zeigen eine Verhaltenstendenz, h{\"a}ufig zwischen Brutbereich und Honiglager hin- und her zu pendeln. Dabei nehmen sie im Honiglager Honig in ihren Kropf auf und f{\"u}ttern mit dieser Nahrung danach Bienen im Brutbereich. Außerdem zeigen die Ergebnisse, dass es einen w{\"a}rmegesteuerten Ausl{\"o}semechanismus gibt, der den Donoren und Rezipienten des trophallaktischen Kontakts dazu verhilft, trotz der Dunkelheit des Stocks praktisch verz{\"o}gerungsfreie Nahrungs{\"u}bertragung am Ort des h{\"o}chsten Energieverbrauchs zu gew{\"a}hrleisten. Das Hervorw{\"u}rgen von Nahrung angesichts einer W{\"a}rmequelle k{\"o}nnte seinen Ursprung in einer Beschwichtigungsgeste haben. Aggressive Tiere zeigen neben sichtbaren aggressiven Verhalten auch durch ihre erh{\"o}hte K{\"o}rpertemperatur, dass sie bereit sind sich auf einen Kampf einzulassen. Die Temperaturerh{\"o}hung eines aggressiven Tieres beruht dabei auf der erh{\"o}hten Muskelaktivit{\"a}t, die vor allem bei Insekten dazu n{\"o}tig ist, einen entsprechende Reaktion im Falle eines Kampfes oder der Flucht zeigen zu k{\"o}nnen. Wird ein Individuum mit Aggression konfrontiert, so bleibt ihm die Wahl sich auf einen Kampf einzulassen, zu fl{\"u}chten oder durch eine Beschwichtigungsgeste eine Deeskalation der Situation einzuleiten. Besonders h{\"a}ufig wird f{\"u}r diesen Zweck Nahrung regurgitiert und dem dominanteren Tier angeboten, um einem Konflikt aus dem Weg zu gehen. Die F{\"a}higkeit, Arbeiterinnen mit kleinen Portionen konzentrierter Nahrung zu versorgen tr{\"a}gt zu einer {\"o}konomischen Verteilung der Ressourcen bei, die mit den physiologischen Bed{\"u}rfnissen der Honigbienen konform geht und die {\"o}kologischen Erfordernisse des Stockes erf{\"u}llt. Das daraus resultierende Managementsystem, welches sparsam mit den Ressourcen haushaltet und auf die individuellen Bed{\"u}rfnisse jeder einzelnen Biene einzugehen vermag, k{\"o}nnte ein Grund f{\"u}r die F{\"a}higkeit der Honigbienen zur Entwicklung mehrj{\"a}hriger Kolonien sein, die, anders als Hummeln oder Wespen, auch den Winter in gem{\"a}ßigten Zonen als Gemeinschaft zu {\"u}berstehen verm{\"o}gen.}, subject = {Biene}, language = {en} } @phdthesis{Jenett2007, author = {Jenett, Arnim}, title = {The Virtual Insect Brain Protocol : development and application of software for the standardization of neuroanatomy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-22297}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Since the fruit fly Drosophila melanogaster entered the laboratories as a model organism, new genetic, physiological, molecular and behavioral techniques for the functional analysis of the brain rapidly accumulated. Nowadays this concerted assault obtains its main thrust form Gal4 expression patterns that can be visualized and provide the means for manipulating -in unrestrained animals- groups of neurons of the brain. To take advantage of these patterns one needs to know their anatomy. This thesis describes the Virtual Insect Brain (VIB) protocol, a software package for the quantitative assessment, comparison, and presentation of neuroanatomical data. It is based on the 3D-reconstruction and visualization software Amira (Mercury Inc.). Its main part is a standardization procedure which aligns individual 3D images (series of virtual sections obtained by confocal microscopy) to a common coordinate system and computes average intensities for each voxel (volume pixel). The VIB protocol facilitates direct comparison of gene expression patterns and describes their interindividual variability. It provides volumetry of brain regions and helps to characterize the phenotypes of brain structure mutants. Using the VIB protocol does not require any programming skills since all operations are carried out at a (near to) self-explanatory graphical user interface. Although the VIB protocol has been developed for the standardization of Drosophila neuroanatomy, the program structure can be used for the standardization of other 3D structures as well. Standardizing brains and gene expression patterns is a new approach to biological shape and its variability. Using the VIB protocol consequently may help to integrate knowledge on the correlation of form and function of the insect brain. The VIB protocol provides a first set of tools supporting this endeavor in Drosophila. The software is freely available at http://www.neurofly.de.}, subject = {Taufliege}, language = {en} } @phdthesis{Spohn1999, author = {Spohn, Gunther}, title = {The transcriptional control of virulence gene expression in Helicobacter pylori}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-2334}, school = {Universit{\"a}t W{\"u}rzburg}, year = {1999}, abstract = {The Gram-negative, spiral-shaped, microaerophilic bacterium Helicobacter pylori is the causative agent of various disorders of the upper gastrointestinal tract, such as chronic superficial gastritis, chronic active gastritis, peptic ulceration and adenocarcinoma. Although many of the bacterial factors associated with disease development have been analysed in some detail in the recent years, very few studies have focused so far on the mechanisms that regulate expression of these factors at the molecular level. In an attempt to obtain an overview of the basic mechanisms of virulence gene expression in H. pylori, three important virulence factors of this pathogen, representative of different pathogenic mechanisms and different phases of the infectious process, are investigated in detail in the present thesis regarding their transcriptional regulation. As an essential factor for the early phase of infection, including the colonisation of the gastric mucosa, the flagella are analysed; the chaperones including the putative adhesion factors GroEL and DnaK are investigated as representatives of the phase of adherence to the gastric epithelium and persistence in the mucus layer; and finally the cytotoxin associated antigen CagA is analysed as representative of the cag pathogenicity island, which is supposed to account for the phenomena of chronic inflammation and tissue damage observed in the later phases of infection. RNA analyses and in vitro transcription demonstrate that a single promoter regulates expression of cagA, while two promoters are responsible for expression of the upstream divergently transcribed cagB gene. All three promoters are shown to be recognised by RNA polymerase containing the vegetative sigma factor sigma 80. Promoter deletion analyses establish that full activation of the cagA promoter requires sequences up to -70 and binding of the C-terminal portion of the alpha subunit of RNA polymerase to an UP-like element located between -40 and -60, while full activation of the major cagB promoter requires sequences upstream of -96 which overlap with the cagA promoter. These data suggest that the promoters of the pathogenicity island represent a class of minimum promoters, that ensure a basic level of transcription, while full activation requires regulatory elements or structural DNA binding proteins that provide a suitable DNA context. Regarding flagellar biosynthesis, a master transcriptional factor is identified that regulates expression of a series of flagellar basal body and hook genes in concert with the alternative sigma factor sigma 54. Evidence is provided that this regulator, designated FlgR (for flagellar regulatory protein), is necessary for motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR is shown to act as a repressor of transcription of the sigma 28-regulated promoter of the flaA gene, while changes in DNA topology are shown to affect transcription of the sigma 54-regulated flaB promoter. These data indicate that the regulatory network that governs flagellar gene expression in H. pylori shows similarities to the systems of both Salmonella spp. and Caulobacter crescentus. In contrast to the flagellar genes which are regulated by three different sigma factors, the three operons encoding the major chaperones of H. pylori are shown to be transcribed by RNA polymerase containing the vegetative sigma factor sigma 80. Expression of these operons is shown to be regulated negatively by the transcriptional repressor HspR, a homologue of a repressor protein of Streptomyces spp., known to be involved in negative regulation of heat shock genes. In vitro studies with purified recombinant HspR establish that the protein represses transcription by binding to large DNA regions centered around the transcription initiation site in the case of one promoter, and around -85 and -120 in the case of the the other two promoters. In contrast to the situation in Streptomyces, where transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible with thermal stimuli. Transcription of two of the three chaperone encoding operons is induced by osmotic shock, while transcription of the third operon, although HspR-dependent, is not affected by salt treatment. Taken together, the analyses carried out indicate that H. pylori has reduced its repertoire of specific regulatory proteins to a basic level that may ensure coordinate regulation of those factors that are necessary during the initial phase of infection including the passage through the gastric lumen and the colonisation of the gastric mucosa. The importance of DNA topology and/or context for transcription of many virulence gene promoters may on the other hand indicate, that a sophisticated global regulatory network is present in H. pylori, which influences transcription of specific subsets of virulence genes in response to changes in the microenvironment.}, subject = {Helicobacter-pylori-Infektion}, language = {en} } @phdthesis{Pusch2015, author = {Pusch, Tobias}, title = {The transcription factor NFATc1 mediates cytotoxic T cell function in vitro and in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123690}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment. Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells. Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade. We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation. Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse.}, subject = {Transkriptionsfaktor}, language = {en} } @phdthesis{Hein2004, author = {Hein, Silke}, title = {The survival of grasshoppers and bush crickets in habitats variable in space and time}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-9140}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {Die zunehmende Nutzung von Landschaften f{\"u}hrt zu einer steigenden Fragmentierung sch{\"u}tzenswerter Fl{\"a}chen. Damit verbunden ist eine Zerschneidung von großen Populationen in Metapopulationen. In solchen F{\"a}llen bestimmt das Gleichgewicht zwischen Aussterben und Besiedlung von Habitaten die regionale {\"U}berlebenswahrscheinlichkeit von Arten. Um diese bestimmen, braucht man ein gutes Verst{\"a}ndnis der Habitatanspr{\"u}che der Arten, sowie Informationen {\"u}ber ihr Ausbreitungsverhalten. Ziel dieser Arbeit war es, geeignete Fl{\"a}chen f{\"u}r Heuschrecken in einer Landschaft identifizieren zu k{\"o}nnen, sowie einen Beitrag zur Quantifizierung der Erreichbarkeit einzelner Fl{\"a}chen durch Individuen zu leisten. Der erste Teil dieser Arbeit besch{\"a}ftigt sich mit der Quantifizierung der Habitateignung von Fl{\"a}chen f{\"u}r Heuschrecken. Dazu habe ich statistische Habitateignungsmodelle mittels logistischer Regression erstellt, evaluiert und validiert. Es zeigte sich, dass die Habitatwahl der Heuschrecken auf einer mittleren r{\"a}umlichen Skalenebene erfolgt. Dies steht mit der beobachteten Ausbreitungsdistanz der Tiere im Einklang. Neben dem nur grob klassifizierten Landschaftsfaktor „Biotoptyp" korrelieren vor allem strukturelle Faktoren sowie abiotische Faktoren mit dem Vorkommen der Heuschreckenarten. Bei der Bestimmung eines gemeinsamen Models f{\"u}r alle drei Heuschreckenarten erwies sich das Model der Art S. lineatus mit den Parametern Biotoptyp und Vegetationsh{\"o}he als am besten geeignet zur Vorhersage der Vorkommen der anderen Heuschreckenarten. Um zu testen, ob auch die Vorkommen von Arten unterschiedlicher Tiergruppen mittels eines gemeinsamen Modells vorhergesagt werden k{\"o}nnen, habe ich sowohl die Heuschreckenmodelle zur Prognose von Faltervorkommen getestet, als auch Modelle f{\"u}r Falter auf Heuschrecken {\"u}bertragen. Dabei erwiesen sich die Heuschreckenmodelle zur Prognose der anderen Arten weniger geeignet als das Modell f{\"u}r das Widderchen Z. carniolica in das der Anteil an geeignetem Habitat sowie die Vorkommen der beiden Saugpflanzen C. jacea und S. columbaria einfließen. Diese Art wird als standorttreu eingestuft und repr{\"a}sentiert damit auch die anderen Arten, die typisch f{\"u}r S{\"a}ume und Halbtrockenrasen sind. Die erh{\"o}hte Mobilit{\"a}t von Z. carniolica im Vergleich zu den Heuschrecken garantiert gleichzeitig auch die Erreichbarkeit aller geeigneten Fl{\"a}chen im Gebiet und damit ein Modell, das nur unwesentlich durch Zufallseffekte bei der Besiedlung beeinflusst wird. Neben der Habitatqualit{\"a}t/-quantit{\"a}t spielt vor allem der Austausch zwischen Fl{\"a}chen eine entscheidende Rolle f{\"u}r das {\"U}berleben der Metapopulation. Im zweiten Teil meiner Arbeit habe ich mich sowohl theoretisch als auch empirisch, mit dem Ausbreitungsverhalten von Heuschrecken besch{\"a}ftigt. In Freilandexperimenten konnte ich zeigen, dass die Annahme eines dichotomen Bewegungsverhaltens f{\"u}r Heuschrecken in einer realen Landschaft nicht zutrifft. Vielmehr wird die Bewegung in einer Fl{\"a}che besser als Kontinuum beschrieben das durch strukturelle Resistenz, Temperatur, Mortalit{\"a}tsrisiko und Ressourcenverf{\"u}gbarkeit bestimmt wird. Die jeweilige Kombination dieser Parameter veranlasst die Tiere dann zu einem entsprechenden Bewegungsmuster, das sich zwischen den beiden Extremen gerichteter und zuf{\"a}lliger Lauf bewegt. In Experimenten zum Grenzverhalten von Heuschrecken best{\"a}tigte sich dieses Ergebnis. F{\"u}r verschiedene Grenzstrukturen konnte ich unterschiedliche {\"U}bertrittswahrscheinlichkeiten nachweisen. Weiterhin konnte ich feststellen, dass Heuschrecken geeignete Habitate aus einer gewissen Entfernung detektieren k{\"o}nnen. Da das Ausbreitungsverhalten von Tieren in theoretischen Modellen eine wichtige Rolle spielt, k{\"o}nnen diese empirischen Daten zur Parametrisierung dieser Modelle verwendet werden. Zus{\"a}tzlich zum Einfluss des Laufmusters der Tiere auf die Erreichbarkeit geeigneter Habitate, zeigte sich in den von mir durchgef{\"u}hrten Simulationsstudien deutlich, dass der landschaftliche Kontext, in dem die Ausbreitung stattfindet, die Erreichbarkeit einzelner Habitate beeinflusst. Dieser Effekt ist zus{\"a}tzlich abh{\"a}ngig von der Mortalit{\"a}tsrate beim Ausbreitungsvorgang. Mit den Ergebnissen aus den Untersuchungen zur Habitateignung lassen sich die f{\"u}r Heuschrecken geeigneten Habitate in einer Landschaft identifizieren. Somit l{\"a}sst sich die potentielle Eignung einer Fl{\"a}che als Habitat, basierend auf Vorhersagen {\"u}ber die {\"A}nderung des Biotoptyps durch ein Managementverfahren, vorhersagen. Diese Information allein reicht aber nicht aus, um die regionale {\"U}berlebenswahrscheinlichkeit einer Art bestimmen zu k{\"o}nnen. Meine Untersuchungen zum Ausbreitungsverhalten zeigen deutlich, dass die Erreichbarkeit geeigneter Fl{\"a}chen von der r{\"a}umlichen Anordnung der Habitate und der Struktur der Fl{\"a}chen, die zwischen Habitaten liegen, abh{\"a}ngt. Zus{\"a}tzlich spielen individuenspezifische Faktoren wie Motivation und physiologische Faktoren eine ausschlaggebende Rolle f{\"u}r die Erreichbarkeit von geeigneten Fl{\"a}chen.}, subject = {Naturschutzgebiet Hohe Wann}, language = {en} } @phdthesis{Schindelin2005, author = {Schindelin, Johannes}, title = {The standard brain of Drosophila melanogaster and its automatic segmentation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15518}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {In this thesis, I introduce the Virtual Brain Protocol, which facilitates applications of the Standard Brain of Drosophila melanogaster. By providing reliable and extensible tools for the handling of neuroanatomical data, this protocol simplifies and organizes the recurring tasks involved in these applications. It is demonstrated that this protocol can also be used to generate average brains, i.e. to combine recordings of several brains with the same features such that the common features are emphasized. One of the most important steps of the Virtual Insect Protocol is the aligning of newly recorded data sets with the Standard Brain. After presenting methods commonly applied in a biological or medical context to align two different recordings, it is evaluated to what extent this alignment can be automated. To that end, existing Image Processing techniques are assessed. I demonstrate that these techniques do not satisfy the requirements needed to guarantee sensible alignments between two brains. Then, I analyze what needs to be taken into account in order to formulate an algorithm which satisfies the needs of the protocol. In the last chapter, I derive such an algorithm using methods from Information Theory, which bases the technique on a solid mathematical foundation. I show how Bayesian Inference can be applied to enhance the results further. It is demonstrated that this approach yields good results on very noisy images, detecting apparent boundaries between structures. The same approach can be extended to take additional knowledge into account, e.g. the relative position of the anatomical structures and their shape. It is shown how this extension can be utilized to segment a newly recorded brain automatically.}, subject = {Taufliege}, language = {en} } @phdthesis{Chan2002, author = {Chan, Gordon}, title = {The Role of Vav-1, Vav-2 and Lsc in NK T cell development and NK cell cytotoxicity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-3645}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2002}, abstract = {The hematopoietic-specific Rho-family GTP exchange factor (GEF) Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, I show that Vav-1 is also expressed in murine NK cells. Vav-1-/- mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as cells from wild type mice. Unexpectedly, IL-2-activated Vav-1-/- NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1-/- NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, these results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK cell natural cytotoxicity. Vav-2, a protein homologous to Vav-1 has also been implicated in NK cell functions. However, NK cells from Vav-2-/- mice have normal cytotoxic activities and NK cells that lack both Vav-1 and Vav-2 exhibit similar defect as Vav-1-/- cells. Thus Vav-2 has no apparent function in the development and the activation of NK cells. Although NK cell development is normal in Vav-1-/- mice, their numbers of NKT cells were dramatically diminished. Furthermore, NKT cells from Vav-1 mutant mice failed to produce IL-4 and IFNg following in vivo CD3 stimulation. A similar loss of NKT cells was observed in Vav-1-/-Vav-2-/- mice, but not in Vav-2-/- mice, suggesting that only Vav-1, and not Vav-2, is an essential regulator of NKT cell development and NK cell cytotoxicity. Similar to Vav-1, Lsc is a Rho GEF that is expressed specifically in the hematopoietic system. It contains a regulator of G-protein signaling (RGS) domain which negatively regulates the Ga12 and Ga13 subunits of G-protein coupled receptors (GPCRs). This study shows that NK and NKT cell development are normal in Lsc-/- mice. However, NK cells from mutant mice display enhanced cytotoxic responses towards a panel of tumor cells. These data implicate for the first time a RGS-containing Rho GEF in cytotoxic responses and suggest that Lsc down-modulate NK cell activation.}, subject = {Maus}, language = {en} } @phdthesis{Blaettner2016, author = {Bl{\"a}ttner, Sebastian}, title = {The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146662}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis. In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70\% of the CA infections. The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur. S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient. In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae. The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria.}, subject = {Staphylococcus aureus}, language = {en} } @phdthesis{LuiblneeHermann2014, author = {Luibl [n{\´e}e Hermann], Christiane}, title = {The role of the neuropeptides NPF, sNPF, ITP and PDF in the circadian clock of Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-93796}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Organisms have evolved endogenous clocks which allow them to organize their behavior, metabolism and physiology according to the periodically changing environmental conditions on earth. Biological rhythms that are synchronized to daily changes in environment are governed by the so-called circadian clock. Since decades, chronobiologists have been investigating circadian clocks in various model organisms including the fruitfly Drosophila melanogaster, which was used in the present thesis. Anatomically, the circadian clock of the fruitfly consists of about 150 neurons in the lateral and dorsal protocerebrum, which are characterized by their position, morphology and neurochemistry. Some of these neurons had been previously shown to contain either one or several neuropeptides, which are thought to be the main signaling molecules used by the clock. The best investigated of these neuropeptides is the Pigment Dispersing Factor (PDF), which had been shown to constitute a synchronizing signal between clock neurons as well as an output factor of the clock. In collaboration with various coworkers, I investigated the roles of three other clock expressed neuropeptides for the generation of behavioral rhythms and the partly published, partly unpublished data are presented in this thesis. Thereby, I focused on the Neuropeptide F (NPF), short Neuropeptide F (sNPF) and the Ion Transport Peptide (ITP). We show that part of the neuropeptide composition within the clock network seems to be conserved among different Drosophila species. However, the PDF expression pattern in certain neurons varied in species deriving from lower latitudes compared to higher latitudes. Together with findings on the behavioral level provided by other people, these data suggest that different species may have altered certain properties of their clocks - like the neuropeptide expression in certain neurons - in order to adapt their behavior to different habitats. We then investigated locomotor rhythms in Drosophila melanogaster flies, in which neuropeptide circuits were genetically manipulated either by cell ablation or RNA interference (RNAi). We found that none of the investigated neuropeptides seems to be of equal importance for circadian locomotor rhythms as PDF. PDF had been previously shown to be necessary for rhythm maintenance in constant darkness (DD) as well as for the generation of morning (M) activity and for the right phasing of the evening (E) activity in entrained conditions. We now demonstrate that NPF and ITP seem to promote E activity in entrained conditions, but are clearly not the only factors doing so. In addition, ITP seems to reduce nighttime activity. Further, ITP and possibly also sNPF constitute weak period shortening components in DD, thereby opposing the effect of PDF. However, neither NPF or ITP, nor sNPF seem to be necessary in the clock neurons for maintaining rhythmicity in DD. It had been previously suggested that PDF is released rhythmically from the dorsal projection terminals. Now we discovered a rhythm in ITP immunostaining in the dorsal projection terminals of the ITP+ clock neurons in LD, suggesting a rhythm in peptide release also in the case of ITP. Rhythmic release of both ITP and PDF seems to be important to maintain rhythmic behavior in DD, since constantly high levels of PDF and ITP in the dorsal protocerebrum lead to behavioral arrhythmicity. Applying live-imaging techniques we further demonstrate that sNPF acts in an inhibitory way on few clock neurons, including some that are also activated by PDF, suggesting that it acts as signaling molecule within the clock network and has opposing effects to PDF. NPF did only evoke very little inhibitory responses in very few clock neurons, suggesting that it might rather be used as a clock output factor. We were not able to apply the same live-imaging approach for the investigation of the clock neuron responsiveness to ITP, but overexpression of ITP with various driver lines showed that the peptide most likely acts mainly in clock output pathways rather than inter-clock neuron communication. Taking together, I conclude that all investigated peptides contribute to the control of locomotor rhythms in the fruitfly Drosophila melanogaster. However, this control is in most aspects dominated by the actions of PDF and rather only fine-tuned or complemented by the other peptides. I assume that there is a high complexity in spatial and temporal action of the different neuropeptides in order to ensure correct signal processing within the clock network as well as clock output.}, subject = {Taufliege}, language = {en} } @phdthesis{Pasch2016, author = {Pasch, Elisabeth}, title = {The role of SUN4 and related proteins in sperm head formation and fertility}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139092}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Spermiogenesis describes the differentiation of haploid germ cells into motile, fertilization-competent spermatozoa. During this fundamental transition the species-specific sperm head is formed, which necessitates profound nuclear restructuring coincident with the assembly of sperm-specific structures and chromatin compaction. In the case of the mouse, it is characterized by reshaping of the early round spermatid nucleus into an elongated sickle-shaped sperm head. This tremendous shape change requires the transduction of cytoskeletal forces onto the nuclear envelope (NE) or even further into the nuclear interior. LINC (linkers of nucleoskeleton and cytoskeleton) complexes might be involved in this process, due to their general function in bridging the NE and thereby physically connecting the nucleus to the peripheral cytoskeleton. LINC complexes consist of inner nuclear membrane integral SUN-domain proteins and outer nuclear membrane KASH-domain counterparts. SUN- and KASH-domain proteins are directly connected to each other within the perinuclear space, and are thus capable of transferring forces across the NE. To date, these protein complexes are known for their essential functions in nuclear migration, anchoring and positioning of the nucleus, and even for chromosome movements and the maintenance of cell polarity and nuclear shape. In this study LINC complexes were investigated with regard to their potential role in sperm head formation, in order to gain further insight into the processes occurring during spermiogenesis. To this end, the behavior and function of the testis-specific SUN4 protein was studied. The SUN-domain protein SUN4, which had received limited characterization prior to this work, was found to be exclusively expressed in haploid stages during germ cell development. In these cell stages, it specifically localized to the posterior NE at regions decorated by the manchette, a spermatid-specific structure which was previously shown to be involved in nuclear shaping. Mice deficient for SUN4 exhibited severely disorganized manchette residues and gravely misshapen sperm heads. These defects resulted in a globozoospermia-like phenotype and male mice infertility. Therefore, SUN4 was not only found to be mandatory for the correct assembly and anchorage of the manchette, but also for the correct localization of SUN3 and Nesprin1, as well as of other NE components. Interaction studies revealed that SUN4 had the potential to interact with SUN3, Nesprin1, and itself, and as such is likely to build functional LINC complexes that anchor the manchette and transfer cytoskeletal forces onto the nucleus. Taken together, the severe impact of SUN4 deficiency on the nucleocytoplasmic junction during sperm development provided direct evidence for a crucial role of SUN4 and other LINC complex components in mammalian sperm head formation and fertility.}, subject = {Maus}, language = {en} } @phdthesis{Pei2000, author = {Pei, Geng}, title = {The Role of Raf-mediated Signalling Pathways for Motoneuron}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1846}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2000}, abstract = {The transmission of proliferative and developmental signals from activated cell-surface receptors to initiation of cellular responses in the nucleus is synergically controlled by the coordinated action of a diverse set of intracellular signalling proteins. The Ras/Raf/MEK/MAPK signalling pathway has been shown to control the expression of genes which are crucial for the physiological regulation of cell proliferation, differentiation and apoptosis. Within this signalling cascade, the Raf protein family of serine/threonine kinases serves as a central intermediate which connects to many of other signal transduction pathways. To elucidate the signalling functions of the different Raf kinases in motoneurons during development, the expression, distribution and subcellular localization of Rafs in the spinal cord and the facial nucleus in brainstem of mice at various embryonic and postnatal stages were investigated. Moreover, we have investigated the intracellular redistribution of Raf molecules in isolated motoneurons from 13 or 14 day old mouse embryos, after addition or withdrawal of neurotrophic factors to induce Raf kinases activation in vitro. Furthermore, in order to investigate the potential anti-apoptotic function of Raf kinases on motoneurons, we isolated motoneurons from B-raf-/- and c-raf-1-/- mouse embryos and analysed the survival and differentiation effects of neurotrophic factors in motoneurons lacking B-Raf and c-Raf-1. We provide evidence here that all three Raf kinases are expressed in mouse spinal motoneurons. Their expression increases during the period of naturally occurring cell death of motoneurons. In sections of embryonic and postnatal spinal cord, motoneurons express exclusively B-Raf and c-Raf-1, but not A-Raf, and subcellularly Raf kinases are obviously colocalized with mitochondria. In isolated motoneurons, most of the B-Raf or c-Raf-1 immunoreactivity is located in the perinuclear space but also in the nucleus, especially after activation by addition of CNTF and BDNF in vitro. We found that c-Raf-1 translocation from the cytosol into the nucleus of motoneurons after its activation by neurotrophic factors is a distinct event. As a central finding of our study, we observed that the viability of isolated motoneurons from B-raf but not c-raf-1 knockout mice is lost even in the presence of CNTF and other neurotrophic factors. This indicates that B-Raf but not c-Raf-1, which is still present in B-raf deficient motoneurons, plays a crucial role in mediating the survival effect of neurotrophic factors during development. In order to prove that B-Raf is an essential player in this scenario, we have re-expressed B-Raf in mutant sensory and motor neurons by transfection. The motoneurons and the sensory neurons from B-raf knockout mouse which were transfected with exogenous B-raf gene revealed the same viability in the presence of neurotrophic factors as primary neurons from wild-type mice. Our results suggest that Raf kinases have important signalling functions in motoneurons in mouse CNS. In vitro, activation causes redistribution of Raf protein kinases, particularly for c-Raf-1, from motoneuronal cytoplasm into the nucleus. This redistribution of c-Raf-1, however, is not necessary for the survival effect of neurotrophic factors, given that B-raf-/- motor and sensory neurons can not survive despite the presence of c-Raf-1. We hypothesize that c-Raf-1 nuclear translocation may play a direct role in transcriptional regulation as a consequence of neurotrophic factor induced phosphorylation and activation of c-Raf-1 in motoneurons. Moreover, the identification of target genes for nuclear translocated c-Raf-1 and of specific cellular functions initiated by this mechanism awaits its characterization.}, subject = {Maus}, language = {en} } @phdthesis{Link2013, author = {Link, Jana}, title = {The role of meiotic nuclear envelope components in chromosome dynamics and meiotic progression}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83540}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {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.}, subject = {Meiose}, language = {en} } @phdthesis{Reichert2008, author = {Reichert, Nina}, title = {The Role of LIN9 in Mouse Development}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-30889}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {LINC, the human homologue of an evolutionary conserved complex, regulates the transcription of a set of genes essential during the G2/M transition (Osterloh et al., 2007; Schmit et al., 2007). One component of the LINC core module is LIN-9. LIN-9 is essential for the transcriptional activation of LINC target genes and also promotes differentiation in association with pRB (Gagrica et al., 2004). However, nothing is known about its function in vivo. Histological and molecular analysis revealed that Lin9 is ubiquitously expressed throughout embryonic development and in all examined adult organs. Additionally, Lin9 mRNA is expressed in ES cells and blastocysts. Moreover the analogous distribution of the other LINC components suggested that they all function in the same cells and most likely in the same pathway. To deeper investigate the role of LIN9 in cell cycle and differentiation in vivo, a Lin9 gene trap mouse model (GT) was successfully generated and examined. Heterozygouse Lin9GT/+ mice were inconspicuous and develop normally. However, homozygouse knockout embryos were never obtained. The Lin9GT/GT embryos die at peri-implantation, probably due to a defect in the development of the epiblast, which could be shown with in situ hybridization with specific lineage markers. In vitro, the ICM of Lin9-deficient blastocysts did not develop properly. These data suggest that the loss of Lin9 leads to embryonic lethality at peri-implantation, and indicates that LIN9 is required for proper formation of the epiblast. In parallel, the first conditional Lin9 mouse model based on the Cre-loxP technology was generated. The Lin9fl/fl allele can be deleted by Cre-recombinase, in vivo and in vitro. Therefore an inducible system with Lin9fl/fl mice harboring Cre-ERT2 was established. The MEFs generated from these transgenic mice carried a nearly complete knockout upon induction with tamoxifen. Deletion of LIN9 in MEFs had a major impact upon the cell cycle and growth rates. Specifically, they arrested in G2/M phase and stopped to proliferate. Taken together, I was able to generate a lin9 gene trap and a lin9 conditional knockout mouse model. All results obtained so far demonstrate, that Lin9 is an essential gene for embryonic development and cell cycle control. It will be of great interest to further investigate Lin9-deficiency to gain insights into the mechanism of cell cycle control in early embryonic development and cell differentiation.}, subject = {Zellzyklus}, language = {en} } @phdthesis{Herold2003, author = {Herold, Andrea}, title = {The role of human and Drosophila NXF proteins in nuclear mRNA export}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-5601}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2003}, abstract = {A distinguishing feature of eukaryotic cells is the spatial separation of the site of mRNA synthesis (nucleus) from the site of mRNA function (cytoplasm) by the nuclear envelope. As a consequence, mRNAs need to be actively exported from the nucleus to the cytoplasm. At the time when this study was initiated, both human TAP and yeast Mex67p had been proposed to play a role in this process. Work presented in this thesis (section 2.1) revealed that TAP and Mex67p belong to an evolutionarily conserved family of proteins which are characterized by a conserved modular domain organization. This family was termed nuclear export factor (NXF) family. While the yeast genome encodes only one NXF protein (Mex67p), the genomes of higher eukaryotes encode several NXF proteins. There are two nxf genes in C. elegans and A. gambiae, four in D. melanogaster, and at least four in H. sapiens and M. musculus. It was unclear whether, apart from TAP and Mex67p, other members of this family would also be involved in mRNA export. In the first part of this thesis (2.1), several human NXF members were tested for a possible function in nuclear mRNA export. They were analyzed for their interaction with RNA, nucleoporins and other known TAP partners in vitro, and tested for their ability to promote nuclear export of a reporter mRNA in vivo. Using these assays, human NXF2, NXF3 and NXF5 were all shown to interact with the known NXF partner p15. NXF2 and NXF5 were also found to bind directly to RNA, but only NXF2 was able to bind directly to nucleoporins and to promote the nuclear export of an (untethered) reporter mRNA. Thus NXF2 possesses many and NXF3 and NXF5 possess some of the features required to serve as an export receptor for cellular mRNAs. As NXF2 and NXF3 transcripts were mainly found in testis, and the closest orthologue of NXF5 in mouse has the highest levels of expression in brain, these NXF members could potentially serve as tissue-specific mRNA export receptors. In the second part of this work (2.2), the role of different Drosophila NXF proteins and other export factors in mRNA export was investigated using double-stranded RNA interference (RNAi) in Drosophila Schneider cells. Three of the four predicted Drosophila NXF members (NXF1-3) were found to be expressed in this cell line and could be targeted by RNAi. Depletion of endogenous NXF1 inhibited growth and resulted in the nuclear accumulation of polyadenylated RNA. Fluorescence in situ hybridization revealed that export of both heat shock and non-heat shock mRNAs, including intron-containing and intronless mRNAs, was inhibited. Depleting endogenous NXF2 or NXF3 had no apparent phenotype. These results suggested that NXF1 (but not NXF2-NXF4) mediates the export of bulk mRNA in Drosophila cells. We and others have shown that human NXF proteins function as heterodimers bound to the small protein p15. Accordingly, silencing of Drosophila p15 resulted in a block of mRNA export which was indistinguishable from the export inhibition seen after targeting NXF1. These observations indicated that neither NXF1 nor p15 can promote export in the absence of the other subunit of the heterodimer. NXF1:p15 heterodimers are implicated in late steps of mRNA export, i.e. in the translocation of mRNP export cargoes across the nuclear pore complex. The mechanism by which NXF1:p15 dimers are recruited to the mRNA is unclear. A protein that is thought to play a role in this process is the putative RNA helicase UAP56. Similar to NXF1 and p15, UAP56 was shown to be essential for mRNA export in Drosophila. UAP56 is recruited cotranscriptionally to nascent transcripts and was suggested to facilitate the interaction of NXF1:p15 with mRNPs. Even though both NXF1:p15 heterodimers and UAP56 had been implicated in general mRNA export, it was unclear whether there are classes of mRNAs that require NXF1:p15, but not UAP56 or vice versa. It was also unclear what fraction of cellular mRNAs is exported by NXF1:p15 dimers and UAP56, and whether mRNAs exist that reach the cytoplasm through alternative routes, i.e. by recruiting other export receptors. To address these issues we performed a genome-wide analysis of nuclear mRNA export pathways using microarray technology (2.2.2). We analyzed the relative abundance of nearly half of the Drosophila transcriptome in the cytoplasm of Drosophila Schneider cells depleted of different export factors by RNAi. We showed that the vast majority of transcripts were underrepresented in the cytoplasm of cells depleted of NXF1, p15 or UAP56 as compared to control cells. Only a small number of mRNAs were apparently not affected by the depletions. These observations, together with the wide and similar effects on mRNA levels caused by the depletion of NXF1, p15 or UAP56, indicate that these proteins define the major mRNA export pathway in these cells. We also identified a small subset of mRNAs which appeared to be exported by NXF1:p15 dimers independently of UAP56. In contrast, no significant changes in mRNA expression profiles were observed in cells depleted of NXF2 or NXF3, suggesting that neither NXF2 nor NXF3 play an essential role in mRNA export in Drosophila Schneider cells. Crm1 is a transport receptor implicated in the export of a variety of non-mRNA and protein cargoes. In addition, human Crm1 has been suggested to be involved in the export of a specific mRNA species, serving as a "specialized" mRNA export receptor. A role of human Crm1 in the export of bulk mRNA is considered unlikely. We analyzed the role of Drosophila Crm1 in mRNA export by inhibiting Crm1 with the drug leptomycin B in Schneider cells. Subsequent microarray analysis demonstrated that the inactivation of Crm1 resulted in decreased cytoplasmic levels of less than 1\% of all mRNAs, indicating that Crm1 is indeed not a major mRNA export receptor. The genome-wide analysis also revealed a feedback loop by which a block to mRNA export triggers the upregulation of genes involved in this process. This thesis also includes two sections describing projects in which I participated during my Ph.D., but which were not the main focus of this thesis. In section 2.3, the role of the different TAP/NXF1 domains in nuclear mRNA export is discussed. Section 2.4 describes results that were obtained as part of a collaboration using the RNAi technique in Schneider cells to study the function of Cdc37.}, subject = {Zellkern}, language = {en} } @phdthesis{Iltzsche2017, author = {Iltzsche, Fabian}, title = {The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154108}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.}, subject = {Nicht-kleinzelliges Bronchialkarzinom (NSCLC)}, language = {en} } @phdthesis{Dornhaus2002, author = {Dornhaus, Anna}, title = {The role of communication in the foraging process of social bees}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-3468}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2002}, abstract = {In the various groups of social bees, different systems of communication about food sources occur. These communication systems are different solutions to a common problem of social insects: efficiently allocating the necessary number of workers first to the task of foraging and second to the most profitable food sources. The solution chosen by each species depends on the particular ecological circumstances as well as the evolutionary history of that species. For example, the outstanding difference between the bumble bee and the honey bee system is that honey bees can communicate the location of profitable food sources to nestmates, which bumble bees cannot. To identify possible selection pressures that could explain this difference, I have quantified the benefits of communicating location in honey bees. I show that these strongly depend on the habitat, and that communicating location might not benefit bees in temperate habitats. This could be due to the differing spatial distributions of resources in different habitats, in particular between temperate and tropical regions. These distributions may be the reason why the mostly temperate-living bumble bees have never evolved a communication system that allows them to transfer information on location of food sources, whereas most tropical social bees (all honey bees and many stingless bees) are able to recruit nestmates to specific points in their foraging range. Nevertheless, I show that in bumble bees the allocation of workers to foraging is also regulated by communication. Successful foragers distribute in the nest a pheromone which alerts other bees to the presence of food. This pheromone stems from a tergite gland, the function of which had not been identified previously. Usage of a pheromone in the nest to alert other individuals to forage has not been described in other social insects, and might constitute a new mode of communicating about food sources. The signal might be modulated depending on the quality of the food source. Bees in the nest sample the nectar that has been brought into the nest. Their decision whether to go out and forage depends not only on the pheromone signal, but also on the quality of the nectar they have sampled. In this way, foraging activity of a bumble bee colony is adjusted to foraging conditions, which means most bees are allocated to foraging only if high-quality food sources are available. In addition, foraging activity is adjusted to the amount of food already stored. In a colony with full honeypots, no new bees are allocated to foraging. These results help us understand how the allocation of workers to the task of food collection is regulated according to external and internal nest conditions in bumble bees.}, subject = {Hummel}, language = {en} } @phdthesis{Krones2022, author = {Krones, David}, title = {The Role of Acid Sphingomyelinase in \(Staphylococcus\) \(aureus\) Infection of Endothelial Cells}, doi = {10.25972/OPUS-29049}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290492}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Staphylococcus aureus is a human bacterial pathogen responsible for a variety of diseases including bacterial pneumonia and sepsis. Recent studies provided an explanation, how S. aureus and its exotoxins contribute to the degradation of endothelial junction proteins and damage lung tissue [4]. Previous findings were indicating an involvement of acid sphingomyelinase (ASM) activity in cell barrier degradation [5]. In the presented study the impact of singular virulence factors, such as staphylococcal α-toxin, on in vitro cell barrier integrity as well as their ability to elicit an activation of ASM were investigated. Experiments with bacterial supernatants performed on human endothelial cells demonstrated a rapid dissociation after treatment, whereas murine endothelial cells were rather resistant against cell barrier degradation. Furthermore, amongst all tested staphylococcal toxins it was found that only α-toxin had a significant impact on endothelial junction proteins and ASM activity. Ablation of this single toxin was sufficient to protect endothelial cells from cell barrier degradation and activation of ASM was absent. In this process it was verified, that α-toxin induces a recruitment of intracellular ASM, which is accompanied by rapid and oscillating changes in cytoplasmic Ca2+ concentration and an increased exposure of Lysosomal associated membrane protein 1 (LAMP1) on the cell surface. Recruitment of lysosomal ASM is associated, among other aspects, to plasma membrane repair and was previously described to be involved with distinct pathogens as well as other pore forming toxins (PFT). However, with these findings a novel feature for α-toxin has been revealed, indicating that the staphylococcal PFT is able to elicit a similar process to previously described plasma membrane repair mechanisms. Increased exposure and intake of surface membrane markers questioned the involvement of ASM activity in S. aureus internalization by non-professional phagocytes such as endothelial cells. By modifying ASM expression pattern as well as application of inhibitors it was possible to reduce the intracellular bacterial count. Thus, a direct connection between ASM activity and S. aureus infection mechanisms was observed, therefore this study exemplifies how S. aureus is able to exploit the host cell sphingolipid metabolism as well as benefit of it for invasion into non-professional phagocytic cells}, subject = {Staphylococcus aureus}, language = {en} } @phdthesis{Froese2008, author = {Froese, Alexander}, title = {The Popeye domain containing gene 2 (Popdc2): Generation and functional characterization of a null mutant in mice and promoter analysis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-26473}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {In the present study a knockout mouse model of the Popeye domain containing gene 2 (Popdc2) was generated and functionally characterized. The Popdc2 null mutants were viable with an apparent normal life span. ß-galactosidase staining to visualize the expression of the Popdc2-LacZ transgene revealed the presence of the Popdc2 in heart, bladder, smooth and skeletal muscles. In the heart LacZ was found to be present in cardiac myocytes with elevated levels in the myocytes of the cardiac conduction system. Holter ECGs records of the heart function of the 8 months (but not in 3 and 6 months) old mutant and WT littermates revealed a pronounced sinus bradycardia in the mutant mice in response to three different stress regimens: isoproterenol infusion, mental stress and a physical exercise. Histological examination of the Popdc2 null mutants SAN revealed structural alterations as was detected by HCN4 staining. Moreover, volume measurements using 3-D reconstructions of serial sections stained with HCN4 antibody revealed a volume reduction of about 30\% in the mutant SAN. Taken together data presented in this study suggest that the Popdc2 KO mouse line may serve as an animal model of human sick sinus syndrome. In the second part of this thesis the Popdc2 gene promoter was analyzed. Three transcription factors binding sites were predicted in the promoter region and characterized.}, subject = {Herz}, language = {en} } @phdthesis{Grue1999, author = {Grue, Pernille}, title = {The physiological role of the two isoforms of DNA topoisomerase II in human cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1369}, school = {Universit{\"a}t W{\"u}rzburg}, year = {1999}, abstract = {Unique functions of DNA topoisomerase IIalpha and IIbeta have been suggested. A human cell line which carries a homozygeous mutation of the nuclear localization sequence of the topoisomerase IIalpha gene expresses the isoform outside the nucleus at the onset of mitosis. At mitosis topoisomerase IIbeta diffused away from the chromatin despite the nuclear lack of the IIalpha-form. Chromosome condensation and disjunction was performed with the aid of cytosolic topoisomerase IIalpha which bound to the mitotic chromatin with low affinity. Consequently an increased rate of nondisjunction is observed in these cells. It is concluded that high affinity chromatin binding of topoisomerase IIalpha is essential for chromosome condensation/disjunction and that topoisomerase IIbeta does not adopt these functions. A centrosomal protein was recognized by topoisomerase IIalpha. This topoisomerase IIalpha-like protein resembles a modified form of topoisomerase IIalpha with an apparent size of 205 kDa compared to 170 kDa. The expression of the protein is constant in all stages of the cell cycle and it appears in proliferating as well as in resting cells. If there is not sufficient topoisomerase IIalpha present at mitosis the centrosomal proteins might adopt the function and a mitotic catastrophe in the cells could therefore be prevented.}, subject = {DNS-Gyrase}, language = {en} } @phdthesis{Kelber2009, author = {Kelber, Christina}, title = {The olfactory system of leafcutting ants: neuroanatomy and the correlation to social organization}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-47769}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {In leaf-cutting ants (genera Atta and Acromyrmex), the worker caste exhibits a pronounced size-polymorphism, and division of labor is largely dependent on worker size (alloethism). Behavioral studies have shown a rich diversity of olfactory-guided behaviors, and the olfactory system seems to be highly developed and very sensitive. To allow fine-tuned behavioral responses to different tasks, adaptations within the olfactory system of different sized workers are expected. In a recent study, two different phenotypes of the antennal lobe of Atta vollenweideri workers were found: MG- and RG-phenotype (with and without a macroglomerulus, MG). The existence of the macroglomerulus is correlated to the body size of workers, with small workers showing the RG-phenotype and large workers showing the MG-phenotype. In the MG, the information about the releaser component of the trail-pheromone is processed. In the first part of my PhD-project, I focus on quantifying behavioral differences between different sized workers in Atta vollenweideri. The study analyzes the trail following behavior; which can be generally performed by all workers. An artificial trail consisting of the releaser component of the trail-pheromone in decreasing concentration was used to test the trail-following performance of individual workers. The trail-following performance of the polymorphic workers is depended of the existence of the MG in the antennal lobe. Workers possessing the MG-phenotype were significantly better in following a decreasing trail then workers showing the RG-phenotype. In the second part I address the question if there are more structural differences, besides the MG, in the olfactory system of different sized workers. Therefore I analyze whether the glomerular numbers are related to worker size. The antennal lobes of small workers contain ~390 glomeruli (low-number; LN-phenotype), and in large workers I found a substantially higher number of ~440 glomeruli (high-number; HN-phenotype). All LN-phenotype workers and some of the small HN-phenotype workers do not possess an MG (LN-RG-phenotype and HN-RG-phenotype) at all, whereas the remaining majority of HN-phenotype workers do possess an MG (HN-MG-phenotype). Mass-stainings of antennal olfactory receptor neurons revealed that the sensory tracts divide the antennal lobe into six clusters of glomeruli (T1-T6). In the T4-cluster ~50 glomeruli are missing in the LN-phenotype workers. Selective staining of single sensilla and their associated receptor neurons showed that T4-glomeruli are innervated by receptor neurons from the main type of olfactory sensilla, the Sensilla trichodea curvata which are also projecting to glomeruli in all other clusters. The other type of olfactory sensilla, the Sensilla basiconica, exclusively innervates T6-glomeruli. Quantitative analyses revealed a correlation between the number of Sensilla basiconica and the volume of T6 glomeruli in different sized workers. The results of both behavioral and neuroanatomical studies in Atta vollenweideri suggest that developmental plasticity of antennal-lobe phenotypes promotes differences in olfactory-guided behavior which may underlie task specialization within ant colonies. The last part of my project focuses on the evolutionary origin of the macroglomerulus and the number of glomeruli in the antennal lobe. I compared the number, volumes and position of the glomeruli of the antennal lobe of 25 different species from all three major Attini groups (lower, higher and leaf-cutting Attini). The antennal lobes of all investigated Attini comprise a high number of glomeruli (257-630). The highest number was found in Apterostigma cf. mayri. This species is at a basal position within the Attini phylogeny, and a high number of glomeruli might have been advantageous in the evolution of the advanced olfactory systems of this Taxa. The macroglomerulus can be found in all investigated leaf-cutting Attini, but in none of the lower and higher Attini species. It is found only in large workers, and is located close to the entrance of the antennal nerve in all investigated species. The results indicate that the presence of a macroglomerulus in large workers of leaf-cutting Attini is a derived overexpression of a trait in the polymorphic leaf-cutting species. It presumably represents an olfactory adaptation to elaborate foraging and mass recruitment systems, and adds to the complexity of division of labor and social organization known for this group.}, subject = {Gehirn}, language = {en} } @phdthesis{Cook2012, author = {Cook, Mandy}, title = {The neurodegenerative Drosophila melanogaster AMPK mutant loechrig}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72027}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {In dieser Doktorarbeit wird die Drosophila Mutante loechrig (loe), die progressive Degeneration des Nervensystems aufweist, weiter beschrieben. In der loe Mutante fehlt eine neuronale Isoform der γ- Untereinheit der Proteinkinase AMPK (AMP-activated protein kinase). Die heterotrimere AMPK (auch als SNF4Aγ bekannt) kontrolliert das Energieniveau der Zelle, was st{\"a}ndiges Beobachten des ATP/AMP- Verh{\"a}ltnis erfordert. AMPK wird durch niedrige Energiekonzentrationen und Beeintr{\"a}chtigungen im Metabolismus, wie zum Beispiel Sauerstoffmangel, aktiviert und reguliert mehrere wichtige Signaltransduktionswege, die den Zellmetabolismus kontrollieren. Jedoch ist die Rolle von AMPK im neuronalen {\"U}berleben noch unklar. Eines der Proteine, dass von AMPK reguliert wird, ist HMGR (hydroxymethylglutaryl-CoA- reductase), ein Schl{\"u}sselenzym in der Cholesterin- und Isoprenoidsynthese. Es wurde gezeigt, dass wenn die Konzentration von HMGR manipuliert wird, auch der Schweregrad des neurodegenerativen Ph{\"a}notyps in loe beeinflusst wird. Obwohl die regulatorische Rolle von AMPK auf HMGR in Drosophila konserviert ist, k{\"o}nnen Insekten Cholesterin nicht de novo synthetisieren. Dennoch ist der Syntheseweg von Isoprenoiden zwischen Vertebraten und Insekten evolution{\"a}r konserviert. Isoprenylierung von Proteinen, wie zum Beispiel von kleinen G-Proteinen, stellt den Proteinen einen hydophobischen Anker bereit, mit denen sie sich an die Zellmembran binden k{\"o}nnen, was in anschließender Aktivierung resultieren kann. In dieser Doktorarbeit wird gezeigt, dass die loe Mutation die Prenylierung von Rho1 und den LIM-Kinasesignalweg beeinflusst, was eine wichtige Rolle im Umsatz von Aktin und axonalem Auswachsen spielt. Die Ergebnisse weisen darauf hin, dass die Mutation in LOE, Hyperaktivit{\"a}t des Isoprenoidsynthesewegs verursacht, was zur erh{\"o}hten Farnesylierung von Rho1 und einer dementsprechend h{\"o}heren Konzentration von Phospho- Cofilin f{\"u}hrt. Eine Mutation in Rho1 verbessert den neurodegenerativen Ph{\"a}notyp und die Lebenserwartung von loe. Der Anstieg vom inaktiven Cofilin in loe f{\"u}hrt zu einer Zunahme von filament{\"o}sen Aktin. Aktin ist am Auswachen von Neuronen beteiligt und Experimente in denen loe Neurone analysiert wurden, gaben wertvolle Einblicke in eine m{\"o}gliche Rolle die AMPK, und dementsprechend Aktin, im Neuronenwachstum spielt. Des Weiteren wurde demonstriert, dass Neurone, die von der loe Mutante stamen, einen verlangsamten axonalen Transport aufweisen, was darauf hinweist dass Ver{\"a}nderungen, die durch den Einfluss von loe auf den Rho1 Signalweg im Zytoskelettnetzwerk hervorgerufen wurden, zur St{\"o}rung des axonalen Transports und anschließenden neuronalen Tod f{\"u}hren. Es zeigte außerdem, dass Aktin nicht nur am neuronalen Auswachsen beteiligt ist, sondern auch wichtig f{\"u}r die Aufrechterhaltung von Neuronen ist. Das bedeutet, dass {\"A}nderungen der Aktindynamik zur progressiven Degeneration von Neuronen f{\"u}hren kann. Zusammenfassend unterstreichen diese Ergebnisse die wichtige Bedeutung von AMPK in den Funktionen und im {\"U}berleben von Neuronen und er{\"o}ffnen einen neuartigen funktionellen Mechanismus in dem {\"A}nderungen in AMPK neuronale Degeneration hervorrufen kann.}, subject = {Taufliege}, language = {en} } @phdthesis{Paul2001, author = {Paul, J{\"u}rgen}, title = {The Mouthparts of Ants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-1179130}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2001}, abstract = {Ant mandible movements cover a wide range of forces, velocities and precision. The key to the versatility of mandible functions is the mandible closer muscle. In ants, this muscle is generally composed of distinct muscle fiber types that differ in morphology and contractile properties. Volume proportions of the fiber types are species-specific and correlate with feeding habits. Two biomechanical models explain how the attachment angles are optimized with respect to force and velocity output and how filament-attached fibers help to generate the largest force output from the available head capsule volume. In general, the entire mandible closer muscle is controlled by 10-12 motor neurons, some of which exclusively supply specific muscle fiber groups. Simultaneous recordings of muscle activity and mandible movement reveal that fast movements require rapid contractions of fast muscle fibers. Slow and accurate movements result from the activation of slow muscle fibers. Forceful movements are generated by simultaneous co-activation of all muscle fiber types. For fine control, distinct fiber bundles can be activated independently of each other. Retrograde tracing shows that most dendritic arborizations of the different sets of motor neurons share the same neuropil in the suboesophageal ganglion. In addition, some motor neurons invade specific parts of the neuropil. The labiomaxillary complex of ants is essential for food intake. I investigated the anatomical design of the labiomaxillary complex in various ant species focusing on movement mechanisms. The protraction of the glossa is a non muscular movement. Upon relaxation of the glossa retractor muscles, the glossa protracts elastically. I compared the design of the labiomaxillary complex of ants with that of the honey bee, and suggest an elastic mechanism for glossa protraction in honey bees as well. Ants employ two different techniques for liquid food intake, in which the glossa works either as a passive duct (sucking), or as an up- and downwards moving shovel (licking). For collecting fluids at ad libitum food sources, workers of a given species always use only one of both techniques. The species-specific feeding technique depends on the existence of a well developed crop and on the resulting mode of transporting the fluid food. In order to evaluate the performance of collecting liquids during foraging, I measured fluid intake rates of four ant species adapted to different ecological niches. Fluid intake rate depends on sugar concentration and the associated fluid viscosity, on the species-specific feeding technique, and on the extent of specialization on collecting liquid food. Furthermore, I compared the four ant species in terms of glossa surface characteristics and relative volumes of the muscles that control licking and sucking. Both probably reflect adaptations to the species-specific ecological niche and determine the physiological performance of liquid feeding. Despite species-specific differences, single components of the whole system are closely adjusted to each other according to a general rule.}, subject = {Ameisen}, language = {en} } @phdthesis{Schuecker2018, author = {Sch{\"u}cker, Katharina}, title = {The molecular architecture of the meiotic chromosome axis as revealed by super-resolution microscopy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144199}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {During meiosis proteins of the chromosome axis are important for monitoring chromatin structure and condensation, for pairing and segregation of chromosomes, as well as for accurate recombination. They include HORMA-domain proteins, proteins of the DNA repair system, synaptonemal complex (SC) proteins, condensins and cohesins. To understand more about their function in shaping the meiotic chromosome it is crucial to establish a defined model of their molecular architecture. Up to now their molecular organization was analysed using conventional methods, like confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). Unfortunately, these techniques are limited either by their resolution power or their localization accuracy. In conclusion, a lot of data on the molecular organization of chromosome axis proteins stays elusive. For this thesis the molecular structure of the murine synaptonemal complex (SC) and the localization of its proteins as well as of three cohesins was analysed with isotropic resolution, providing new insights into their architecture and topography on a nanoscale level. This was done using immunofluorescence labelling in combination with super-resolution microscopy, line profiles and average position determination. The results show that the murine SC has a width of 221.6 nm ± 6.1 nm including a central region (CR) of 148.2 nm ± 2.6 nm. In the CR a multi-layered organization of the central element (CE) proteins was verified by measuring their strand diameters and strand distances and additionally by imaging potential anchoring sites of SYCP1 (synaptonemal complex protein 1) to the lateral elements (LEs). We were able to show that the two LEs proteins SYCP2 and SYCP3 do co-localize alongside their axis and that there is no significant preferential localization towards the inner LE axis of SYCP2. The presented results also predict an orderly organization of murine cohesin complexes (CCs) alongside the chromosome axis in germ cells and support the hypothesis that cohesins in the CR of the SC function independent of CCs. In the end new information on the molecular organization of two main components of the murine chromosome axis were retrieved with nanometer precision and previously unknown details of their molecular architecture and topography were unravelled.}, subject = {Meiose}, language = {en} } @phdthesis{Beck2005, author = {Beck, Jan}, title = {The macroecology of Southeast-Asian hawkmoths (Lepidoptera: Sphingidae)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-13001}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {This study investigates the abundance and geographic distribution of the hawkmoth species (Lepidoptera: Sphingidae) of Southeast-Asia and analyses the resulting patterns of biodiversity, biogeography and macroecology. Data on the distribution of species were retrieved from published and unpublished faunal lists and museum collections (in close cooperation with the Natural History Museum, London). Over 34,500 records of the global distribution of the 380 species that occur in Southeast-Asia (including New Guinea and the Solomon Islands) were used for a GIS-supported estimate of distributional ranges, which can be accessed at http://www.sphingidae-sea.biozentrum.uni-wuerzburg.de, an Internet site that also provides pictures of the species and checklists for 114 islands of the Malesian region. The abundance of species in local assemblages was assessed from nightly collections at artificial light sources. Using a compilation of own samples as well as published and unpublished data from other sources, local abundance data on 93 sites were used for analysis, covering 159 species or 17,676 specimens.}, language = {en} } @phdthesis{Saverschek2010, author = {Saverschek, Nicole}, title = {The influence of the symbiotic fungus on foraging decisions in leaf-cutting ants - Individual behavior and collective patterns}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-52087}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Foraging behavior is a particularly fascinating topic within the studies of social insects. Decisions made by individuals have effects not only on the individual level, but on the colony level as well. Social information available through foraging in a group modulates individual preferences and shapes the foraging pattern of a colony. Identifying parameters influencing foraging behavior in leaf-cutting ants is especially intriguing because they do not harvest for themselves, but for their symbiotic fungus which in turn influences their plant preferences after the incorporation of the substrate. To learn about the substrates' unsuitability for the fungus, ants need to be able to identify the incorporated substrate and associate it with detrimental effects on the fungus. Odor is an important plant characteristic known to be used as recognition key outside the nest in the context of foraging. Chapter 1 shows that foragers are able to recall information about the unsuitability of a substrate through odor alone and consequently reject the substrate, which leads to the conclusion that inside the nest, odor might be enough to indentify incorporated substrate. Identification of plant species is a key factor in the foraging success of leaf-cutting ants as they harvest a multitude of different plant species in a diverse environment and host plant availability and suitability changes throughout the year. Fixed plant preferences of individuals through innate tendencies are therefore only one factor influencing foraging decisions. On the individual as well as the colony level, foraging patterns are flexible and a result of an intricate interplay between the different members involved in the harvesting process: foragers, gardeners and the symbiotic fungus. In chapter 2 I identified several conditions necessary for na{\"i}ve foragers to learn about the unsuitability of substrate inside the nest. In order to exchange of information about the unsuitability of a substrate, the plant in question must be present in the fungus garden. Foragers can learn without own foraging experience and even without experiencing the effects of the substrate on the fungus, solely through the presence of experienced gardeners. The presence of experienced foragers alone on the other hand is not enough to lower the acceptance of substrate by na{\"i}ve foragers in the presence of na{\"i}ve gardeners, even if experienced foragers make up the majority of the workforce inside the nest. Experienced foragers are also able to reverse their previous negative experience and start accepting the substrate again. The individual behavior of foragers and gardeners with different experiential backgrounds in the presence of suitable or unsuitable substrate inside the fungus chamber was investigated in chapter 3 to shed some light on possible mechanisms involved in the flow of information about substrate suitability from the fungus to the ants. Gardeners as well as foragers are involved in the leaf processing and treatment of the applied leaf patches on the fungus. If the plant material is unsuitable, significantly more ants treat the plant patches, but foragers are less active overall. Contacts between workers initiated by either gardeners or foragers occur significantly more frequent and last longer if the substrate is unsuitable. Even though experienced gardeners increase na{\"i}ve foragers' contact rates and duration with other workers in the presence of suitable plant patches, na{\"i}ve foragers show no differences in the handling of the plant patches. This suggests that foragers gain information about plant suitability not only indirectly through the gardening workers, but might also be able to directly evaluate the effects of the substrate on the fungus themselves. Outside the nest, foragers influence each other the trail (chapter 4). Foraging in a group and the presence of social information is a decisive factor in the substrate choice of the individual and leads to a distinct and consentaneous colony response when encountering unfamiliar or unsuitable substrates. As leaf-cutting ants harvest different plant species simultaneously on several trails, foragers gain individual experiences concerning potential host plants. Preferences might vary among individuals of the same colony to the degree that foragers on the same trail perceive a certain substrate as either suitable or unsuitable. If the majority of foragers on the trail perceives one of the currently harvested substrates as unsuitable, na{\"i}ve foragers lower their acceptance within 4 hours. In the absence of a cue in the fungus, na{\"i}ve foragers harvesting by themselves still eventually (within 6 hours) reject the substrate as they encounter experienced gardeners during visits to the nest within foraging bouts. As foraging trails can be up to 100 m long and foragers spend a considerable amount of time away from the nest, learning indirectly from experienced foragers on the trail accelerates the distribution of information about substrate suitability. The level of rejection of a formerly unsuitable substrate after eight hours of foraging by na{\"i}ve foragers correlates with the average percentage of unladen experienced foragers active on the trail. This suggests that unladen experienced foragers might actively contact laden na{\"i}ve workers transmitting information about the unsuitability of the load they carry. Results from experiments were I observed individual laden foragers on their way back to the nest backed up this assumption as individuals were antennated and received bites into the leaf disk they carried. Individuals were contacted significantly more often by nestmates that perceived the carried leaf disk as unsuitable due to previous experience than by nestmates without this experience (chapter 6). Leaf-cutting ants constantly evaluate, learn and re-evaluate the suitability of harvested substrate and adjust their foraging activity accordingly. The importance of the different sources of information within the colony and their effect on the foraging pattern of the colony depend on the presence or absence of each of them as e.g. experienced foragers have a bigger influence on the plant preferences of na{\"i}ve foragers in the absence of a cue in the fungus garden.}, subject = {Blattschneiderameisen}, language = {en} } @phdthesis{Boetzl2022, author = {B{\"o}tzl, Fabian Alexander}, title = {The influence of crop management and adjacent agri-environmental scheme type on natural pest control in differently structured landscapes}, doi = {10.25972/OPUS-24140}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-241400}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Summary Chapters I \& II: General Introduction \& General Methods Agriculture is confronted with a rampant loss of biodiversity potentially eroding ecosystem service potentials and adding up to other stressors like climate change or the consequences of land-use change and intensive management. To counter this 'biodiversity crisis', agri-environment schemes (AES) have been introduced as part of ecological intensification efforts. These AES combine special management regimes with the establishment of tailored habitats to create refuges for biodiversity in agricultural landscapes and thus ensure biodiversity mediated ecosystem services such as pest control. However, little is known about how well different AES habitats fulfil this purpose and whether they benefit ecosystem services in adjacent crop fields. Here I investigated how effective different AES habitats are for restoring biodiversity in different agricultural landscapes (Chapter V) and whether they benefit natural pest control in adjacent oilseed rape (Chapter VI) and winter cereal fields (Chapter VII). I recorded biodiversity and pest control potentials using a variety of different methods (Chapters II, V, VI \& VII). Moreover, I validated the methodology I used to assess predator assemblages and predation rates (Chapters III \& IV). Chapter III: How to record ground dwelling predators? Testing methodology is critical as it ensures scientific standards and trustworthy results. Pitfall traps are widely used to record ground dwelling predators, but little is known about how different trap types affect catches. I compared different types of pitfall traps that had been used in previous studies in respect to resulting carabid beetle assemblages. While barrier traps collected more species and deliver more complete species inventories, conventional simple pitfall traps provide reliable results with comparatively little handling effort. Placing several simple pitfall traps in the field can compensate the difference while still saving handling effort.   Chapter IV: How to record predation rates? A plethora of methods has been proposed and used for recording predation rates, but these have rarely been validated before use. I assessed whether a novel approach to record predation, the use of sentinel prey cards with glued on aphids, delivers realistic results. I compared different sampling efforts and showed that obtained predation rates were similar and could be linked to predator (carabid beetle) densities and body-sizes (a proxy often used for food intake rates). Thus, the method delivers reliable and meaningful predation rates. Chapter V: Do AES habitats benefit multi-taxa biodiversity? The main goal of AES is the conservation of biodiversity in agricultural landscapes. I investigated how effectively AES habitats with different temporal continuity fulfil this goal in differently structured landscapes. The different AES habitats investigated had variable effects on local biodiversity. Temporal continuity of AES habitats was the most important predictor with older, more temporally continuous habitats harbouring higher overall biodiversity and different species assemblages in most taxonomic groups than younger AES habitats. Results however varied among taxonomic groups and natural enemies were equally supported by younger habitats. Semi-natural habitats in the surrounding landscape and AES habitat size were of minor importance for local biodiversity and had limited effects. This stresses that newly established AES habitats alone cannot restore farmland biodiversity. Both AES habitats as well as more continuous semi-natural habitats synergistically increase overall biodiversity in agricultural landscapes. Chapter VI: The effects of AES habitats on predators in adjacent oilseed rape fields Apart from biodiversity conservation, ensuring ecosystem service delivery in agricultural landscapes is a crucial goal of AES. I therefore investigated the effects of adjacent AES habitats on ground dwelling predator assemblages in oilseed rape fields. I found clear distance decay effects from the field edges into the field centres on both richness and densities of ground dwelling predators. Direct effects of adjacent AES habitats on assemblages in oilseed rape fields however were limited and only visible in functional traits of carabid beetle assemblages. Adjacent AES habitats doubled the proportion of predatory carabid beetles indicating a beneficial role for pest control. My results show that pest control potentials are largest close to the field edges and beneficial effects are comparably short ranged. Chapter VII: The effects of AES habitats on pest control in adjacent cereal fields Whether distance functions and potential effects of AES habitats are universal across crops is unknown. Therefore, I assessed distance functions of predators, pests, predation rates and yields after crop rotation in winter cereals using the same study design as in the previous year. Resulting distance functions were not uniform and differed from those found in oilseed rape in the previous year, indicating that the interactions between certain adjacent habitats vary with habitat and crop types. Distance functions of cereal-leaf beetles (important cereal pests) and parasitoid wasps were moreover modulated by semi-natural habitat proportion in the surrounding landscapes. Field edges buffered assemblage changes in carabid beetle assemblages over crop rotation confirming their important function as refuges for natural enemies. My results emphasize the beneficial role of field edges for pest control potentials. These findings back the calls for smaller field sizes and more diverse, more heterogeneously structured agricultural landscapes. Chapter VIII: General Discussion Countering biodiversity loss and ensuring ecosystem service provision in agricultural landscapes is intricate and requires strategic planning and restructuring of these landscapes. I showed that agricultural landscapes could benefit maximally from (i) a mixture of AES habitats and semi-natural habitats to support high levels of overall biodiversity and from (ii) smaller continuously managed agricultural areas (i.e. smaller field sizes or the insertion of AES elements within large fields) to maximize natural pest control potentials in crop fields. I propose a mosaic of younger AES habitats and semi-natural habitats to support ecosystem service providers and increase edge density for ecosystem service spillover into adjacent crops. The optimal extent and density of this network as well as the location in which AES and semi-natural habitats interact most beneficially with adjacent crops need further investigation. My results provide a further step towards more sustainable agricultural landscapes that simultaneously allow biodiversity to persist and maintain agricultural production under the framework of ecological intensification.}, subject = {{\"O}kologie}, language = {en} } @phdthesis{Eck2016, author = {Eck, Saskia}, title = {The impact of thermogenetic depolarizations of specific clock neurons on Drosophila melanogaster's circadian clock}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137118}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms' behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly's circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock's interneuronal communication. Drosophila melanogaster's circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly's locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature. Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs). The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs \& l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day. To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner. However, the molecular link between clock neuron depolarization and shifts in the molecular clock's cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock. Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.}, subject = {Chronobiologie}, language = {en} } @phdthesis{HornneeBunz2020, author = {Horn [n{\´e}e Bunz], Melanie}, title = {The impact of Drosophila melanogaster`s endogenous clock on fitness: Influence of day length, humidity and food composition}, doi = {10.25972/OPUS-21141}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211415}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {We are living in a system that underlies permanent environmental changes due to the rotation of our planet. These changes are rhythmic with the most prominent one having a period of about 24 hours, but also shorter and longer rhythms characterize our environment. To cope with the ever-changing environmental conditions, it is thought to be beneficial if an organism can track and anticipate these changes. The so called endogenous clocks enable this and might provide a fitness advantage. To investigate and unravel the mechanism of endogenous clocks Chronobiologists have used different model organisms. In this thesis Drosophila melanogaster was used as model organism with its about 150 clock neurons representing the main endogenous clock of the fly in the central brain. The molecular mechanisms and the interlocked feedback loops with the main circadian key players like period, timeless, clock or cycle are under investigation since the 1970s and are characterized quite well so far. But the impact of a functional endogenous clock in combination with diverse factors and the resulting fitness advantages were analysed in only a few studies and remains for the most part unknown. Therefore the aim of this thesis was to unravel the impact of Drosophila melanogaster`s endogenous clock on the fitness of the fly. To achieve this goal different factors - like day length, humidity and food composition - were analyzed in wild type CS and three different period mutants, namely perL, perS and per01, that carry a point mutation altering or abolishing the free-running period of the fruit fly as well as a second arrhythmic strain, clkAR. In competition assay experiments wild type and clock mutant flies competed for up to 63 generations under a normal 24 hour rhythm with 12 hours light/day and 12 hours darkness/night (LD12:12) or T-cycles with 19 or 29 hours, according to the mutants free-running period, or constant light (LL) in case of the arrhythmic mutant as well as under natural-like outdoor conditions in two consecutive years. Overall the wild type CS strain was outcompeting the clock mutant strains independent of the environmental conditions. As the perL fly strain elongated their free-running period, the competition experiments were repeated with naturally cantonized new fly strains. With these experiments it could be shown that the genetic background of the fly strains - which are kept for decades in the lab, with backcrosses every few years - is very important and influences the fitness of flies. But also the day length impacts the fitness of the flies, enabling them to persist in higher percentage in a population under competition. Further factors that might influence the survival in a competing population were investigated, like e.g. mating preferences and locomotor activity of homo- and heterozygous females or sperm number of males transferred per mating. But these factors can still not explain the results in total and play no or only minor roles and show the complexity of the whole system with still unknown characteristics. Furthermore populations of flies were recorded to see if the flies exhibit a common locomotor activity pattern or not and indeed a population activity pattern could be recorded for the first time and social contact as a Zeitgeber could be verified for Drosophila melanogaster. In addition humidity and its impact on the flies´ fitness as well as a potential Zeitgeber was examined in this thesis. The flies experienced different relative humidities for eclosion and wing expansion and humidity cycle phase shifting experiments were performed to address these two different questions of fitness impact and potential Zeitgeber. The fruit fly usually ecloses in the morning hours when the relative humidity is quite high and the general assumption was that they do so to prevent desiccation. The results of this thesis were quite clear and demonstrate that the relative humidity has no great effect on the fitness of the flies according to successful eclosion or wing expansion and that temperature might be the more important factor. In the humidity cycle phase shifting experiments it could be revealed that relative humidity cannot act as a Zeitgeber for Drosophila melanogaster, but it influences and therefore masks the activity of flies by allowing or surpressing activity at specific relative humidity values. As final experiments the lifespan of wild type and clock mutant flies was investigated under different day length and with different food qualities to unravel the impact of these factors on the fitness and therefore survival of the flies on the long run. As expected the flies with nutrient-poor minimum medium died earlier than on the nutrient-rich maximum medium, but a small effect of day length could also be seen with flies living slightly longer when they experience environmental day length conditions resembling their free-running period. The experiments also showed a fitness advantage of the wild type fly strain against the clock mutant strains for long term, but not short term (about the first 2-3 weeks). As a conclusion it can be said that genetic variation is important to be able to adapt to changing environmental conditions and to optimize fitness and therefore survival. Having a functional endogenous clock with a free-running period of about 24 hours provides fitness advantages for the fruit fly, at least under competition. The whole system is very complex and many factors - known and unknown ones - play a role in this system by interacting on different levels, e.g. physiology, metabolism and/or behavior.}, subject = {Taufliege}, language = {en} } @phdthesis{Seida2012, author = {Seida, Ahmed Adel}, title = {The Immunomodulatory Role of Endogenous Glucocorticoids in Ovarian Cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73901}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Ovarian cancer currently causes ~6,000 deaths per year in Germany alone. Since only palliative treatment is available for ovarian carcinomas that have developed resistance against platinum-based chemotherapy and paclitaxel, there is a pressing medical need for the development of new therapeutic approaches. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. The research of our group thus aims at overcoming tumour immune escape by counteracting immunosuppressive mechanisms in the tumour microenvironment. In this context, we found that tumour-infiltrating myeloid-derived suppressor cells (MDSC) or tumour associated macrophages (TAM) which are abundant in ovarian cancer express high levels of the enzyme 11β-hydroxysteroid dehydrogenase1 (11-HSD1). This oxido-reductase enzyme is essential for the conversion of biologically inactive cortisone into active cortisol. In line with this observation, high endogenous cortisol levels could be detected in serum, ascitic fluid and tumour exudates from ovarian cancer patients. Considering that cortisol exerts strong anti-inflammatory and immunosuppressive effects on immune cells, it appears likely that high endogenous cortisol levels contribute to immune escape in ovarian cancer. We thus hypothesised that local activation of endogenous glucocorticoids could suppress beneficial immune responses in the tumour microenvironment and thereby prevent a successful immunotherapy. To investigate the in vivo relevance of this postulated immune escape mechanism, irradiated PTENloxP/loxP loxP-Stop-loxP-krasG12D mice were reconstituted with hematopoietic stem cells from either glucocorticoid receptor (GR) expressing mice (GRloxP/loxP) or from mice with a T cell-specific glucocorticoid receptor knock-out (lck-Cre GRloxP/loxP) mice. In the host mice, the combination of a conditional PTEN knock-out with a latent oncogenic kras leads to tumour development when a Cre-encoding adenovirus is injected into the ovarian bursa. Using this model, mice that had been reconstituted with GC-insensitive T cells showed better intratumoural T cell infiltration than control mice that had received functionally unaltered GRloxP/loxP cells via adoptive transfer. However, tumour-infiltrating T cells mostly assumed a Foxp3+ (regulatory) phenotype and survival was even shortened in mice with cortisol-insensitive T cells. Thus, endogenous cortisol seems to inhibit immune cell infiltration in ovarian cancer, but productive anti-tumour immune responses might still be prevented by further factors from the tumour microenvironment. Thus, our data did not provide a sufficiently strong rationale to further pursue the antagonisation of glucocorticoid signalling in ovarian cancer patients, Moreover, glucocorticoids are frequently administered to cancer patients to reduce inflammation and swelling and to prevent chemotherapy-related toxic side effects like nausea or hypersensitivity reactions associated with paclitaxel therapy. Thus, we decided to address the question whether specific signalling pathways in innate immune cells, preferentially in NK cells, could still be activated even in the presence of GC. A careful investigation of the various activating NK cell receptors (i.e. NKp30, NKp44, NKp46), DNAM-1 and NKG2D) was thus performed which revealed that NKp30, NKp44 and NKG2D are all down-regulated by cortisol whereas NKp46 is actually induced by cortisol. Interestingly, NKp46 is the only known receptor that is strictly confined to NK cells. Its activation via crosslinking leads to cytokine release and activation of cytotoxic activity. Stimulation of NK cells via NKp46 may contribute to immune-mediated tumour destruction by triggering the lysis of tumour cells and by altering the cytokine pattern in the tumour microenvironment, thereby generating more favourable conditions for the recruitment of antigen-specific immune cells. Accordingly, our observation that even cortisol-treated NK cells can still be activated via NKp46 and CD2 might become valuable for the design of immunotherapies that can still be applied in the presence of endogenous or therapeutically administered glucocorticoids.}, subject = {Cortison}, language = {en} } @phdthesis{Kupper2016, author = {Kupper, Maria}, title = {The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142534}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {The evolutionary success of insects is believed to be at least partially facilitated by symbioses between insects and prokaryotes. Bacterial endosymbionts confer various fitness advantages to their hosts, for example by providing nutrients lacking from the insects' diet thereby enabling the inhabitation of new ecological niches. The Florida carpenter ant Camponotus floridanus harbours endosymbiotic bacteria of the genus Blochmannia. These primary endosymbionts mainly reside in the cytoplasm of bacteriocytes, specialised cells interspersed into the midgut tissue, but they were also found in oocytes which allows their vertical transmission. The social lifestyle of C. floridanus may facilitate the rapid spread of infections amongst genetically closely related animals living in huge colonies. Therefore, the ants require an immune system to efficiently combat infections while maintaining a "chronic" infection with their endosymbionts. In order to investigate the immune repertoire of the ants, the Illumina sequencing method was used. The previously published genome sequence of C. floridanus was functionally re-annotated and 0.53\% of C. floridanus proteins were assigned to the gene ontology (GO) term subcategory "immune system process". Based on homology analyses, genes encoding 510 proteins with possible immune function were identified. These genes are involved in microbial recognition and immune signalling pathways but also in cellular defence mechanisms, such as phagocytosis and melanisation. The components of the major signalling pathways appear to be highly conserved and the analysis revealed an overall broad immune repertoire of the ants though the number of identified genes encoding pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs) is comparatively low. Besides three genes coding for homologs of thioester-containing proteins (TEPs), which have been shown to act as opsonins promoting phagocytosis in other insects, six genes encoding the AMPs defesin-1 and defensin-2, hymenoptaecin, two tachystatin-like peptides and one crustin-like peptide are present in the ant genome. Although the low number of known AMPs in comparison to 13 AMPs in the honey bee Apis mellifera and 46 AMPs in the wasp Nasonia vitripennis may indicate a less potent immune system, measures summarised as external or social immunity may enhance the immune repertoire of C. floridanus, as it was discussed for other social insects. Also, the hymenoptaecin multipeptide precursor protein may be processed to yield seven possibly bioactive peptides. In this work, two hymenoptaecin derived peptides were heterologously expressed and purified. The preliminary antimicrobial activity assays indicate varying bacteriostatic effects of different hymenoptaecin derived peptides against Escherichia coli D31 and Staphylococcus aureus which suggests a functional amplification of the immune response further increasing the antimicrobial potency of the ants. Furthermore, 257 genes were differentially expressed upon immune challenge of C. floridanus and most of the immune genes showing differential expression are involved in recognition of microbes or encode immune effectors rather than signalling components. Additionally, genes coding for proteins involved in storage and metabolism were downregulated upon immune challenge suggesting a trade-off between two energy-intensive processes in order to enhance effectiveness of the immune response. The analysis of gene expression via qRT-PCR was used for validation of the transcriptome data and revealed stage-specific immune gene regulation. Though the same tendencies of regulation were observed in larvae and adults, expression of several immune-related genes was generally more strongly induced in larvae. Immune gene expression levels depending on the developmental stage of C. floridanus are in agreement with observations in other insects and might suggest that animals from different stages revert to individual combinations of external and internal immunity upon infection. The haemolymph proteome of immune-challenged ants further established the immune-relevance of several proteins involved in classical immune signalling pathways, e.g. PRRs, extracellularly active proteases of the Toll signalling pathway and effector molecules such as AMPs, lysozymes and TEPs. Additionally, non-canonical proteins with putative immune function were enriched in immune-challenged haemolymph, e.g. Vitellogenins, NPC2-like proteins and Hemocytin. As known from previous studies, septic wounding also leads to the upregulation of genes involved in stress responses. In the haemolymph, proteins implicated in protein stabilisation and in the protection against oxidative stress and insecticides were enriched upon immune challenge. In order to identify additional putative immune effectors, haemolymph peptide samples from immune-challenged larvae and adults were analysed. The analysis in this work focussed on the identification of putative peptides produced via the secretory pathway as previously described for neuropeptides of C. floridanus. 567 regulated peptides derived from 39 proteins were identified in the larval haemolymph, whereas 342 regulated peptides derived from 13 proteins were found in the adult haemolymph. Most of the peptides are derived from hymenoptaecin or from putative uncharacterised proteins. One haemolymph peptide of immune-challenged larvae comprises the complete amino acid sequence of a predicted peptide derived from a Vitellogenin. Though the identified peptide lacks similarities to any known immune-related peptide, it is a suitable candidate for further functional analysis. To establish a stable infection with the endosymbionts, the bacteria have to be transmitted to the next generation of the ants. The vertical transmission of B. floridanus is guaranteed by bacterial infestation of oocytes. This work presents the first comprehensive and detailed description of the localisation of the bacterial endosymbionts in C. floridanus ovaries during oogenesis. Whereas the most apical part of the germarium, which contains the germ-line stem cells, is not infected by the bacteria, small somatic cells in the outer layers of each ovariole were found to be infected in the lower germarium. Only with the beginning of cystocyte differentiation, endosymbionts are exclusively transported from follicle cells into the growing oocytes, while nurse cells were never infected with B. floridanus. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. A previous study suggested a down-modulation of the immune response in the midgut tissue which may promote endosymbiont tolerance. Therefore, the expression of several potentially relevant immune genes was analysed in the ovarial tissue by qRT-PCR. The relatively low expression of genes involved in Toll and IMD signalling, and the high expression of genes encoding negative immune regulators, such as PGRP-LB, PGRP-SC2, and tollip, strongly suggest that a down-modulation of the immune response may also facilitate endosymbiont tolerance in the ovaries and thereby contribute to their vertical transmission. Overall, the present thesis improves the knowledge about the immune repertoire of C. floridanus and provides new candidates for further functional analyses. Moreover, the involvement of the host immune system in maintaining a "chronic" infection with symbiotic bacteria was confirmed and extended to the ovaries.}, subject = {Camponotus floridanus}, language = {en} } @phdthesis{Jordan2001, author = {Jordan, Bruce}, title = {The identification of NRAGE}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-1180090}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2001}, abstract = {The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and signalling pathways in order to fulfil their anti-apoptotic role. In order to investigate in detail how the avian homologue ITA interfered with both TNF induced apoptosis and the NGF mediated differentiation in PC12 cells, a two hybrid screen was performed with a PC12 library using ITA as a bait. The screen resulted in the identification of several overlapping fragments of a previously unknown gene. The complete cDNA for this gene was isolated, the analysis of which revealed a high homology with a large family of tumour antigens known as MAGE (melanoma associated antigens). This newly identified member of the MAGE family, which was later named NRAGE, exhibited some unique characteristics that suggested for the first time a role in normal cellular physiology for this protein family. MAGE proteins are usually restricted in their expression to malignant or tumour cells, however NRAGE was also expressed in terminally differentiated adult tissue. NRAGE also interacted with the human XIAP in direct two-hybrid tests. The interactions observed in yeast cells were confirmed in mammalian cell culture, employing both coimmunoprecipitation and mammalian two-hybrid methods. Moreover, the results of the coimmunoprecipitation experiments indicated that this interaction requires the RING domain. The widely studied 32D cell system was chosen to investigate the effect of NRAGE on apoptosis. NRAGE was stably transduced in 32D cells, and found to augment cell death induced by the withdrawal of Interleukin-3. One reason for this reduced cell viability in NRAGE expressing cells could be the binding of endogenous XIAP, which occurred inducibly after growth factor withdrawal. Interestingly, NRAGE was able to overcome the protection afforded to 32D cells by the exogenous expression of human Bcl-2. Thus NRAGE was identified during this research doctorate as a novel pro-apoptotic, IAP-interacting protein, able to accelerate apoptosis in a pathway independent of Bcl-2 cell protection.}, subject = {Apoptosis}, language = {en} } @phdthesis{Pinkert2008, author = {Pinkert, Stefan}, title = {The human proteome is shaped by evolution and interactions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-35566}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Das menschliche Genom ist seit 2001 komplett sequenziert. Ein Großteil der Proteine wurde mittlerweile beschrieben und t{\"a}glich werden bioinformatische Vorhersagen praktisch best{\"a}tigt. Als weiteres Großprojekt wurde k{\"u}rzlich die Sequenzierung des Genoms von 1000 Menschen gestartet. Trotzdem ist immer noch wenig {\"u}ber die Evolution des gesamten menschlichen Proteoms bekannt. Proteindom{\"a}nen und ihre Kombinationen sind teilweise sehr detailliert erforscht, aber es wurden noch nicht alle Dom{\"a}nenarchitekturen des Menschen in ihrer Gesamtheit miteinander verglichen. Der verwendete große hochqualitative Datensatz von Protein-Protein-Interaktionen und Komplexen stammt aus dem Jahr 2006 und erm{\"o}glicht es erstmals das menschliche Proteom mit einer vorher nicht m{\"o}glichen Genauigkeit analysieren zu k{\"o}nnen. Hochentwickelte Cluster Algorithmen und die Verf{\"u}gbarkeit von großer Rechenkapazit{\"a}t bef{\"a}higen uns neue Information {\"u}ber Proteinnetzwerke ohne weitere Laborarbeit zu gewinnen. Die vorliegende Arbeit analysiert das menschliche Proteom auf drei verschiedenen Ebenen. Zuerst wurde der Ursprung von Proteinen basierend auf ihrer Dom{\"a}nenarchitektur analysiert, danach wurden Protein-Protein-Interaktionen untersucht und schließlich erfolgte Einteilung der Proteine nach ihren vorhandenen und fehlenden Interaktionen. Die meisten bekannten Proteine enthalten mindestens eine Dom{\"a}ne und die Proteinfunktion ergibt sich aus der Summe der Funktionen der einzelnen enthaltenen Dom{\"a}nen. Proteine, die auf der gleichen Dom{\"a}nenarchitektur basieren, das heißt die die gleichen Dom{\"a}nen in derselben Reihenfolge besitzen, sind homolog und daher aus einem gemeinsamen urspr{\"u}nglichen Protein entstanden. Die Dom{\"a}nenarchitekturen der urspr{\"u}nglichen Proteine wurden f{\"u}r 750000 Proteine aus 1313 Spezies bestimmt. Die Gruppierung von Spezies und ihrer Proteine ergibt sich aus taxonomischen Daten von NCBI-Taxonomy, welche mit zus{\"a}tzlichen Informationen basierend auf molekularen Markern erg{\"a}nzt wurden. Der resultierende Datensatz, bestehend aus 5817 Dom{\"a}nen und 32868 Dom{\"a}nenarchitekturen, war die Grundlage f{\"u}r die Bestimmung des Ursprungs der Proteine aufgrund ihrer Dom{\"a}nenarchitekturen. Es wurde festgestellt, dass nur ein kleiner Teil der neu evolvierten Dom{\"a}nenarchitekturen eines Taxons gleichzeitig auch im selben Taxon neu entstandene Proteindom{\"a}nen enth{\"a}lt. Ein weiteres Ergebnis war, dass Dom{\"a}nenarchitekturen im Verlauf der Evolution l{\"a}nger und komplexer werden, und dass so verschiedene Organismen wie der Fadenwurm, die Fruchtfliege und der Mensch die gleiche Menge an unterschiedlichen Proteinen haben, aber deutliche Unterschiede in der Anzahl ihrer Dom{\"a}nenarchitekturen aufweisen. Der zweite Teil besch{\"a}ftigt sich mit der Frage wie neu entstandene Proteine Bindungen mit dem schon bestehenden Proteinnetzwerk eingehen. In fr{\"u}heren Arbeiten wurde gezeigt, dass das Protein-Interaktions-Netzwerk ein skalenfreies Netz ist. Skalenfreie Netze, wie zum Beispiel das Internet, bestehen aus wenigen Knoten mit vielen Interaktionen, genannt Hubs, und andererseits aus vielen Knoten mit wenigen Interaktionen. Man vermutet, dass zwei Mechanismen zur Entstehung solcher Netzwerke f{\"u}hren. Erstens m{\"u}ssen neue Proteine um auch Teil des Proteinnetzwerkes zu werden mit Proteinen interagieren, die bereits Teil des Netzwerkes sind. Zweitens interagieren die neuen Proteine, gem{\"a}ß der Theorie der bevorzugten Bindung, mit h{\"o}herer Wahrscheinlichkeit mit solchen Proteinen im Netzwerk, die schon an zahlreichen weiteren Protein-Interaktionen beteiligt sind. Die Human Protein Reference Database stellt ein auf Informationen aus in-vivo Experimenten beruhendes Proteinnetzwerk f{\"u}r menschliche Proteine zur Verf{\"u}gung. Basierend auf den in Kapitel I gewonnenen Informationen wurden die Proteine mit dem Ursprungstaxon ihrer Dom{\"a}nenarchitekturen versehen. Dadurch wurde gezeigt, dass ein Protein h{\"a}ufiger mit Proteinen, die im selben Taxon entstanden sind, interagiert, als mit Proteinen, die in anderen Taxa neu aufgetreten sind. Es stellte sich heraus, dass diese Interaktionsraten f{\"u}r alle Taxa deutlich h{\"o}her waren, als durch das Zufallsmodel vorhergesagt wurden. Alle Taxa enthalten den gleichen Anteil an Proteinen mit vielen Interaktionen. Diese zwei Ergebnisse sprechen dagegen, dass die bevorzugte Bindung der alleinige Mechanismus ist, der zum heutigen Aufbau des menschlichen Proteininteraktion-Netzwerks beigetragen hat. Im dritten Teil wurden Proteine basierend auf dem Vorhandensein und der Abwesenheit von Interaktionen in Gruppen eingeteilt. Proteinnetzwerke k{\"o}nnen in kleine hoch vernetzte Teile zerlegt werden, die eine spezifische Funktion aus{\"u}ben. Diese Gruppen k{\"o}nnen mit hoher statistischer Signifikanz berechnet werden, haben meistens jedoch keine biologische Relevanz. Mit einem neuen Algorithmus, welcher zus{\"a}tzlich zu Interaktionen auch Nicht-Interaktionen ber{\"u}cksichtigt, wurde ein Datensatz bestehend aus 8,756 Proteinen und 32,331 Interaktionen neu unterteilt. Eine Einteilung in elf Gruppen zeigte hohe auf Gene Ontology basierte Werte und die Gruppen konnten signifikant einzelnen Zellteilen zugeordnet werden. Eine Gruppe besteht aus Proteinen, welche wenige Interaktionen miteinander aber viele Interaktionen zu zwei benachbarten Gruppen besitzen. Diese Gruppe enth{\"a}lt eine signifikant erh{\"o}hte Anzahl an Transportproteinen und die zwei benachbarten Gruppen haben eine erh{\"o}hte Anzahl an einerseits extrazellul{\"a}ren und andererseits im Zytoplasma und an der Membran lokalisierten Proteinen. Der Algorithmus hat damit unter Beweis gestellt das die Ergebnisse nicht bloß statistisch sondern auch biologisch relevant sind. Wenn wir auch noch weit vom Verst{\"a}ndnis des Ursprungs der Spezies entfernt sind, so hat diese Arbeit doch einen Beitrag zum besseren Verst{\"a}ndnis der Evolution auf dem Level der Proteine geleistet. Im Speziellen wurden neue Erkenntnisse {\"u}ber die Beziehung von Proteindom{\"a}nen und Dom{\"a}nenarchitekturen, sowie ihre Pr{\"a}ferenzen f{\"u}r Interaktionspartner im Interaktionsnetzwerk gewonnen.}, subject = {Evolution}, language = {en} } @phdthesis{Pietsch2005, author = {Pietsch, Christof}, title = {The genetics of species differences within the genus Nasonia ASHMEAD 1904 (Hymenoptera: Pteromalidae)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-14348}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {The genetics of species differences is an outstanding question in evolutionary biology. How do species evolve to become phenotypically distinct and how is the genetic architecture organized that underlie species differences? Phenotypic diverged traits are supposed to be frequently involved in prezygotic isolation, i.e. they prevent the formation of hybrids, whereas postzygotic isolation occurs when hybrids experience a fitness reduction. The parasitic wasp genus Nasonia represents an appropriate model system to investigate the genetics of species differences as well as the genetics of postzygotic isolation. The genus consists of three species N. vitripennis, N. longicornis and N. giraulti that differ particularly in male traits that are assumed to posses an adaptive significance: courtship behaviour and wing size differences. The courtship behaviour consists of cyclically repeated series of head nods that are separated by pauses. The stereotypic performance allowed to split up the display into distinct courtship components. Males of N. vitripennis bear vestigial forewings and are incapable of flight, whereas N. longicornis wear intermediate sized wings and N. giraulti is fully capable of flying. Nasonia species can produce interspecific hybrids after removing Wolbachia bacteria induced hybrid incompatibilities with antibiotics. Postzygotic isolation occurs to different extent and is asymmetric among reciprocal crosses, e.g. inviability is stronger in the N. vitripennis (\&\#9792;) x N. longicornis (\&\#9794;) cross than in the N. longicornis (\&\#9792;) x N. vitripennis (\&\#9794;) cross. The formation of hybrids allow to study the genetic of species differences in QTL (quantitative trait locus) analyses as well as the genetics of postzygotic isolation causing hybrid inviability. The aim of the study was to investigate the genetic architecture of differences in courtship behaviour and wing size between N. vitripennis and N. longicornis and to assess the genetics of postzygotic isolation to gain clues about the evolutionary processes underlying trait divergence and establishment of reproductive isolation between taxa. In a QTL analysis based on 94 F2-hybrid individuals of an LV cross only few QTL for wing size differences have been found with relatively large effects, although a large proportion of the phenotypic variance remained unexplained. The QTL on courtship behaviour analysis based on 94-F2 hybrid males revealed a complex genetic architecture of courtship behaviour with QTL of large phenotypic effects that explained more than 40 \% of the phenotypic variance in one case. Additionally, an epistatic analysis (non-additive interlocus interaction) of courtship QTL revealed frequent genetic interchromsomal relations leading in some instances to hybrid specific effects, e.g. reversion of phenotypic effects or the transgression of phenotypes. A QTL analysis based on a threefold sample size revealed, however, an overestimation of QTL effects in the analysis based on smaller sample size pointing towards a genetic architecture of many loci with small effects governing the phenotypic differences in courtship behaviour. Furthermore, the the study comprised the analysis of postzygotic isolation in the reciprocal crosses N. vitripennis (\&\#9792;) x N. longicornis (\&\#9794;) versus N. longicornis (\&\#9792;) x N. vitripennis (\&\#9794;) located several loci distributed over different chromosomes that are involved in hybrid incompatibility. The mapping of hybrid incompatibility regions reproduced for the first time the observed asymmetries in the strength of postzygotic isolation in reciprocal crosses of between the more distant related taxa within the genus Nasonia. Stronger postzygotic incompatibilities in the VL cross are supposed to result from the superposition of nuclear-nuclear incompatibilities with nuclear-cytoplasmic incompatibilities, whereas the coincidences of these to types of incompatibilities were found to be much weaker in the reciprocal LV cross.}, subject = {Pteromalidae}, language = {en} } @phdthesis{Grob2022, author = {Grob, Robin}, title = {The Function of Learning Walks of \({Cataglyphis Ants}\): Behavioral and Neuronal Analyses}, doi = {10.25972/OPUS-29017}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290173}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Humans and animals alike use the sun, the moon, and the stars to guide their ways. However, the position of celestial cues changes depending on daytime, season, and place on earth. To use these celestial cues for reliable navigation, the rotation of the sky has to be compensated. While humans invented complicated mechanisms like the Antikythera mechanism to keep track of celestial movements, animals can only rely on their brains. The desert ant Cataglyphis is a prime example of an animal using celestial cues for navigation. Using the sun and the related skylight polarization pattern as a compass, and a step integrator for distance measurements, it can determine a vector always pointing homewards. This mechanism is called path integration. Since the sun's position and, therefore, also the polarization pattern changes throughout the day, Cataglyphis have to correct this movement. If they did not compensate for time, the ants' compass would direct them in different directions in the morning and the evening. Thus, the ants have to learn the solar ephemeris before their far-reaching foraging trips. To do so, Cataglyphis ants perform a well-structured learning-walk behavior during the transition phase from indoor worker to outdoor forager. While walking in small loops around the nest entrance, the ants repeatedly stop their forward movements to perform turns. These can be small walked circles (voltes) or tight turns about the ants' body axes (pirouettes). During pirouettes, the ants gaze back to their nest entrance during stopping phases. These look backs provide a behavioral read-out for the state of the path integrator. The ants "tell" the observer where they think their nest is, by looking back to it. Pirouettes are only performed by Cataglyphis ants inhabiting an environment with a prominent visual panorama. This indicates, that pirouettes are performed to learn the visual panorama. Voltes, on the other hand, might be used for calibrating the celestial compass of the ants. In my doctoral thesis, I employed a wide range of state-of-the-art techniques from different disciplines in biology to gain a deeper understanding of how navigational information is acquired, memorized, used, and calibrated during the transition phase from interior worker to outdoor forager. I could show, that celestial orientation cues that provide the main compass during foraging, do not guide the ants during the look-backbehavior of initial learning walks. Instead Cataglyphis nodus relies on the earth's magnetic field as a compass during this early learning phase. While not guiding the ants during their first walks outside of the nest, excluding the ants from perceiving the natural polarization pattern of the skylight has significant consequences on learning-related plasticity in the ants' brain. Only if the ants are able to perform their learning-walk behavior under a skylight polarization pattern that changes throughout the day, plastic neuronal changes in high-order integration centers are induced. Especially the mushroom bogy collar, a center for learning and memory, and the central complex, a center for orientation and motor control, showed an increase in volume after learning walks. This underlines the importance of learning walks for calibrating the celestial compass. The magnetic compass might provide the necessary stable reference system for the ants to calibrate their celestial compass and learn the position of landmark information. In the ant brain, visual information from the polarization-sensitive ocelli converge in tight apposition with neuronal afferents of the mechanosensitive Johnston's organ in the ant's antennae. This makes the ants' antennae an interesting candidate for studying the sensory bases of compass calibration in Cataglyphis ants. The brain of the desert navigators is well adapted to successfully accomplish their navigational needs. Females (gynes and workers) have voluminous mushroom bodies, and the synaptic complexity to store large amount of view-based navigational information, which they acquire during initial learning walks. The male Cataglyphis brain is better suited for innate behaviors that support finding a mate. The results of my thesis show that the well adapted brain of C. nodus ants undergoes massive structural changes during leaning walks, dependent on a changing celestial polarization pattern. This underlies the essential role of learning walks in the calibration of orientation systems in desert ants.}, subject = {Cataglyphis}, language = {en} } @phdthesis{Fraune2014, author = {Fraune, Johanna}, title = {The evolutionary history of the mammalian synaptonemal complex}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-100043}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Der Synaptonemalkomplex (SC) ist eine hochkonservierte Proteinstruktur. Er weist eine dreiteili-ge, leiter{\"a}hnliche Organisation auf und ist f{\"u}r die stabile Paarung der homologen Chromosomen w{\"a}hrend der Prophase der ersten meiotischen Teilung verantwortlich, die auch als Synpase be-zeichnet wird. Fehler w{\"a}hrend der Synpase f{\"u}hren zu Aneuploidie oder Apoptose der sich entwi-ckelnden Keimzellen. Seit 1956 ist der SC Gegenstand intensiver Forschung. Seine Existenz wurde in zahlreichen Orga-nismen von der Hefe bis zum Menschen beschrieben. Seine Struktur aus zwei parallel verlaufen-den Lateralelementen (LE), die durch eine Vielzahl von sogenannten Transversalfilamenten (TF) verbunden werden und dem Zentralen Element (CE) in der Mitte des SC ist dabei offensichtlich {\"u}ber die Millionen von Jahren der Evolution erhalten geblieben. Einzelne Proteinkomponenten des SC wurden jedoch nur in wenigen Modelorganismen charakterisiert, darunter Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Ceanorhabditis elegans und Mus mus-culus. Unerwarteter Weise gelang es bei dieser Charakterisierung nicht, eine evolution{\"a}re Ver-wandtschaft, d.h. eine Homologie zwischen den Proteinsequenzen der verschiedenen SCs nach-zuweisen. Diese Tatsache sprach gegen die grunds{\"a}tzliche Annahme, dass der SC in der Evolution nur einmal entstanden sei. Diese Arbeit hat sich nun der Aufgabe gewidmet, die Diskrepanz zwischen der hochkonservierten Struktur des SC und seiner augenscheinlich nicht-homologen Proteinzusammensetzung zu l{\"o}sen. Dabei beschr{\"a}nkt sie sich auf die Analyse des Tierreichs. Es ist die erste Studie zur Evolution des SC in Metazoa und demonstriert die Monophylie der S{\"a}uger SC Proteinkomponenten im Tierreich. Die Arbeit zeigt, dass mindestens vier von sieben SC Proteinen der Maus sp{\"a}testens im letzten gemeinsamen Vorfahren der Gewebetiere (Eumetazoa) enstanden sind und auch damals Teil ei-nes urspr{\"u}nglichen SC waren, wie er heute in dem Nesseltier Hydra zu finden ist. Dieser SC weist die typische Struktur auf und besitzt bereits alle notwendigen Komponenten, um die drei Dom{\"a}-nen - LE, TF und CE - zu assemblieren. Dar{\"u}ber hinaus ergaben die einzelnen Phylogenien der verschiedenen SC Proteine der Maus, dass der SC eine sehr dynamische Evolutionsgeschichte durchlaufen hat. Zus{\"a}tzliche Proteine wurden w{\"a}hrend der Entstehung der Bilateria und der Wir-beltiere in den SC integriert, w{\"a}hrend andere urspr{\"u}ngliche Komponenten m{\"o}glicherweise Gen-Duplikationen erfuhren bzw. besonders in der Linie der H{\"a}utungstiere verloren gingen oder sich stark ver{\"a}nderten. Es wird die These aufgestellt, dass die auf den ersten Blick nicht-homologen SC Proteine der Fruchtfliege und des Fadenwurms tats{\"a}chlich doch von den urspr{\"u}nglichen Prote-inenkomponenten abstammen, sich aber aufgrund der rasanten Evolution der Arthropoden und der Nematoden bis zu deren Unkenntlichkeit diversifizierten. Zus{\"a}tzlich stellt die Arbeit Hydra als alternatives wirbelloses Modellsystem f{\"u}r die Meiose- und SC-Forschung zu den {\"u}blichen Modellen D. melanogaster und C. elegans vor. Die k{\"u}rzlich gewon-nenen Erkenntnisse {\"u}ber den Hydra SC sowie der Einsatz der Standard-Methoden in diesem Orga-nismus werden in dem abschließenden Kapitel zusammengefasst und diskutiert.}, subject = {Synaptinemal-Komplex}, language = {en} } @phdthesis{Mitesser2006, author = {Mitesser, Oliver}, title = {The evolution of insect life history strategies in a social context}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-22576}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {This thesis extends the classical theoretical work of Macevicz and Oster (1976, expanded by Oster and Wilson, 1978) on adaptive life history strategies in social insects. It focuses on the evolution of dynamic behavioural patterns (reproduction and activity) as a consequence of optimal allocation of energy and time resources. Mathematical modelling is based on detailed empirical observations in the model species Lasioglossum malachurum (Halictidae; Hymenoptera). The main topics are field observations, optimisation models for eusocial life histories, temporal variation in life history decisions, and annual colony cycles of eusocial insects.}, subject = {Schmalbienen}, language = {en} } @phdthesis{Classen2021, author = {Claßen, Alexandra}, title = {The ERK-cascade in the pathophysiology of cardiac hypertrophy}, doi = {10.25972/OPUS-22966}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229664}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {ERK1/2 are known key players in the pathophysiology of heart failure, but the members of the ERK cascade, in particular Raf1, can also protect the heart from cell death and ischemic injury. An additional autophosphorylation (ERK1 at Thr208, ERK2 at Thr188) empowers ERK1/2 translocation to the nucleus and phosphorylation of nuclear targets which take part in the development of cardiac hypertrophy. Thereby, targeting this additional phosphorylation is a promising pharmacological approach. In this thesis, an in silico model of ERK cascade in the cardiomyocyte is introduced. The model is a semi-quantitive model and its behavior was tested with different softwares (SQUAD and CellNetAnalyzer). Different phosphorylation states of ERK1/2 as well as different stimuli can be reproduced. The different types of stimuli include hypertrophic as well as non-hypertrophic stimuli. With the introduced in-silico model time courses and synergistic as well as antagonistic receptor stimuli combinations can be predicted. The simulated time courses were experimentally validated. SQUAD was mainly used to make predictions about time courses and thresholds, whereas CNA was used to analyze steady states and feedback loops. Furthermore, new targets of ERK1/2 which partially contribute, also in the formation of cardiac hypertrophy, were identified and the most promising of them were illuminated. Important further targets are Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 and SPIN90. Cardiomyocyte gene expression data sets were analyzed to verify involved components and to find further significantly altered genes after induced hypertrophy with TAC (transverse aortic constriction). Changes in the ultrastructure of the cardiomyocyte are the final result of induced hypertrophy.}, subject = {Herzhypertrophie}, language = {en} } @phdthesis{BergmannBorges2023, author = {Bergmann Borges, Alyssa}, title = {The endo-lysosomal system of \(Trypanosoma\) \(brucei\): insights from a protist cell model}, doi = {10.25972/OPUS-32924}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-329248}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain. The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis. Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group's macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.}, subject = {Endocytose}, language = {en} } @phdthesis{Vogel2022, author = {Vogel, Cassandra Ezra}, title = {The effects of land-use and agroecological practices on biodiversity and ecosystem services in tropical smallholder farms}, doi = {10.25972/OPUS-29066}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290661}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Biodiversity is in rapid decline worldwide. These declines are more pronounced in areas that are currently biodiversity rich, but economically poor - essentially describing many tropical regions in the Global South where landscapes are dominated by smallholder agriculture. Agriculture is an important driver of biodiversity decline, through habitat destruction and unsustainable practices. Ironically, agriculture itself is dependent on a range of ecosystem services, such as pollination and pest control, provided by biodiversity. Biodiversity on fields and the delivery of ecosystem services to crops is often closely tied to the composition of the surrounding landscape - complex landscapes with a higher proportion of (semi-)natural habitats tend to support a high abundances and biodiversity of pollinators and natural enemies that are beneficial to crop production. However, past landscape scale studies have focused primarily on industrialized agricultural landscapes in the Global North, and context dependent differences between regions and agricultural systems are understudied. Smallholder agriculture supports 2 billion people worldwide and contributes to over half the world's food supply. Yet smallholders, particularly in sub-Saharan Africa, are underrepresented in research investigating the consequences of landscape change and agricultural practices. Where research in smallholder agriculture is conducted, the focus is often on commodity crops, such as cacao, and less on crops that are directly consumed by smallholder households, though the loss of services to these crops could potentially impact the most vulnerable farmers the hardest. Agroecology - a holistic and nature-based approach to agriculture, provides an alternative to unsustainable input-intensive agriculture. Agroecology has been found to benefit smallholders through improved agronomical and food-security outcomes. Co-benefits of agroecological practices with biodiversity and ecosystem services are assumed, but not often empirically tested. In addition, the local and landscape effects on biodiversity and ecosystem services are more commonly studied in isolation, but their potentially interactive effects are so far little explored. Our study region in northern Malawi exemplifies many challenges experienced by smallholder farmers throughout sub-Saharan Africa and more generally in the Global South. Malawi is located in a global biodiversity hotspot, but biodiversity is threatened by rapid habitat loss and a push for input-intensive agriculture by government and other stakeholders. In contrast, agroecology has been effectively promoted and implemented in the study region. We investigated how land-use differences and the agroecological practices affects biodiversity and ecosystem services of multiple taxa in a maize-bean intercropping system (Chapter 2), and pollination of pumpkin (Chapter 3) and pigeon pea (Chapter 4). Additionally, the effects of local and landscape scale shrub- to farmland habitat conversion was investigated on butterfly communities, as well as the potential for agroecology to mitigate these effects (Chapter 5).}, language = {en} } @phdthesis{Heidrich2021, author = {Heidrich, Lea}, title = {The effect of environmental heterogeneity on communities}, doi = {10.25972/OPUS-22178}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221781}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {How diversity of life is generated, maintained, and distributed across space and time is the central question of community ecology. Communities are shaped by three assembly processes: (I) dispersal, (II) environ-mental, and (III) interaction filtering. Heterogeneity in environmental conditions can alter these filtering processes, as it increases the available niche space, spatially partitions the resources, but also reduces the effective area available for individual species. Ultimately, heterogeneity thus shapes diversity. However, it is still unclear under which conditions heterogeneity has positive effects on diversity and under which condi-tions it has negative or no effects at all. In my thesis, I investigate how environmental heterogeneity affects the assembly and diversity of diverse species groups and whether these effects are mediated by species traits. In Chapter II, I first examine how much functional traits might inform about environmental filtering pro-cesses. Specifically, I examine to which extent body size and colour lightness, both of which are thought to reflect the species thermal preference, shape the distribution and abundance of two moth families along elevation. The results show, that assemblages of noctuid moths are more strongly driven by abiotic filters (elevation) and thus form distinct patterns in colour lightness and body size, while geometrid moths are driven by biotic filters (habitat availability), and show no decline in body size nor colour lightness along elevation. Thus, one and the same functional trait can have quite different effects on community assembly even between closely related taxonomic groups. In Chapter III, I elucidate how traits shift the relative importance of dispersal and environmental filtering in determining beta diversity between forests. Environmental filtering via forest heterogeneity had on aver-age higher independent effects than dispersal filtering within and among regions, suggesting that forest heterogeneity determines species turnover even at country-wide extents. However, the relative importance of dispersal filtering increased with decreasing dispersal ability of the species group. From the aspects of forest heterogeneity covered, variations in herb or tree species composition had overall stronger influence on the turnover of species than forest physiognomy. Again, this ratio was influenced by species traits, namely trophic position, and body size, which highlights the importance of ecological properties of a taxo-nomic group in community assembly. In Chapter IV, I assess whether such ecological properties ultimately determine the level of heterogeneity which maximizes species richness. Here, I considered several facets of heterogeneity in forests. Though the single facets of heterogeneity affected diverse species groups both in positive and negative ways, we could not identify any generalizable mechanism based on dispersal nor the trophic position of the species group which would dissolve these complex relationships. In Chapter V, I examine the effect of environmental heterogeneity of the diversity of traits itself to evalu-ate, whether the effects of environmental heterogeneity on species richness are truly based on increases in the number of niches. The results revealed that positive effects of heterogeneity on species richness are not necessarily based on an increased number of niches alone, but proposedly also on a spatially partition of resources or sheltering effects. While ecological diversity increased overall, there were also negative trends which indicate filtering effects via heterogeneity. In Chapter VI, I present novel methods in measuring plot-wise heterogeneity of forests across continental scales via Satellites. The study compares the performance of Sentinel-1 and LiDar-derived measurements in depicting forest structures and heterogeneity and to their predictive power in modelling diversity. Senti-nel-1 could match the performance of Lidar and shows high potential to assess free yet detailed infor-mation about forest structures in temporal resolutions for modelling the diversity of species. Overall, my thesis supports the notion that heterogeneity in environmental conditions is an important driv-er of beta-diversity, species richness, and ecological diversity. However, I could not identify any general-izable mechanism which direction and form this effect will have.}, subject = {Heterogenit{\"a}t}, language = {en} } @phdthesis{Drescher2011, author = {Drescher, Jochen}, title = {The Ecology and Population structure of the invasive Yelllow Crazy Ant Anoplolepis gracilipes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-57332}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {The invasive Yellow Crazy Ant Anoplolepis gracilipes is a widespread tropical ant species which is particularly common in anthropogenically disturbed habitats in South-East Asia and the Indopacific region. Its native range is unknown, and there is little information concerning its social structure as a potential mechanism facilitating invasion as well as its ecology in one of the putative native ranges, South-East Asia. Using mitochondrial DNA sequences, I demonstrated that the majority of the current Indopacific colonies were likely introduced from South-East Asian populations, which in turn may have been introduced much earlier from a yet unidentified native range. By conducting behavioral, genetic and chemical analyses, I found that A. gracilipes supercolonies contain closely related individuals, thus resembling enlarged versions of monogynous, polydomous colonies of other ant species. Furthermore, mutually aggressive A. gracilipes supercolonies were highly differentiated both genetically and chemically, suggesting limited or even absent gene flow between supercolonies. Intranidal mating and colony-budding are most likely the predominant, if not the exclusive mode of reproduction and dispersal strategy of A. gracilipes. Consequently, a positive feedback between genetic, chemical and behavioral traits may further enhance supercolony differentiation though genetic drift and neutral evolution. This potential scenario led to the hypothesis that absent gene flow between different A. gracilipes supercolonies may drive them towards different evolutionary pathways, possibly including speciation. Thus, I examined one potential way by which gene flow between supercolonies of an ant species without nuptial flights may be maintained, i.e. the immigration of sexuals into foreign supercolonies. The results suggest that this option of maintaining gene flow between different supercolonies is likely impaired by severe aggression of workers towards allocolonial sexuals. Moreover, breeding experiments involving males and queens from different supercolonies suggest that A. gracilipes supercolonies may already be on the verge of reproductive isolation, which might lead to the diversification of A. gracilipes into different species. Regarding the ecological consequences of its potential introduction to NE-Borneo, I could show that A. gracilipes supercolonies may affect the local ant fauna. The ant community within supercolonies was less diverse and differed in species composition from areas outside supercolonies. My data suggest that the ecological dominance of A. gracilipes within local ant communities was facilitated by monopolization of food sources within its supercolony territory, achieved by a combination of rapid recruitment, numerical dominance and pronounced interspecific aggression. A. gracilipes' distribution is almost exclusively limited to anthropogenically altered habitat, such as residential and agricultural areas. The rate at which habitat conversion takes place in NE-Borneo will provide A. gracilipes with a rapidly increasing abundance of suitable habitats, thus potentially entailing significant population growth. An potentially increasing population size and ecological dominance, however, are not features that are limited to invasive alien species, but may also occur in native species that become 'pests' in an increasing abundance of anthropogenically altered habitat. Lastly, I detected several ant guests in supercolonies of A. gracilipes. I subsequently describe the relationship between one of them (the cricket Myrmecophilus pallidithorax) and its ant host. By conducting behavioral bioassays and analyses of cuticular hydrocarbon (CHC) profiles, I revealed that although M. pallidithorax is attacked and consumed by A. gracilipes whenever possible, it may evade aggression from its host by a combination of supreme agility and, possibly, chemical deception. This thesis adds to our general understanding of biological invasions by contributing species-specific data on a previously understudied invasive organism, the Yellow Crazy Ant Anoplolepis gracilipes. Introductions which may have occurred a long time ago may make it difficult to determine whether a given species is an introduced invader or a native pest species, as both may have pronounced ecological effects in native species communities. Furthermore, this thesis suggests that supercolonialism in invasive ants may not be an evolutionary dead end, but that it may possibly give rise to new species due to reproductive boundaries between supercolonies evoked by peculiar mating and dispersal strategies.}, subject = {Dem{\"o}kologie}, language = {en} } @phdthesis{Kropf2018, author = {Kropf, Jan}, title = {The Dual Olfactory Pathway in the Honeybee Brain: Sensory Supply and Electrophysiological Properties}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108369}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {The olfactory sense is of utmost importance for honeybees, Apis mellifera. Honeybees use olfaction for communication within the hive, for the identification of nest mates and non-nest mates, the localization of food sources, and in case of drones (males), for the detection of the queen and mating. Honeybees, therefore, can serve as excellent model systems for an integrative analysis of an elaborated olfactory system. To efficiently filter odorants out of the air with their antennae, honeybees possess a multitude of sensilla that contain the olfactory sensory neurons (OSN). Three types of olfactory sensilla are known from honeybee worker antennae: Sensilla trichoidea, Sensilla basiconica and Sensilla placodea. In the sensilla, odorant receptors that are located in the dendritic arborizations of the OSNs transduce the odorant information into electrical information. Approximately 60.000 OSN axons project in two parallel bundles along the antenna into the brain. Before they enter the primary olfactory brain center, the antennal lobe (AL), they diverge into four distinct tracts (T1-T4). OSNs relay onto ~3.000-4.000 local interneurons (LN) and ~900 projection neurons (PN), the output neurons of the AL. The axons of the OSNs together with neurites from LNs and PNs form spheroidal neuropil units, the so-called glomeruli. OSN axons from the four AL input tracts (T1-T4) project into four glomerular clusters. LNs interconnect the AL glomeruli, whereas PNs relay the information to the next brain centers, the mushroom body (MB) - associated with sensory integration, learning and memory - and the lateral horn (LH). In honeybees, PNs project to the MBs and the LH via two separate tracts, the medial and the lateral antennal-lobe tract (m/lALT) which run in parallel in opposing directions. The mALT runs first to the MB and then to the LH, the lALT runs first to the LH and then to the MB. This dual olfactory pathway represents a feature unique to Hymenoptera. Interestingly, both tracts were shown to process information about similar sets of odorants by extracting different features. Individual mALT PNs are more odor specific than lALT PNs. On the other hand, lALT PNs have higher spontaneous and higher odor response action potential (AP) frequencies than mALT PNs. In the MBs, PNs form synapses with ~184.000 Kenyon cells (KC), which are the MB intrinsic neurons. KCs, in contrast to PNs, show almost no spontaneous activity and employ a spatially and temporally sparse code for odor coding. In manuscript I of my thesis, I investigated whether the differences in specificity of odor responses between m- and lALT are due to differences in the synaptic input. Therefore, I investigated the axonal projection patterns of OSNs housed in S. basiconica in honeybee workers and compared them with S. trichoidea and S. placodea using selective anterograde labeling with fluorescent tracers and confocal- microscopy analyses of axonal projections in AL glomeruli. Axons of S. basiconica-associated OSNs preferentially projected into the T3 input-tract cluster in the AL, whereas the two other types of sensilla did not show a preference for a specific glomerular cluster. T3- associated glomeruli had previously been shown to be innervated by mALT PNs. Interestingly, S. basiconica as well as a number of T3 glomeruli lack in drones. Therefore I set out to determine whether this was associated with the reduction of glomeruli innervated by mALT PNs. Retrograde tracing of mALT PNs in drones and counting of innervated glomeruli showed that the number of mALT-associated glomeruli was strongly reduced in drones compared to workers. The preferential projections of S. basiconica-associated OSNs into T3 glomeruli in female workers together with the reduction of mALT-associated glomeruli in drones support the presence of a female-specific olfactory subsystem that is partly innervated by OSNs from S. basiconica and is associated with mALT projection neurons. As mALT PNs were shown to be more odor specific, I suppose that already the OSNs in this subsystem are more odor specific than lALT associated OSNs. I conclude that this female-specific subsystem allows the worker honeybees to respond adequately to the enormous variety of odorants they experience during their lifetime. In manuscript II, I investigated the ion channel composition of mALT and lALT PNs and KCs in situ. This approach represents the first study dealing with the honeybee PN and KC ion channel composition under standard conditions in an intact brain preparation. With these recordings I set out to investigate the potential impact of intrinsic neuronal properties on the differences between m- and lALT PNs and on the sparse odor coding properties of KCs. In PNs, I identified a set of Na+ currents and diverse K+ currents depending on voltage and Na+ or Ca2+ that support relatively high spontaneous and odor response AP frequencies. This set of currents did not significantly differ between mALT and lALT PNs, but targets for potential modulation of currents leading to differences in AP frequencies were found between both types of PNs. In contrast to PNs, KCs have very prominent K+ currents, which are likely to contribute to the sparse response fashion observed in KCs. Furthermore, Ca2+ dependent K+ currents were found, which may be of importance for coincidence detection, learning and memory formation. Finally, I conclude that the differences in odor specificity between m- and lALT PNs are due to their synaptic input from different sets of OSNs and potential processing by LNs. The differences in spontaneous activity between the two tracts may be caused by different neuronal modulation or, in addition, also by interaction with LNs. The temporally sparse representation of odors in KCs is very likely based on the intrinsic KC properties, whereas general excitability and spatial sparseness are likely to be regulated through GABAergic feedback neurons.}, subject = {Voltage-Clamp-Methode}, language = {en} } @phdthesis{Hsieh2011, author = {Hsieh, Samuel Yu-Lung}, title = {The diversity and ecology of the spider communities of European beech canopy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-66966}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Ein wesentliches Ziel {\"o}kologischer Forschung ist es, die Frage zu beantworten, wie Arten koexistieren k{\"o}nnen und die biologische Vielfalt erhalten bleibt. Um zu verstehen, wie dabei Gemeinschaften in unterschiedlichen r{\"a}umlich-zeitlichen Dimensionen interagieren, um die biologische Vielfalt zu erhalten, ist ein umfassendes prozessorientiertes Wissen erforderlich. Demzufolge konzentrierte sich meine Studie im Wesentlichen auf die Biodiversit{\"a}t und die sie beeinflussenden raum-zeitlichen {\"o}kologischen Prozesse. Vergleicht man die {\"A}hnlich- bzw. Un{\"a}hnlichkeit der in verschieden alten Best{\"a}nden lebenden Spinnengemeinschaften der Buchen (Fagus sylvatica L.), dann zeigt sich, dass die {\"a}lteste Baumkohorte offensichtlich einzigartige Ressourcen besitzt, welche die Zusammensetzung der Spinnengemeinschaften deutlich pr{\"a}gen. {\"U}ber das Jahr hin zeigten die Spinnengemeinschaften trotz der jahreszeitlich unterschiedlich {\"o}kologischen Randbedingungen eine sich wiederholende, vorhersehbare Dynamik. Der Vergleich {\"u}ber die Jahre ergab, dass das "Neutrale Modell" und das "Nischen-Modell" gleichzeitig funktionieren k{\"o}nnen. Beide sind notwendig, um die Dynamik der in den Buchenkronen der verschiedenen Altersklassen lebenden Spinnengemeinschaften vollst{\"a}ndig erkl{\"a}ren zu k{\"o}nnen.}, subject = {Spinnen}, language = {en} } @phdthesis{Heisswolf2006, author = {Heisswolf, Annette}, title = {The distribution of leaf beetles on multiple spatial scales : causes and consequences}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18945}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Herbivorous insects are the major link between primary producers and a multitude of animals at higher trophic levels. Elucidating the causes and consequences of their distribution patterns in the "green world" is thus essential for our understanding of numerous ecological processes on multiple spatial scales. We can ask where and why a certain herbivore can be found in the landscape, within the habitat, on which plant within the habitat and finally, where on that plant. Depending on spatial scale the distribution of herbivores is shaped by different processes (fitness considerations, physiological abilities, population dynamics, dispersal behavior, history of the landscape etc.). Scaling down from fragmented landscapes to individual host plants this thesis analyzes the distribution patterns of the strictly monophagous herbivore Cassida canaliculata Laich. (Coleoptera: Chrysomelidae), which feeds and oviposits exclusively on meadow sage, Salvia pratensis L. (Lamiales: Lamiaceae), and compares it to those of the polyphagous tansy leaf beetle Galeruca tanaceti L. (Coleoptera: Chrysomelidae), which does not oviposit on its host plants, but on dry non-host structures. The specialist Cassida canaliculata depended on all spatial scales (fragmented landscape, microhabitat and host plant individual) mainly on the distribution and quality of its single host plant species Salvia pratensis, whereas enemy-free-space - i.e. avoidance of parasitism and predation of egg clutches, larvae, and pupae - seemed to influence oviposition site choice only on the scale of the host plant individual. On this spatial scale, offspring of Cassida canaliculata had a higher chance of survival on large host plant individuals, which were also preferred for oviposition by the females. In contrast, the distribution patterns of the generalist Galeruca tanaceti was shaped by the interaction with its parasitoid regarding both microhabitat choice and egg distribution within individual host plants. On the microhabitat scale, beetles could escape from their parasitoids by ovipositing into high and dense vegetation. Regarding oviposition site choice within a host plant individual, females oviposited as high as possible in the vegetation and could thus reduce both the risk of parasitism and the probability of winter mortality. The results of my thesis show that the degree of specificity of a herbivore is of central importance for the resulting egg distribution pattern on all spatial scales.}, subject = {Blattk{\"a}fer}, language = {en} } @phdthesis{Mishra2011, author = {Mishra, Dushyant}, title = {The content of olfactory memory in larval Drosophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-66316}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. 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 olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade.}, subject = {Drosophila}, language = {en} } @phdthesis{Ruf2016, author = {Ruf, Franziska}, title = {The circadian regulation of eclosion in \(Drosophila\) \(melanogaster\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146265}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Eclosion is the emergence of an adult insect from the pupal case at the end of development. In the fruit fly Drosophila melanogaster, eclosion is a circadian clock-gated event and is regulated by various peptides. When studied on the population level, eclosion reveals a clear rhythmicity with a peak at the beginning of the light-phase that persists also under constant conditions. It is a long standing hypothesis that eclosion gating to the morning hours with more humid conditions is an adaption to reduce water loss and increase the survival. Eclosion behavior, including the motor pattern required for the fly to hatch out of the puparium, is orchestrated by a well-characterized cascade of peptides. The main components are ecdysis-triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). The molt is initiated by a peak level and pupal ecdysis by a subsequent decline of the ecdysteroid ecdysone. Ecdysteroids are produced by the prothoracic gland (PG), an endocrine tissue that contains a peripheral clock and degenerates shortly after eclosion. Production and release of ecdysteroids are regulated by the prothoracicotropic hormone (PTTH). Although many aspects of the circadian clock and the peptidergic control of the eclosion behavior are known, it still remains unclear how both systems are interconnected. The aim of this dissertation research was to dissect this connection and evaluate the importance of different Zeitgebers on eclosion rhythmicity under natural conditions. Potential interactions between the central clock and the peptides regulating ecdysis motor behavior were evaluated by analyzing the influence of CCAP on eclosion rhythmicity. Ablation and silencing of CCAP neurons, as well as CCAP null-mutation did not affect eclosion rhythmicity under either light or temperature entrainment nor under natural conditions. To dissect the connection between the central and the peripheral clock, PTTH neurons were ablated. Monitoring eclosion under light and temperature entrainment revealed that eclosion became arrhythmic under constant conditions. However, qPCR expression analysis revealed no evidence for cycling of Ptth mRNA in pharate flies. To test for a connection with pigment-dispersing factor (PDF)-expressing neurons, the PDF receptor (PDFR) and short neuropeptide F receptor (sNPFR) were knocked down in the PTTH neurons. Knockdown of sNPFR, but not PDFR, resulted in arrhythmic eclosion under constant darkness conditions. PCR analysis of the PTTH receptor, Torso, revealed its expression in the PG and the gonads, but not in the brain or eyes, of pharate flies. Knockdown of torso in the PG lead to arrhythmicity under constant conditions, which provides strong evidence for the specific effect of PTTH on the PG. These results suggest connections from the PDF positive lateral neurons to the PTTH neurons via sNPF signaling, and to the PG via PTTH and Torso. This interaction presumably couples the period of the peripheral clock in the PG to that of the central clock in the brain. To identify a starting signal for eclosion and possible further candidates in the regulation of eclosion behavior, chemically defined peptidergic and aminergic neurons were optogenetically activated in pharate pupae via ChR2-XXL. This screen approach revealed two candidates for the regulation of eclosion behavior: Dromyosuppressin (DMS) and myo-inhibitory peptides (MIP). However, ablation of DMS neurons did not affect eclosion rhythmicity or success and the exact function of MIP must be evaluated in future studies. To assess the importance of the clock and of possible Zeitgebers in nature, eclosion of the wildtype Canton S and the clock mutant per01 and the PDF signaling mutants pdf01 and han5304 was monitored under natural conditions. For this purpose, the W{\"u}rzburg eclosion monitor (WEclMon) was developed, which is a new open monitoring system that allows direct exposure of pupae to the environment. A general decline of rhythmicity under natural conditions compared to laboratory conditions was observed in all tested strains. While the wildtype and the pdf01 and han5304 mutants stayed weakly rhythmic, the per01 mutant flies eclosed mostly arrhythmic. PDF and its receptor (PDFR encoded by han) are required for the synchronization of the clock network and functional loss can obviously be compensated by a persisting synchronization to external Zeitgebers. The loss of the central clock protein PER, however, lead to a non-functional clock and revealed the absolute importance of the clock for eclosion rhythmicity. To quantitatively analyze the effect of the clock and abiotic factors on eclosion rhythmicity, a statistical model was developed in cooperation with Oliver Mitesser and Thomas Hovestadt. The modelling results confirmed the clock as the most important factor for eclosion rhythmicity. Moreover, temperature was found to have the strongest effect on the actual shape of the daily emergence pattern, while light has only minor effects. Relative humidity could be excluded as Zeitgeber for eclosion and therefore was not further analyzed. Taken together, the present dissertation identified the so far unknown connection between the central and peripheral clock regulating eclosion. Furthermore, a new method for the analysis of eclosion rhythms under natural conditions was established and the necessity of a functional clock for rhythmic eclosion even in the presence of multiple Zeitgebers was shown.}, subject = {Taufliege}, language = {en} } @phdthesis{Kay2018, author = {Kay, Janina}, title = {The circadian clock of the carpenter ant \(Camponotus\) \(floridanus\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158061}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Due to the earth´s rotation around itself and the sun, rhythmic daily and seasonal changes in illumination, temperature and many other environmental factors occur. Adaptation to these environmental rhythms presents a considerable advantage to survival. Thus, almost all living beings have developed a mechanism to time their behavior in accordance. This mechanism is the endogenous clock. If it fulfills the criteria of (1) entraining to zeitgebers (2) free-running behavior with a period of ~ 24 hours (3) temperature compensation, it is also referred to as "circadian clock". Well-timed behavior is crucial for eusocial insects, which divide their tasks among different behavioral castes and need to respond to changes in the environment quickly and in an orchestrated fashion. Circadian rhythms have thus been studied and observed in many eusocial species, from ants to bees. The underlying mechanism of this clock is a molecular feedback loop that generates rhythmic changes in gene expression and protein levels with a phase length of approximately 24 hours. The properties of this feedback loop are well characterized in many insects, from the fruit fly Drosophila melanogaster, to the honeybee Apis mellifera. Though the basic principles and components of this loop are seem similar at first glance, there are important differences between the Drosophila feedback loop and that of hymenopteran insects, whose loop resembles the mammalian clock loop. The protein PERIOD (PER) is thought to be a part of the negative limb of the hymenopteran clock, partnering with CRYPTOCHROME (CRY). The anatomical location of the clock-related neurons and the PDF-network (a putative in- and output mediator of the clock) is also well characterized in Drosophila, the eusocial honeybee as well as the nocturnal cockroach Leucophea maderae. The circadian behavior, anatomy of the clock and its molecular underpinnings were studied in the carpenter ant Camponotus floridanus, a eusocial insect Locomotor activity recordings in social isolation proved that the majority of ants could entrain to different LD cycles, free-ran in constant darkness and had a temperature-compensated clock with a period slightly shorter than 24 hours. Most individuals proved to be nocturnal, but different types of activity like diurnality, crepuscularity, rhythmic activity during both phases of the LD, or arrhythmicity were also observed. The LD cycle had a slight influence on the distribution of these activities among individuals, with more diurnal ants at shorter light phases. The PDF-network of C. floridanus was revealed with the anti-PDH antibody, and partly resembled that of other eusocial or nocturnal insects. A comparison of minor and major worker brains, only revealed slight differences in the number of somata and fibers crossing the posterior midline. All in all, most PDF-structures that are conserved in other insects where found, with numerous fibers in the optic lobes, a putative accessory medulla, somata located near the proximal medulla and many fibers in the protocerebrum. A putative connection between the mushroom bodies, the optic lobes and the antennal lobes was found, indicating an influence of the clock on olfactory learning. Lastly, the location and intensity of PER-positive cell bodies at different times of a 24 hour day was established with an antibody raised against Apis mellifera PER. Four distinct clusters, which resemble those found in A. mellifera, were detected. The clusters could be grouped in dorsal and lateral neurons, and the PER-levels cycled in all examined clusters with peaks around lights on and lowest levels after lights off. In summary, first data on circadian behavior and the anatomy and workings of the clock of C. floridanus was obtained. Firstly, it´s behavior fulfills all criteria for the presence of a circadian clock. Secondly, the PDF-network is very similar to those of other insects. Lastly, the location of the PER cell bodies seems conserved among hymenoptera. Cycling of PER levels within 24 hours confirms the suspicion of its role in the circadian feedback loop.}, subject = {Chronobiologie}, language = {en} } @phdthesis{Schubert2019, author = {Schubert, Frank Klaus}, title = {The circadian clock network of \(Drosophila\) \(melanogaster\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157136}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism. The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons. To further study the functional hierarchy within the clock network and to monitor the "ticking clock" over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.}, subject = {Taufliege}, language = {en} } @phdthesis{Lapuente2021, author = {Lapuente, Juan M.}, title = {The Chimpanzees of the Como{\´e} National Park, Ivory Coast. Status, distribution, ecology and behavior}, doi = {10.25972/OPUS-22318}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223180}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Although wild chimpanzees (Pan troglodytes) have been studied intensely for more than 50 years, there are still many aspects of their ecology and behavior that are not well understood. Every time that a new population of chimpanzees has been studied, new behaviors and unknown aspects of their ecology have been discovered. All this accumulated knowledge is helping us to piece together a model of how could last human and chimpanzee common ancestors have lived and behaved between seven and five million years ago. Como{\´e} chimpanzees had never been studied in depth, until we started our research in October 2014, only a few censuses had been realized. The last surveys prior our work, stated that the population was so decimated that was probably functionally extinct. When we started this research, we had to begin with a new intensive survey, using new methods, to ascertain the real status and distribution of the chimpanzees living in Como{\´e} National Park (CNP). During the last five years, we have realized a deep study aiming to know more about their ecology and behavior. We combined transects and reconnaissance marches (recces) with the use of camera traps, for the first time in CNP, obtaining a wealth of data that is not fully comprised in this dissertation. With this research, we determined that there is a sustainable continuous population of Western chimpanzees (Pan troglodytes verus) in CNP and the adjacent area of Mont Tingui, to the West, with a minimum of 127 weaned chimpanzees living in our main 900 km2 study area, SW of CNP. We found that this population is formed by a minimum of eight different chimpanzee communities, of which we studied seven, four of them more in detail. These chimpanzees spent much more time in the forest than in the savanna habitats. We also found that Como{\´e} chimpanzees consumed at least 58 different food items in their dit, which they obtained both from forest and savanna habitats. Another finding was that insectivory had an important role in their diet, with at least four species of ants, three of termites and some beetle larvae. These chimpanzees also hunted at least three species of monkeys and maybe rodents and duikers and occasionally consumed the big land snails of genus Achatina. We found that, during the fruit scarcity period in the late rainy season, they intensely consumed the cambium of Ceiba pentandra, as fallback food, much more than the bark or cambium of any other tree species. Another interesting finding was that all the chimpanzees in the studied area realized this particular bark-peeling behavior and had been repeatedly peeling the trees of this species for years. This did not increase tree mortality and the damage caused to the trees was healed in two years, not reducing the growth, thus being a sustainable use of the trees. We found that Como{\´e} chimpanzees produced and used a great variety of tools, mainly from wooden materials, but also from stone and herbaceous vegetation. Their tool repertory included stick tools to dip for Dorylus burmeisteri ants, to fish for Camponotus and Crematogaster ants, to dip for honey, mainly from Meliponini stingless bees, but sometimes from honey bees (Apis mellifera). It also included the use of stick tools to fish termites of Macrotermes subhyalinus and Odontotermes majus (TFTs), to dip for water from tree holes and investigatory probes for multiple purposes. Additionally, these chimpanzees used leaf-sponges to drink from tree holes and to collect clayish water from salt-licks. They also used stones to hit the buttresses of trees during displays, the so called accumulative stone throwing behavior and probably used stones as hammers, to crack open hard-shelled Strichnos spinosa and Afraegle paniculata fruits and Achatina snails. The chimpanzees also used objects that are not generally accepted as animal tools, for being attached to the substrate, with different purposes: they drummed buttresses of trees with hands and/or feet to produce sound during male displays and they pounded open hard-shelled fruits, Achatina snails and Cubitermes termite mounds on stone or root anvils. We finally measured the stick tools and found significant differences between them suggesting that they were specialized tools made specifically for every purpose. We studied more in detail the differences between apparently similar tools, the honey dipping tools and the water dipping tools, often with brushes made at their tips to collect the fluids. These last tools were exclusive from Como{\´e} and have not been described at any other site. We found that total length, diameter and brush length were significantly different, suggesting that they were specialized tools. We concluded that Como{\´e} chimpanzees had a particular culture, different from those of other populations of Western chimpanzees across Africa. Efficient protection, further research and permanent presence of research teams are required to avoid that this unique population and its culture disappears by the poaching pressure and maybe by the collateral effects of climate change.}, subject = {Parc National de la Como{\´e}}, language = {en} } @phdthesis{Triphan2009, author = {Triphan, Tilman}, title = {The Central Control of Gap Climbing Behaviour in Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-43666}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {In this work, a behavioural analysis of different mutants of the fruit fly Drosophila melanogaster has been carried out. Primarily, the gap climbing behaviour (Pick \& Strauss, 2005) has been assayed as it lends itself for the investigation of decision making processes and the neuronal basis of adaptive behaviour. Furthermore it shows how basic motor actions can be combined into a complex motor behaviour. Thanks to the neurogenetic methods, Drosophila melanogaster has become an ideal study object for neurobiological questions. Two different modules of climbing control have been examined in detail. For the decision making, the mutant climbing sisyphus was analysed. While wild-type flies adapt the initiation of climbing behaviour to the width of the gap and the probability for a successful transition. climbing sisyphus flies initiate climbing behaviour even at clearly insurmountable gap widths. The climbing success itself is not improved in comparison to the wild-type siblings. The mutant climbing sisyphus is a rare example of a hyperactive mutant besides many mutants that show a reduced activity. Basic capabilities in vision have been tested in an optomotor and a distance-estimation paradigm. Since they are not affected, a defect in decision making is most probably the cause of this behavioural aberration. A second module of climbing control is keeping up orientation towards the opposite side of the gap during the execution of climbing behaviour. Mutants with a structural defect in the protocerebral bridge show abnormal climbing behaviour. During the climbing attempt, the longitudinal body axis does not necessarily point into the direction of the opposite side. Instead, many climbing events are initiated at the side edge of the walking block into the void and have no chance to ever succeed. The analysed mutants are not blind. In one of the mutants, tay bridge1 (tay1) a partial rescue attempt used to map the function in the brain succeeded such that the state of the bridge was restored. That way, a visual targeting mechanism has been activated, allowing the flies to target the opposite side. When the visibility of the opposing side was reduced, the rescued flies went back to a tay1 level of directional scatter. The results are in accord with the idea that the bridge is a central constituent of the visual targeting mechanism. The tay1 mutant was also analysed in other behavioural paradigms. A reduction in walking speed and walking activity in this mutant could be rescued by the expression of UAS-tay under the control of the 007Y-GAL4 driver line, which concomitantly restores the structure of the protocerebral bridge. The separation of bridge functions from functions of other parts of the brain of tay1 was accomplished by rescuing the reduced optomotor compensation in tay1 by the mb247-GAL4>UAS-tay driver. While still having a tay1-like protocerebral bridge, mb247-GAL4 rescue flies are able to compensate at wild-type levels. An intact compensation is not depended on the tay expression in the mushroom bodies, as mushroom body ablated flies with a tay1 background and expression of UAS-tay under the control of mb247-GAL4 show wild-type behaviour as well. The most likely substrate for the function are currently unidentified neurons in the fan-shaped body, that can be stained with 007Y-GAL4 and mb247-GAL4 as well.}, subject = {Taufliege}, language = {en} } @phdthesis{Kohl2023, author = {Kohl, Patrick Laurenz}, title = {The buzz beyond the beehive: population demography, parasite burden and limiting factors of wild-living honeybee colonies in Germany}, doi = {10.25972/OPUS-33032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-330327}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The western honeybee (Apis mellifera) is widely known as the honey producer and pollinator managed by beekeepers but neglected as a wild bee species. Central European honeybee populations have been anthropogenically disturbed since about 1850 through introgression and moderate artificial selection but have never been truly domesticated due to a lack of mating control. While their decline in the wild was historically attributed to the scarcity of nesting cavities, a contemporary view considers the invasion of the parasitic mite Varroa destructor in the 1970s as the major driver. However, there are no longitudinal population data available that could substantiate either claim. Based on the insight that introduced European honeybees form viable wild populations in eastern North America and reports on the occurrence of wild-living colonies from various European countries, we systematically studied the ecology of wild-living honeybees in Germany. First, we investigated whether wild-living honeybees colonising German forests form a self-sustaining population. Second, we asked how the parasite burden of wild-living colonies relates to that of managed colonies. And third, we explored whether the winter mortality of wild-living colonies is associated with parasite burden, nest depredation, or the lack of resources on the landscape scale. Between 2017 and 2021, we monitored listed trees with black woodpecker cavities for honeybees in the managed forests of three study regions (Swabian Alb, counties Coburg and Lichtenfels, county Weilheim-Schongau). Continuity of occupation was determined using microsatellite genetic markers. Wild-living colonies predictably colonised forests in summer, when about 10\% of all cavities were occupied. The annual colony survival rate and colony lifespan (based on N=112 colonies) were 10.6\% and 0.6 years, with 90\% of colonies surviving summer (July-September), 16\% surviving winter (September-April), and 72\% surviving spring (April-July). The average maximum and minimum colony densities were 0.23 (July) and 0.02 (April) colonies per km^2. During the (re-)colonisation of forests in spring, swarms preferred cavities that had already been occupied by other honeybee colonies. We estimate the net reproductive rate of the population to be R0= 0.318, meaning that it is currently not self-sustaining but maintained by the annual immigration of swarms from managed hives. The wild-living colonies are feral in a behavioural sense. We compared the occurrence of 18 microparasites among feral colonies (N=64) and managed colonies (N=74) using qPCR. Samples were collected in four regions (the three regions mentioned above and the city of Munich) in July 2020; they consisted of 20 workers per colony captured at flight entrances. We distinguished five colony types representing differences in colony age and management histories. Besides strong regional variation, feral colonies consistently hosted fewer microparasite taxa (median: 5, range 1-8) than managed colonies (median: 6, range 4-9) and had different parasite communities. Microparasites that were notably less prevalent among feral colonies were Trypanosomatidae, Chronic bee paralysis virus, and Deformed wing viruses A and B. In the comparison of five colony types, parasite burden was lowest in newly founded feral colonies, intermediate in overwintered feral colonies and managed nucleus colonies, and highest in overwintered managed colonies and hived swarms. This suggests that the natural mode of colony reproduction by swarming, which creates pauses in brood production, and well-dispersed nests, which reduce horizontal transmission, explain the reduced parasite burden in feral compared to managed colonies. To explore the roles of three potential drivers of feral colony winter mortality, we combined colony observations gathered during the monitoring study with data on colony-level parasite burden, observations and experiments on nest depredation, and landscape analyses. There was no evidence for an effect of summertime parasite burden on subsequent winter mortality: colonies that died (N=57) did not have a higher parasite burden than colonies that survived (N=10). Camera traps (N=15) installed on cavity trees revealed that honeybee nests are visited by a range of vertebrate species throughout the winter at rates of up to 10 visits per week. Four woodpecker species, great tits, and pine martens acted as true nest depredators. The winter survival rate of colonies whose nest entrances were protected by screens of wire mesh (N=32) was 50\% higher than that of colonies with unmanipulated entrances (N=40). Analyses of land cover maps revealed that the landscapes surrounding surviving colonies (N=19) contained on average 6.4 percentage points more resource-rich cropland than landscapes surrounding dying colonies (N=94). We estimate that tens of thousands of swarms escape from apiaries each year to occupy black woodpecker cavities and other hollow spaces in Germany and that feral colonies make up about 5\% of the regional honeybee populations. They are unlikely to contribute disproportionately to the spread of bee diseases. Instead, by spatially complementing managed colonies, they contribute to the pollination of wild plants in forests. Honeybees occupying tree cavities likely have various effects on forest communities by acting as nest site competitors or prey, and by accumulating biomass in tree holes. Nest depredation (a consequence of a lack of well-protected nest sites) and food resource limitation seem to be more important than parasites in hampering feral colony survival. The outstanding question is how environmental and intrinsic factors interact in preventing population establishment. Nest boxes with movable frames could be used to better study the environmental drivers of feral colonies' mortality. Pairs of wild (self-sustaining) and managed populations known to exist outside Europe could provide answers to whether modern apiculture creates honeybee populations maladapted to life in the wild. In Europe, large continuous forests might represent evolutionary refuges for wild honeybees.}, subject = {Biene }, language = {en} } @phdthesis{Brink2007, author = {Brink, Andreas}, title = {The biological significance of chemically-induced DNA adducts in relation to background DNA damage}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-23850}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {No abstract available}, subject = {DNS-Sch{\"a}digung}, language = {en} } @phdthesis{Kaiser2014, author = {Kaiser, Dorkas}, title = {Termites and ants in BURKINA FASO (WEST AFRICA): taxonomic and functional diversity along land-use gradients; ecosystem services of termites in the traditional ZA{\"I} SYSTEM}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107001}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The consequences of habitat change for human well-being are assumed to be especially extreme in Burkina Faso. The country is located in a highly drought-sensitive zone of West Africa, and small-scale subsistence farmers may be especially affected if losses of biodiversity lead to changes in ecosystem functioning; many depend on more or less degraded lands for agricultural production. The overall aim of the present thesis consequently was to characterize the functional traits of soil-organisms which are crucial for a productive and balanced soil environment in the study region - termites and ants. They are true ecosystem engineers whose activity alters the habitat. Through soil-turnover in the course of constructing biogenic structures of varying size and nature (mounds, nests, galleries, soil-sheetings, foraging-holes), they bioturbate huge amounts of soil masses and exert massive effects on soil structure, positively influencing the fertility, stability, aeration and water infiltration rate into soils; and they provide habitats for other species. In sub-Saharan Africa, ants and termites are the only active soil macrofauna during the long dry season; in the sub-Sahel zone of Burkina Faso, termites even represent the only active, quantitatively remarkable decomposers all year round. Since no information was available about the actual diversity of the focal arthropods, I divided the thesis in two main parts: In the first part, a baseline study, I assessed the local termite and ant fauna, and investigated their quantitative and qualitative response to changing habitat parameters resulting from increasing human impact ('functional response traits'). In the second and applied part, I addressed the impact of the biogenic structures which are important for the restoration of degraded soils ('functional effect traits'). Two traditional agricultural systems characteristic for the study region were selected. Each system represented a land-use intensification gradient comprising four distinct habitats now differing in the magnitude of human intervention but formerly having the same initial state. The first disturbance gradient, the temporal cross-section of a traditional soil water conservation technique to restore degraded heavily encrusted, barren soil named Za{\"i} in Ouahigouya (Yatenga province, sub-Sahel zone); the second disturbance gradient, an agriculture type using crop rotation and fallow as nutrient management techniques near Fada N'Gourma (Gourma province, North-Sudanese zone). No standard protocol existed for the assessment of termite and ant diversity in semi-arid (agro-) ecosystems; two widely accepted standard protocols provided the basis for the newly revised and combined rapid assessment protocol 'RAP': the ALL protocol for leaf litter ants of Agosti and Alonso (2000), and the transect protocol for termites in tropical forests of Jones and Eggleton (2000). In each study site, three to four replicate transects were conducted during the rainy seasons (2004—2008). The RAP-protocol turned out to be very effective to characterize, compare and monitor the taxonomic and functional diversity of termites and ants; between 70\% and 90\% of the estimated total species richness were collected on all levels (transects, habitats, regions). Together in both regions, 65 ant species (25 genera) and 39 termite species (13 genera) were collected. These findings represent the first records for Burkina Faso. The data indicate a high sensitivity of termites and ants to land-use intensification. The diversity strongly decreased with increasing anthropogenic impact in the North-Sudan region. In total, 53 ant species (23 genera) and 31 termite species (12 genera) were found. Very promising results concerning the recovery potential of the soil-arthropods' diversity were gathered in the Za{\"i} system. The diversity of both taxa strongly increased with increasing habitat rehabilitation - in total, 41 ant species (16 genera) and 33 termite species (11 genera) were collected. For both taxa significant differences could be noted in the shape of the density variations along the gradient. For instance termites: Fungus-growers showed the greatest adaptability to different management practices. The greatest variations between the habitats were observed in soil and grass-feeding termites. Whole functional groups were missing in heavily impacted habitats, e.g. soil-, grass-, and wood-feeders were absent in the degraded site in the sub-Sahel zone. Several environmental parameters could be identified which significantly explained a great part of the variations in the composition of the arthropods' communities; they indicate the importance of the habitats' structural complexity (vegetation structure) and concomitant effects on diurnal temperature and moisture fluctuations, the availability of food sources, and the soil-structure. The diversity of termites in the sub-Sahel region was strongly correlated with the crown-cover percentages, the topsoils' sand-content, and the availability of litter; in the North-Sudan region with the cumulated woody plant basal area, the topsoils' clay- and organic matter-content. The parameters identified for ant communities in the Za{\"i} system, were the height of trees, the topsoils' clay-content and air humidity; in the North-Sudan region the habitats' crown-cover percentages, the quantity of litter and again the height of trees. In the second part of the thesis, I first rapidly assessed the (natural) variations in the amount of epigeal soil-structures along the two disturbance gradients in order to judge the relative importance of termites and ants for soil-turnover. The results illustrated impressively that a) in all study sites, termites were the main bioturbators while ant structures were of minor importance for soil turn-over; b) earthworms and grass-feeding termites contributed significantly to soil turn-over in the more humid North-Sudan region; and c) the bioturbated soil mass varied between seasons and years, however, the relative importance of the different taxa seemed to be fairly constant. In the sub-Sahel zone, fungus-growing Odontotermes and Macrotermes species fully take over the important function of bioturbation, leading to the transport of huge amounts of fine-textured soil material to the surface; with increasing habitat restoration, coarse fragments decreased in the upper horizons and became concentrated deeper along the soil profile. Consequently, in the applied part, I concentrated on the bioturbation activity of fungus-growing termites in the four main stages of the Za{\"i} system: crusted bare soil (initial stage), millet field, young and old forest. In each of the four Za{\"i} sites nine experimental blocks (each comprising four plots of 1m2) were used to stimulate the foraging activity of fungus-growing termites with different, locally available organic materials (Aristida kerstingii hay, Bombax costatum wooden blocks, compost and a control without any organic amendment). The experiment was conducted twice for the duration of four weeks (rainy season 2005, dry season 2006). The plots were regularly checked and the increase of the area covered by sheetings chronologically followed. After four weeks a) all sheeting-soil was collected, air dried and separately weighed according to the different genera, and b) the foraging-holes were counted and their diameter measured. Additionally, c) ponded water infiltration was measured in selected plots, and d) the physicochemical properties of sheeting-soil were analyzed. In case of complete consumption of the offered hay during the experimental 4-weeks-duration, the same procedure (a, b) was followed before adding new hay to the respective plot. The comparison between the different plots, sites and seasons revealed clearly that hay was the most attractive bait; for each gram of hay removed, Odontotermes brought about 12 g soil to the surface, Macrotermes 4 g. Odontotermes was the only genus attracted by organic material to the degraded area, and was therefore the decisive primary physical ecosystem engineer in the Za{\"i} system, initiating the restoration process. The mass of soil bioturbated in the course of foraging increased strongly from the degraded, barren towards the most rehabilitated reforested site. Combining all 36 experimental plots per Za{\"i} stage, Odontotermes bioturbated 31.8 tons of soil per hectare and month dry season in the degraded area, and 32.4 tons ha-1 mon-1 in the millet fields; both genera moved 138.9 tons ha-1 mon-1 in the young and 215.5 tons ha-1 mon-1 in the old Za{\"i} forest. Few comparable figures were found in the literature. In northern Burkina Faso, both genera constructed 20 tons of sheetings ha-1 mon-1 after mulching with a straw-wood mixture (Mando \& Miedema 1997), and in Senegal, around 10 tons ha-1 mon-1 were moved in heavily foraged plots (Rouland et al. 2003). Within a site, soil turn-over and the number of foraging holes created was always highest in hay, followed by compost, then by wood and in the end control. The fungus-growers' foraging-activity was leading to an enormous increase in surface pore space - after one month of induced foraging activity in hay-plots, the median number of foraging-holes increased from 142 m-2 in the degraded site up to 921 m-2 in the old Za{\"i} forest. The creation of subterranean galleries and macropores significantly increased the water infiltration rate by a mean factor 2-4. Laboratory analyses revealed that sheeting-soil differed strongly from the respective control soil as well as between the seasons, the food-type covered, and the two genera. Odontotermes-sheetings differed in more parameters than Macrotermes-sheetings, and dry season sheetings differed in more parameters (and more strongly) than rainy season sheetings. In the present study, soil organic matter, carbon and nitrogen contents were significantly increased in all dry season sheetings; in the rainy season mainly in those built on compost. Texture analysis pointed out that both genera used topsoil and soil from deeper horizons in varying mixture ratios, thereby supporting findings of Jouquet et al. (2006). To summarize, the present thesis contributes to a better understanding of the functional response traits of termites and ants to changing environmental parameters resulting from increasing human impact. The RAP-protocol represents an easy-to-learn and very effective method to representatively characterize, compare and monitor the taxonomic and functional diversity of termites and ants. The experiment has provided conclusive evidence of the importance of the consideration of fungus-growing termites (particularly Odontotermes and Macrotermes species) when aiming to restore infertile, degraded and crusted soils and to maintain a sustainable agricultural production in the Sahel-Sudanese zone of West Africa.}, subject = {Termiten}, language = {en} } @phdthesis{Grohmann2010, author = {Grohmann, Constanze}, title = {Termite mediated heterogeneity of soil and vegetation patternsin a semi-arid savanna ecosystem in Namibia}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54318}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Termites are the most important soil ecosystem engineers of semi-arid and arid habitats. They enhance decomposition processes as well as the subsequent mineralisation of nutrients by bacteria and fungi. Through their construction of galleries, nests and mounds, they promote soil turnover and influence the distribution of nutrients and also alter texture and hydrological properties of soils, thereby affecting the heterogeneity of their ecosystem. The main aim of the present thesis was to define the impact of termites on ecosys-tem functioning in a semi-arid ecosystem. In a baseline study, I assessed the diversity of termite taxa in relation to the amount of precipitation, the vegetation patterns and the land use systems at several sites in Namibia. Subsequently, I focussed on a species that is highly abundant in many African savannas, the fungus growing and mound building species Macro-termes michaelseni (Sj{\"o}stedt, 1914). I asked how this species influences the spatial hetero-geneity of soil and vegetation patterns. From repeated samplings at 13 sites in Namibia, I obtained 17 termite taxa of 15 genera. While the type of land use seems to have a minor effect on the termite fauna, the mean annual precipitation explained 96\% and the Simpson index of vascular plant diversity 81\% of the variation in taxa diversity. The number of termite taxa increased with both of these explanation variables. In contrast to former studies on Macrotermes mounds in several regions of Africa that I reviewed, soil analyses from M. michaelseni mounds in the central Namibian savanna revealed that they contain much higher nitrogen contents when compared to their parent material. Further analyses revealed that nitrate forms a major component of the nitrogen content in termite mounds. As nitrate solves easily in water, evaporation processes are most probably responsible for the transport of solved nitrates to the mound surface and their accumulation there. The analysed mounds in central Namibia contained higher sand propor-tions compared to the mounds of the former studies. Through the higher percentage of coarse and middle sized pores, water moves more easily in sandy soils compared to more clayey soils. In consequence, evaporation-driven nitrate accumulation can occur in the studied mounds at high rates. Hochgerechnet auf den Gesamtumfang der H{\"u}gel bedeckte das pro Jahr von einem bewohnten H{\"u}gel erodierte Material theoretisch einen 1 m breiten Kreisring um den Schwemmkegel des H{\"u}gels 2,4 mm hoch. Der entsprechende Wert f{\"u}r unbewohnte H{\"u}gel betrug 1,0 mm. To assess the amount of soil that erodes from termite mounds, I fastened four strong, 65 cm wide plastic bags at 14 mounds each and collected the soil that eroded during five rainfall events. Projected to the total mound circumference, the amount of soil eroded covers theoretically a 1 m wide circular ring around the pediment of an inhabited mound up to a height of 2.4 mm per year. For uninhabited mounds, the height of this soil layer would be 1.0 mm. Per hectare, roughly 245 kg eroded per year from the mounds. However, as the erosion rate depends on several factors such as rainfall intensity, soil texture and point of time within the rainy season, this is only a vague estimate. In order to determine up to which distance the soil erosion from the mounds still influences the chemical characteristics of the adjacent topsoil, I took samples from depth of 0-10 cm at 1, 5 and 25 m distances, respectively, from four different mounds and from the mounds themselves. The non-metric multidimensional scaling of the soil properties showed strong differences between mound and off-mound samples. Soil characteristics within the samples from the mounds did not differ largely. Similarly, I found no strong differences between the samples taken from the different distances from the mound. From these results I conclude that through the construction of foraging galleries and sheetings (soil constructions with which some termite species cover their food items), the soil eroding from termite mounds is quickly mixed with deeper soil layers. In consequence, mound material does not accumulate in the mound's vicinity. In order to reveal how plant growth is influenced by termite mound material, we assessed the number of grass and herb individuals as well as the biomass of plants growing in situ on the base of mounds compared to adjacent sites. While the numbers of both grass and herb individuals were significantly lower compared to adjacent sites, the total biomass of plants growing on the base of mounds was significantly higher. Reverse results were obtained by pot experiments with radish (Raphanus sativus subsp. sativus) and sorghum (Sorghum sp.) growth. Both species grew significantly weaker on mound soil compared to adjacent soil. The contradictory results concerning the biomass of in situ and pot experi-ments are most probably caused by the disturbance of the original soil structure during the potting process. The material was subsequently compacted through watering the plants. In contrast, Macrotermes mounds are pervaded by many macropores which seem to be essential for the plant roots to penetrate the soil. In the last part of this thesis, I posed the question how mounds of M. michaelseni are distributed and what factors might be responsible for this pattern. Former studies showed that mound size is correlated with the size of its inhabiting colony. With several multi-scale analyses, I revealed that larger inhabited mounds were regularly distributed. Additionally, mounds which were closer together tended to be smaller than on average. This indicates that intraspecific competition controls the distribution and size of colonies and their mounds. Former studies concerning Odontotermes mounds substantiated that they are local hotspots of primary productivity and animal abundance. Based on these findings, simulations revealed that a regular distribution of these mounds leads to a greater ecosystem-wide productivity compared to a random arrangement. As in the present study, plant biomass was higher at the mounds compared to off-mound sites, this might hold true for M. michaelseni mounds. From the results of this thesis, I draw the conclusion that through their mound building activities, M. michaelseni strongly influences the distribution patterns of soil nutrients within the central Namibian savanna. These termites create sharp contrasts in nutrient levels and vegetation patterns between mound soils and off-mound soils and enhance the heterogeneity of their habitats. Former studies revealed that habitat hetero-geneity is important in generating species diversity and species richness in turn is correlated positively with biomass production and positively affects ecosystem services. In conclusion, the present thesis underlines the importance of M. michaelseni for ecosystem functioning of the central Namibian savanna.}, subject = {Termiten}, language = {en} } @phdthesis{Mildner2018, author = {Mildner, Stephanie}, title = {Temporal organization in \(Camponotus\) \(ants\): endogenous clocks and zeitgebers responsible for synchronization of task-related circadian rhythms in foragers and nurses}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149382}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {The rotation of the earth around its axis causes recurring and predictable changes in the environment. To anticipate those changes and adapt their physiology and behavior accordingly, most organisms possess an endogenous clock. The presence of such a clock has been demonstrated for several ant species including Camponotus ants, but its involvement in the scheduling of daily activities within and outside the ant nest is fairly unknown. Timing of individual behaviors and synchronization among individuals is needed to generate a coordinated collective response and to maintain colony function. The aim of this thesis was to investigate the presence of a circadian clock in different worker castes, and to determine the daily timing of their behavioral tasks within the colonies of two nectar-collecting Camponotus species. In chapter I, I describe the general temporal organization of work throughout the worker life in the species Camponotus rufipes. Continuous tracking of behavioral activity of individually- marked workers for up to 11 weeks in subcolonies revealed an age-dependent division of labor between interior and exterior workers. After eclosion, the fairly immobile young ants were frequently nurtured by older nurses, yet they started nursing the brood themselves within the first 48 hours of their life. Only 60\% of workers switched to foraging at an age range of one to two weeks, likely because of the reduced needs within the small scale of the subcolonies. Not only the transition rates varied between subcolonies, but also the time courses of the task sequences between workers did, emphasizing the timed allocation of workers to different tasks in response to colony needs. Most of the observed foragers were present outside the nest only during the night, indicating a distinct timing of this behavioral activity on a daily level as well. As food availability, humidity and temperature levels were kept constant throughout the day, the preference for nocturnal activity seems to be endogenous and characteristic for C. rufipes. The subsequent monitoring of locomotor activity of workers taken from the subcolonies revealed the presence of a functional endogenous clock already in one-day old ants. As some nurses displayed activity rhythms in phase with the foraging rhythm, a synchronization of these in-nest workers by social interactions with exterior workers can be hypothesized. Do both castes use their endogenous clock to schedule their daily activities within the colony? In chapter II, I analyzed behavioral activity of C. rufipes foragers and nurses within the social context continuously for 24 hours. As time-restricted access to food sources may be one factor affecting daily activities of ants under natural conditions, I confronted subcolonies with either daily pulses of food availability or ad libitum feeding. Under nighttime and ad libitum feeding, behavioral activity of foragers outside the nest was predominantly nocturnal, confirming the results from the simple counting of exterior workers done in chapter I. Foragers switched to diurnality during daytime feeding, demonstrating the flexible and adaptive timing of a daily behavior. Because they synchronized their activity with the short times of food availability, these workers showed high levels of inactivity. Nurses, in contrast, were active all around the clock independent of the feeding regime, spending their active time largely with feeding and licking the brood. After the feeding pulses, however, a short bout of activity was observed in nurses. During this time period, both castes increasingly interacted via trophallaxis within the nest. With this form of social zeitgeber, exterior workers were able to entrain in-nest workers, a phenomenon observed already in chapter I. Under the subsequent monitoring of locomotor activity under LD conditions the rhythmic workers of both castes were uniformly nocturnal independent of the feeding regime. This endogenous activity pattern displayed by both worker castes in isolation was modified in the social context in adaption to task demands. Chapter III focuses on the potential factors causing the observed plasticity of daily rhythms in the social context in the ant C. rufipes. As presence of brood and conspecifics are likely indicators of the social context, I tested the effect of these factors on the endogenous rhythms of otherwise isolated individuals. Even in foragers, the contact to brood triggered an arrhythmic activity pattern resembling the arrhythmic behavioral activity pattern seen in nurses within the social context. As indicated in chapter I and II, social interaction could be one crucial factor for the synchronization of in nest activities. When separate groups were entrained to phase-shifted light-dark-cycles and monitored afterwards under constant conditions in pairwise contact through a mesh partitioning, both individuals shifted parts of their activity towards the activity period of the conspecific. Both social cues modulated the endogenous rhythms of workers and contribute to the context dependent plasticity in ant colonies. Although most nursing activities are executed arrhythmically throughout the day (chapter II), previous studies reported rhythmic translocation events of the brood in Camponotus nurses. As this behavior favors brood development, the timing of the translocations within the dark nest seems to be crucial. In chapter IV, I tracked translocation activity of all nurses within subcolonies of C. mus. Under the confirmed synchronized conditions of a LD-cycle, the daily pattern of brood relocation was based on the rhythmic, alternating activity of subpopulations with preferred translocation direction either to the warm or to the cold part of the temperature gradient at certain times of the day. Although the social interaction after pulse feeding had noticeable effects on the in-nest activity in C. rufipes (chapter I and II), it was not sufficient to synchronize the brood translocation rhythm of C. mus under constant darkness (e.g. when other zeitgebers were absent). The free-running translocation activity in some nurses demonstrated nevertheless the involvement of an endogenous clock in this behavior, which could be entrained under natural conditions by other potential non-photic zeitgebers like temperature and humidity cycles. Daily cycling of temperature and humidity could not only be relevant for in-nest activities, but also for the foraging activity outside the nest. Chapter V focuses on the monitoring of field foraging rhythms in the sympatric species C. mus and C. rufipes in relation to abiotic factors. Although both species had comparable critical thermal limits in the laboratory, foragers in C. mus were strictly diurnal and therefore foraged under higher temperatures than the predominant nocturnal foragers in C. rufipes. Marking experiments in C. rufipes colonies with higher levels of diurnal activity revealed the presence of temporally specialized forager subpopulations. These results suggest the presence of temporal niches not only between the two Camponotus species, but as well between workers within colonies of the same species. In conclusion, the temporal organization in colonies of Camponotus ants involves not only the scheduling of tasks performed throughout the worker life, but also the precise timing of daily activities. The necessary endogenous clock is already functioning in all workers after eclosion. Whereas the light-dark cycle and food availability seem to be the prominent zeitgebers for foragers, nurses may rely more on non-photic zeitgeber like social interaction, temperature and humidity cycles.}, subject = {circadian clocks}, language = {en} } @phdthesis{Roth2021, author = {Roth, Nicolas M{\´e}riadec Max Andr{\´e}}, title = {Temporal development of communities with a focus on insects, in time series of one to four decades}, doi = {10.25972/OPUS-23549}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235499}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Changes and development are fundamental principles in biocenoses and can affect a multitude of ecological processes. In insect communities phenological and density changes, changes in species richness and community composition, as well as interactions between those changes, are the most important macro processes. However, climate change and other factors like habitat degradation and loss alter these processes leading to shifts and general biodiversity declines. Even though knowledge about insect decline in central Europe increased during the last decades, there are significant knowledge gaps about the development of insect communities in certain habitats and taxa. For example, insect communities in small lentic as well as in forested habitats are under-sampled and reported to be less endangered than communities in other habitats. Furthermore, the changes within habitats and taxa are additionally influenced by certain traits, like host or feeding specialization. To disentangle these influences and to increase the knowledge about the general long-term development of insect communities, comprehensive long-term monitoring studies are needed. In addition, long-term effects of conservation strategies should also be evaluated on large time scales in order to be able to decide on a scientific base which strategies are effective in promoting possibly declining taxa. Hence, this thesis also tackles the effects of an integrative conservation strategy on wood dependent beetle and fungi, beside the development of water beetle and macro moth communities over multiple decades. In Chapter 2 I present a study on the development of water beetle communities (Dytiscidae, Haliplidae, Noteridae) in 33 water bodies in Southern Germany from 1991 to 2018. Time-standardized capture per waterbody was used during three periods: between 1991 and 1995, 2007 and 2008, and 2017 and 2018. Results showed annual declines in both species number (ca. -1\%) and abundance (ca. -2\%). In addition, community composition shifted over time in part due to changing pH values. Hence, the recorded changes during the 28-year study period partly reflect natural succession processes. However, since also moor-related beetle species decreased significantly, it is likely that water beetles in southern Germany are also threatened by non-successional factors, including desiccation, increased nitrogen input and/or mineralization, as well as the loss of specific habitats. The results suggest, that in small to midsize lentic waterbodies, current development should aim for constant creation of new water bodies and protection of moor waterbodies in order to protect water beetle communities on a landscape scale. In Chapter 3 I present an analysis of the development of nocturnal macro moth species richness, abundance and biomass over four decades in forests of southern Germany. Two local scale data sets featuring a coppiced oak forest as well as an oak high forest were analysed separately from a regional data set representing all forest types in the temperate zone of Central Europe. At the regional scale species richness, abundance and biomass showed annual declines of ca. 1 \%, 1.3 \% and 1.4 \%, respectively. These declines were more pronounced in plant host specialists and in dark coloured species. In contrast, species richness increased by ca. 1.5 \% annually in the coppiced forest, while no significant trends were found in the high forest. In contrast to past assumptions, insect decline apparently affects also hyper diverse insect groups in forests. Since host specialists and dark coloured species were affected more heavily by the decline than other groups, habitat loss and climate change seem to be potential drivers of the observed trends. However, the positive development of species richness in the coppiced oak forest indicates that maintaining complex and diverse forest ecosystems through active management might compensate for negative trends in biodiversity. Chapter 4 features a study specifically aiming to investigate the long-term effect of deadwood enrichment as an integrative conservation strategy on saproxylic beetles and fungi in a central European beech forest at a landscape scale. A before-after control-impact design, was used to compare assemblages and gamma diversities of saproxylic organisms (beetles and fungi) in strictly protected old-growth forest areas (reserves) and previously moderately and intensively managed forest areas. Forests were sampled one year before and a decade after starting a landscape-wide strategy of dead-wood enrichment. Ten years after the start of the dead-wood enrichment, neither gamma diversities of saproxylic organisms nor species composition of beetles did reflect the previous management types anymore. However, fungal species composition still mirrored the previous management gradient. The results demonstrated that intentional enrichment of dead wood at the landscape scale can effectively restore communities of saproxylic organisms and may thus be a suitable strategy in addition to permanent strict reserves in order to protect wood dependent organisms in Europe. In this thesis I showed, that in contrast to what was assumed and partly reported so far, also water beetles in lentic water bodies and macro moths in forests decreased in species richness, abundance and biomass during the last three to four decades. In line with earlier studies, especially dark coloured species and specialists decreased more than light-coloured species and generalists. The reasons for these declines could partly be attributed to natural processes and pollution and possibly to climate change. However, further studies, especially experimental ones, will be needed to achieve a better understanding of the reasons for insect decline. Furthermore, analyses of time series data should be interpreted cautiously especially if the number of sampling years is smaller than ten years. In addition, validation techniques such as left- and right- censoring and cross validation should be used in order to proof the robustness of the analyses. However, the lack of knowledge, we are still facing today, should not prevent scientists and practitioners from applying conservation measures. In order to prove the effectiveness of such measures, long-term monitoring is crucial. Such control of success is essential for evidence based and thus adapted conservation strategies of threatened organisms.}, subject = {climate change}, language = {en} } @phdthesis{Koenig2024, author = {K{\"o}nig, Sebastian Thomas}, title = {Temperature-driven assembly processes of Orthoptera communities: Lessons on diversity, species traits, feeding interactions, and associated faecal microorganisms from elevational gradients in Southern Germany (Berchtesgaden Alps)}, doi = {10.25972/OPUS-35460}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-354608}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Chapter I: Introduction Temperature is a major driver of biodiversity and abundance patterns on our planet, which becomes particularly relevant facing the entanglement of an imminent biodiversity and climate crisis. Climate shapes the composition of species assemblages either directly via abiotic filtering mechanisms or indirectly through alterations in biotic interactions. Insects - integral elements of Earth's ecosystems - are affected by climatic variation such as warming, yet responses vary among species. While species' traits, antagonistic biotic interactions, and even species' microbial mutualists may determine temperature-dependent assembly processes, the lion's share of these complex relationships remains poorly understood due to methodological constraints. Mountains, recognized as hotspots of diversity and threatened by rapidly changing climatic conditions, can serve as natural experimental settings to study the response of insect assemblages and their trophic interactions to temperature variation, instrumentalizing the high regional heterogeneity of micro- and macroclimate. With this thesis, we aim to enhance our mechanistic understanding of temperature-driven assembly processes within insect communities, exemplified by Orthoptera, that are significant herbivores in temperate mountain grassland ecosystems. Therefore, we combined field surveys of Orthoptera assemblages on grassland sites with molecular tools for foodweb reconstruction, primarily leveraging the elevational gradients offered by the complex topography within the Berchtesgaden Alpine region (Bavaria, Germany) as surrogate for temperature variation (space-for-time substitution approach). In this framework, we studied the effects of temperature variation on (1) species richness, abundance, community composition, and interspecific as well as intraspecific trait patterns, (2) ecological feeding specialisation, and (3) previously neglected links to microbial associates found in the faeces. Chapter II: Temperature-driven assembly processes Climate varies at multiple scales. Since microclimate is often overlooked, we assessed effects of local temperature deviations on species and trait compositions of insect communities along macroclimatic temperature gradients in Chapter II. Therefore, we employed joint species distribution modelling to explore how traits drive variation in the climatic niches of Orthoptera species at grassland sites characterized by contrasting micro- and macroclimatic conditions. Our findings revealed two key insights: (1) additive effects of micro- and macroclimate on the diversity, but (2) interactive effects on the abundance of several species, resulting in turnover and indicating that species possess narrower climatic niches than their elevational distributions might imply. This chapter suggests positive effects of warming on Orthoptera, but also highlights that the interplay of macro- and microclimate plays a pivotal role in structuring insect communities. Thus, it underscores the importance of considering both elements when predicting the responses of species to climate change. Additionally, this chapter revealed inter- and intraspecific effects of traits on the niches and distribution of species. Chapter III: Dietary specialisation along climatic gradients A crucial trait linked to the position of climatic niches is dietary specialisation. According to the 'altitudinal niche-breadth hypothesis', species of high-elevation habitats should be less specialized compared to their low-elevation counterparts. However, empirical evidence on shifts in specialization is scarce for generalist insect herbivores and existing studies often fail to control for the phylogeny and abundance of interaction partners. In Chapter III, we used a combination of field observations and amplicon sequencing to reconstruct dietary relationships between Orthoptera and plants along an extensive temperature gradient. We did not find close but flexible links between individual grasshopper and plant taxa in space. While interaction network specialisation increased with temperature, the corrected dietary specialisation pattern peaked at intermediate elevations on assemblage level. These nuanced findings demonstrate that (1) resource availability, (2) phylogenetic relationships, and (3) climate can affect empirical foodwebs intra- and interspecifically and, hence, the dietary specialisation of herbivorous insects. In this context, we discuss that the underlying mechanisms involved in shaping the specialisation of herbivore assemblages may switch along temperature clines. Chapter IV: Links between faecal microbe communities, feeding habits, and climate Since gut microbes affect the fitness and digestion of insects, studying their diversity could provide novel insights into specialisation patterns. However, their association with insect hosts that differ in feeding habits and specialisation has never been investigated along elevational climatic gradients. In Chapter IV, we utilized the dietary information gathered in Chapter III to characterize links between insects with distinct feeding behaviour and the microbial communities present in their faeces, using amplicon sequencing. Both, feeding and climate affected the bacterial communities. However, the large overlap of microbes at site level suggests that common bacteria are acquired from the shared feeding environment, such as the plants consumed by the insects. These findings emphasize the influence of a broader environmental context on the composition of insect gut microbial communities. Chapter V: Discussion \& Conclusions Cumulatively, the sections of this dissertation provide support for the hypothesis that climatic conditions play a role in shaping plant-herbivore systems. The detected variation of taxonomic and functional compositions contributes to our understanding of assembly processes and resulting diversity patterns within Orthoptera communities, shedding light on the mechanisms that structure their trophic interactions in diverse climates. The combined results presented suggest that a warmer climate could foster an increase of Orthoptera species richness in Central European semi-natural grasslands, also because the weak links observed between insect herbivores and plants are unlikely to limit decoupled range shifts. However, the restructuring of Orthoptera communities in response to warmer temperatures depends on species' traits such as moisture preferences or phenology. Notably, we were able to demonstrate a crucial role of microclimate for many species, partly unravelling narrower climatic niches than their elevational ranges suggest. We found evidence that not only Orthoptera community composition, specialisation, and traits varied along elevational gradients, but even microbial communities in the faeces of Orthoptera changed, which is a novel finding. This complex restructuring and reassembly of communities, coupled with the nonlinear specialisation of trophic interactions and a high diversity of associated bacteria, emphasize our currently incomplete comprehension of how ecosystems will develop under future climatic conditions, demanding caution in making simplified predictions for biodiversity change under climate warming. Since these predictions may benefit from including biotic interactions and both, micro- and macroclimate based on our findings, conservation authorities and practitioners must not neglect improving microclimatic conditions to ensure local survival of a diverse set of threatened and demanding species. In this context, mountains can play a pivotal role for biodiversity conservation since these offer heterogeneous microclimatic conditions in proximity that can be utilized by species with distinct niches.}, subject = {Heuschrecken}, language = {en} } @phdthesis{Reintjes2004, author = {Reintjes, Norbert}, title = {Taxonomy, faunistics and life-history traits of Dytiscidae and Noteridae (Coleoptera) in a West African savannah}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-10287}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {The studies inventoried the species of the families Dytiscidae and Noteridae (Coleoptera) in Como{\´e} National Park in northern Ivory Coast, West Africa and investigated the ecological role of temporary and permanent water bodies for the aestivation of these aquatic beetles. The ecological studies focused on the question how the beetles cope with the temporary loss of their aquatic habitats during dry season. The climate in the study area is characterised by a pronounced dry season from about November to March/April, in which the temporary ponds and creeks in the savannah entirely desiccate. The only available water bodies during dry season in Como{\´e} National Park are the Como{\´e} River, pools in some of its tributaries, and a few of the large savannah ponds. The taxonomic and faunistic analysis revealed a high species richness in the study area and yielded a total of twelve species of Noteridae in four genera and 95 species of Dytiscidae in 22 genera. Thirty of these species had not yet been reported from the Ivory Coast. A description of a new species in the genus Laccophilus is given, named L. comoensis in honour of the National Park. Strong incidences exist that the material includes more species yet unknown to science. Concerning the mode of aestivation, observations in pilot studies led to the working hypothesis that the beetles pass the dry season as adults in aquatic habitats. Consequently, presence of adults in aquatic habitats throughout the dry season and cyclic migration of adults between temporary and permanent water bodies was expected. Regular sampling of water bodies throughout the dry season and beginning rainy season yielded 33,705 individuals in 72 species and 26 genera. In all the sample periods Noteridae and / or Dytiscidae were recorded. The number of species per period was between 36 and 58. It is concluded that in Como{\´e} National Park a) at least parts of the populations of the recorded species pass the dry season as adults and b) aquatic habitats serve as a refuge for aestivation of these adult beetles. In a rocky area in the riverbed of the permanent Como{\´e} River four sets of studies were performed during dry and beginning rainy season. According to the working hypothesis beetles should be searching for adequate aquatic habitats as long as temporary savannah waters are becoming inhospitable and are falling dry. Seven rock pools in the riverbed of the Como{\´e} River were artificially filled and thus offered for colonization at the peak of the dry season (end of January). After five days the rock pools were quantitatively sampled by completely emptying them. All the rock pools were colonized by Dytiscidae and / or Noteridae and with a total of 1,507 individuals in 26 species abundance and diversity were high. Habitats for aestivation are needed most, when the majority of the savannah waters are fallen dry. Little precipitation on February 18th 1999 had filled rock pools in the riverbed of the Como{\´e} River but no pools in the savannah, where the rain was immediately absorbed by the very dry soil. An inventory of beetles was performed in 21 naturally filled rock pools five to 20 days after this precipitation. The sampling yielded 8,456 individuals in 41 species. Except the smallest, all rock pools contained beetles. The result showed that Dytiscidae and Noteridae utilise the rock pools as aquatic habitat during dry season. Beetles adapted to a highly seasonal environment like the aquatic system in the study area should be good colonizers. Sampling of four, respectively five rock pools at two occasions within 24 hours after the start of precipitation examined the potential of colonizers at that period (March). Prior to these precipitations the pools had been completely dry. Dytiscidae were already present in all rock pools and a total of 434 Dytiscidae in 14 species was found. The working hypothesis of cyclic migration suggests that the beetles should leave the rock pools at the onset of the rainy season when precipitation had filled temporary water bodies in the savannah. After several precipitation events an inventory of 13 rock pools of the Como{\´e} River in May controlled for adult beetles. Only four species with 126 individuals were still found, of which Yolina chopardi contributed 81.7\%. This species seems to differ from the other recorded species in the use of habitats, since it was never recorded in the savannah. In general, however, diversity and abundance of Dytiscidae and Noteridae in the rock pools, as expected, was low after the onset of the rainy season. During the entire study of the rock pools in the riverbed of the Como{\´e} River 10,523 individuals in 44 species and 18 genera were collected. Thus, more than half of the species recorded in Como{\´e} National Park were found in the rock pools. The results suggest that the Como{\´e} River and the rock pools in the riverbed serve as aquatic retreat for adult Dytiscidae and Noteridae during dry season when temporary water bodies in the savannah are desiccated. The suggested cyclic migration between water bodies predicts that newly formed savannah waters are recolonized by the beetles at the onset of the rainy season. This colonization should be a) by adults and b) airborne. Two artificial ponds in the open savannah were offered only for aerial colonization at the beginning of the rainy season. The ponds were controlled for adult Noteridae and Dytiscidae daily during one continuous phase of eleven and a second one of 16 days (end of March to end of April). On every sampling date Noteridae or Dytiscidae were recorded. In the entire study 2,744 individuals in 44 species and 16 genera were collected. After precipitation, abundance and species richness increased. Thirty-five of the encountered species had been recorded in rock pools of the Como{\´e} River before. The principal species in the artificial savannah ponds had been principal species in samplings of the rock pools as well. The results support the hypothesis of cyclic migration: most species of Dytiscidae and Noteridae of the Como{\´e} National Park fly from desiccating savannah waters to permanent water bodies or water bodies holding water for extended times during dry season. They pass the dry season in these waters and fly back into the savannah after precipitation at the onset of the rainy season. Exceptions from this general rule are discussed.}, subject = {Schwimmk{\"a}fer}, language = {en} } @phdthesis{Jung2016, author = {Jung, Lisa Anna}, title = {Targeting MYC Function as a Strategy for Tumor Therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146993}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues. In this study, OmoMYC's mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC. The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1. In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy.}, subject = {Myc}, language = {en} } @phdthesis{Gotthard2023, author = {Gotthard, Hannes}, title = {Targeting Colorectal Cancer Stem Cells with Hemibodies}, doi = {10.25972/OPUS-30309}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303090}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration.}, subject = {Monoklonaler bispezifischer Antik{\"o}rper}, language = {en} } @phdthesis{Adhikari2024, author = {Adhikari, Bikash}, title = {Targeted degradation of Myc-interacting oncoproteins}, doi = {10.25972/OPUS-31732}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317326}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc's structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel-Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field. In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines. In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs. In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs. Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy.}, subject = {Degradation}, language = {en} } @phdthesis{Bruehlmann2017, author = {Br{\"u}hlmann, David}, title = {Tailoring Recombinant Protein Quality by Rational Media Design}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147345}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing.}, subject = {Zellkultur}, language = {en} } @phdthesis{Boyanova2012, author = {Boyanova, Desislava Veselinova}, title = {Systems biological analysis of the platelet proteome and applications of functional module search in proteome networks}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72165}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Recent development of proteomic approaches and generation of large-scale proteomic datasets calls for new methods for biological interpretation of the obtained results. Systems biological approaches such as integrated network analysis and functional module search have become an essential part of proteomic investigation. Proteomics is especially applied in anucleate cells such as platelets. The underlying molecular mechanisms of platelet activation and their pharmacological modulation are of immense importance for clinical research. Advances in platelet proteomics have provided a large amount of proteomic data, which has not yet been comprehensively investigated in a systems biological perspective. To this end, I assembled platelet specific data from proteomic and transcriptomic studies by detailed manual curation and worked on the generation of a comprehensive human platelet repository for systems biological analysis of platelets in the functional context of integrated networks (PlateletWeb) (http:/PlateletWeb.bioapps.biozentrum.uni-wuerzburg.de). I also added platelet-specific experimentally validated phosphorylation data and generated kinase predictions for 80\% of the newly identified platelet phosphosites. The combination of drug, disease and pathway information with phosphorylation and interaction data makes this database the first integrative platelet platform available for platelet research. PlateletWeb contains more than 5000 platelet proteins, which can also be analyzed and visualized in a network context, allowing identification of all major signaling modules involved in platelet activation and inhibition. Using the wealth of integrated data I performed a series of platelet-specific analyses regarding the platelet proteome, pathways, drug targets and novel platelet phosphorylation events involved in crucial signaling events. I analyzed the statistical enrichment of known pathways for platelet proteins and identified endocytosis as a highly represented pathway in platelets. Further results revealed that highly connected platelet proteins are more often targeted by drugs. Using integrated network analysis offered by PlateletWeb, I analyzed the crucial activation signaling pathway of adenosine diphosphate (ADP), visualizing how the signal flow from receptors to effectors is maintained. My work on integrin inside-out signaling was also based on the integrated network approach and examined new platelet-specific phosphorylation sites and their regulation using kinase predictions. I generated hypothesis on integrin signaling, by investigating the regulation of Ser269 phosphorylation site on the docking protein 1 (DOK1). This phosphorylation site may influence the inhibiting effect of DOK1 on integrin a2bb3. Extending the integrated network approach to further cell lines, I used the assembled human interactome information for the analysis of functional modules in cellular networks. The investigation was performed with a previously developed module detection algorithm, which finds maximum-scoring subgraphs in transcriptomic datasets by using assigned values to the network nodes. We extended the algorithm to qualitative proteomic datasets and enhanced the module search by adding functional information to the network edges to concentrate the solution onto modules with high functional similarity. I performed a series of analyses to validate its performance in small-sized (virus-infected gastric cells) and medium-sized networks (human lymphocytes). In both cases the algorithm extracted characteristic modules of sample proteins with high functional similarity. The functional module search is especially useful in site-specific phosphoproteomic datasets, where kinase regulation of the detected sites is often sparse or lacking. Therefore, I used the module detection algorithm in quantitative phosphoproteomic datasets. In a platelet phosphorylation dataset, I presented a pipeline for network analysis of detected phosphorylation sites. In a second approach, the functional module detecting algorithm was used on a phosphoproteome network of human embryonic stem cells, in which nodes represented the maximally changing phosphorylation sites in the experiment. Additional kinases from the human phosphoproteome in PlateletWeb were included to the network to investigate the regulation of the signal flow. Results indicated important phosphorylation sites and their upstream kinases and explained changes observed in embryonic stem cells during differentiation. This work presents novel approaches for integrated network analysis in cells and introduces for the first time a systematic biological investigation of the human platelet proteome based on the platelet-specific knowledge base PlateletWeb. The extended methods for optimized functional module detection offer an invaluable tool for exploring proteomic datasets and covering gaps in complex large-scale data analysis. By combining exact module detection approaches with functional information data between interacting proteins, characteristic functional modules with high functional resemblance can be extracted from complex datasets, thereby focusing on important changes in the observed networks.}, subject = {Netzwerkanalyse}, language = {en} } @phdthesis{AbdelRahman2003, author = {Abdel Rahman, Faisal Mirghani}, title = {Systematic analysis of genes expressed in the retinal pigment epithelium (RPE) and identification of candidates for genetic susceptibility to age-related macular degeneration (AMD)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-7053}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2003}, abstract = {Age related macular degeneration (AMD) is the leading cause of visual impairment in the elderly and the major cause of blindness in the developed world. To date, the molecular mechanisms underlying the disease are not well understood although in recent years a primary involvement of the retinal pigment epithelium (RPE) has become evident. The aim of the present study is to systematically analyse genes which are differentially expressed in the RPE, and to assess their possible association with mechanisms and pathways likely to be related to retinal disease, in particular AMD. Towards this goal, 2379 expressed sequence tags (ESTs) were established from an inhouse generated RPE cDNA library. This library was constructed by using the suppression subtraction hybridization (SSH) technique which normalises redundant sequences and ensures enrichment of rare transcripts. In a first phase, 1002 ESTs were sequenced and subjected to comprehensive alignment with public nucleotide and protein databases. A search of the 1002 ESTs against the human genome draft sequence yielded 168 known genes, 51 predicted genes, 15 unknown transcripts and 41 clones with no significant similarity. Reverse Northern blot hybridization was performed for 318 EST clusters to identify abundantly expressed genes in the RPE and to prioritize subsequent analyses. Representative clones were spotted onto a nylon membrane and hybridized with cDNA probes of driver (heart and liver) and tester (RPE) used in the cDNA library construction. Subsequently, 107 EST clusters were subjected to Northern blot hybridizations. These analyses identified 7 RPE-specific, 3 retina-specific, 7 RPE/retina-specific, and 7 tissue restricted transcripts, while 29 EST clusters were ubiquitously expressed, and evaluation was not possible for another 54 EST clusters. Of the 24 transcripts with specific or restricted expression, 16 clones were selected for further characterization. The predicted gene MGC2477 and 2 novel isoforms of the human transient receptor potential cation channel, subfamily M, member 3 (TRPM3) were cloned and further described in detail. In addition, polymorphic variations for these 2 genes as well as for the human MT-Protocadherin gene were determined. For MGC2477, 15 single nucleotide polymorphisms (SNPs) were identified, with 13 having a frequency of the minor allele greater than 20\%. 10 of the 15 SNPs have not been reported in so far in public SNP repertoires. Partial assessment of the TRPM3 gene yielded 35 SNPs. Of these, 30 (85.7\%) were highly frequent (0.17-0.5\%), and 14 (40\%) were novel. The MT-Protocadherin gene revealed 35 SNPs, including 28 (80\%) with high frequency of the minor allele. 23 (65.7\%) were novel SNPs. These SNPs will be used to construct the most common haplotypes. These will be used in case/control association studies in 400 AMD patients and 200 ethnically and aged matched controls to assess a possible contribution of these genes in the etiology of AMD.}, subject = {Senile Makuladegeneration / Pigmentepithel / Genexpression}, language = {en} } @phdthesis{Grebinyk2021, author = {Grebinyk, Anna}, title = {Synergistic Chemo- and Photodynamic Treatment of Cancer Cells with C\(_{60}\) Fullerene Nanocomplexes}, doi = {10.25972/OPUS-22207}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-222075}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Recent progress in nanotechnology has attracted interest to a biomedical application of the carbon nanoparticle C60 fullerene (C60) due to its unique structure and versatile biological activity. In the current study the dual functionality of C60 as a photosensitizer and a drug nanocarrier was exploited to improve the efficiency of chemotherapeutic drugs towards human leukemic cells. Pristine C60 demonstrated time-dependent accumulation with predominant mitochondrial localization in leukemic cells. C60's effects on leukemic cells irradiated with high power single chip LEDs of different wavelengths were assessed to find out the most effective photoexcitation conditions. A C60-based noncovalent nanosized system as a carrier for an optimized drug delivery to the cells was evaluated in accordance to its physicochemical properties and toxic effects. Finally, nanomolar amounts of C60-drug nanocomplexes in 1:1 and 2:1 molar ratios were explored to improve the efficiency of cell treatment, complementing it with photodynamic approach. A proposed treatment strategy was developed for C60 nanocomplexes with the common chemotherapeutic drug Doxorubicin, whose intracellular accumulation and localization, cytotoxicity and mechanism of action were investigated. The developed strategy was revealed to be transferable to an alternative potent anticancer drug - the herbal alkaloid Berberine. Hereafter, a strong synergy of treatments arising from the combination of C60-mediated drug delivery and C60 photoexcitation was revealed. Presented data indicate that a combination of chemo- and photodynamic treatments with C60-drug nanoformulations could provide a promising synergetic approach for cancer treatment.}, subject = {cancer}, language = {en} } @phdthesis{Stieb2011, author = {Stieb, Sara Mae}, title = {Synaptic plasticity in visual and olfactory brain centers of the desert ant Cataglyphis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85584}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {W{\"u}stenameisen der Gattung Cataglyphis wurden zu Modellsystemen bei der Erforschung der Navigationsmechanismen der Insekten. Ein altersabh{\"a}ngiger Polyethismus trennt deren Kolonien in Innendienst-Arbeiterinnen und kurzlebige lichtausgesetzte Fourageure. Nachdem die Ameisen in strukturlosem oder strukturiertem Gel{\"a}nde bis zu mehrere hundert Meter weite Distanzen zur{\"u}ckgelegt haben, k{\"o}nnen sie pr{\"a}zise zu ihrer oft unauff{\"a}lligen Nest{\"o}ffnung zur{\"u}ckzukehren. Um diese enorme Navigationsleistung zu vollbringen, bedienen sich die Ameisen der sogenannten Pfadintegration, welche die Informationen aus einem Polarisationskompass und einem Entfernungsmesser verrechnet; des Weiteren orientieren sie sich an Landmarken und nutzen olfaktorische Signale. Im Fokus dieser Arbeit steht C. fortis, welche in Salzpfannen des westlichen Nordafrikas endemisch ist - einem Gebiet, welches vollst{\"a}ndig von anderen Cataglyphis Arten gemieden wird. Die Tatsache, dass Cataglyphis eine hohe Verhaltensflexibilit{\"a}t aufweist, welche mit sich drastisch {\"a}ndernden sensorischen Anforderungen verbunden ist, macht diese Ameisen zu besonders interessanten Studienobjekten bei der Erforschung synaptischer Plastizit{\"a}t visueller und olfaktorischer Gehirnzentren. Diese Arbeit fokussiert auf plastische {\"A}nderungen in den Pilzk{\"o}rpern (PK) - sensorischen Integrationszentren, die mutmaßlich an Lern- und Erinnerungsprozessen, und auch vermutlich am Prozess des Landmarkenlernens beteiligt sind - und auf plastische {\"A}nderungen in den synaptischen Komplexen des Lateralen Akzessorischen Lobus (LAL) - einer bekannten Relaisstation in der Polarisations-Leitungsbahn. Um die strukturelle synaptische Plastizit{\"a}t der PK in C. fortis zu quantifizieren, wurden mithilfe immunozytochemischer F{\"a}rbungen die pr{\"a}- und postsynaptischen Profile klar ausgepr{\"a}gter synaptischer Komplexe (Mikroglomeruli, MG) der visuellen Region (Kragen) und der olfaktorischen Region (Lippe) der PK-Kelche visualisiert. Die Ergebnisse legen dar, dass eine Volumenzunahme der PK-Kelche w{\"a}hrend des {\"U}bergangs von Innendiensttieren zu Fourageuren von einer Abnahme der MG-Anzahl im Kragen und, mit einem geringeren Anteil, in der Lippe - dieser Effekt wird als Pruning bezeichnet - und einem gleichzeitigen Auswachsen an Dendriten PK-intrinsischer Kenyonzellen begleitet wird. Im Dunkeln gehaltene Tiere unterschiedlichen Alters zeigen nach Lichtaussetzung den gleichen Effekt und im Dunkel gehaltene, den Fourageuren altersm{\"a}ßig angepasste Tiere weisen eine vergleichbare MG-Anzahl im Kragen auf wie Innendiensttiere. Diese Ergebnisse deuten darauf hin, dass die immense strukturelle synaptische Plastizit{\"a}t in der Kragenregion der PK-Kelche haupts{\"a}chlich durch visuelle Erfahrungen ausgel{\"o}st wird und nicht ausschließlich mit Hilfe eines internen Programms abgespielt wird. Ameisen, welche unter Laborbedingungen bis zu einem Jahr alt wurden, zeigen eine vergleichbare Plastizit{\"a}t. Dies deutet darauf hin, dass das System {\"u}ber die ganze Lebensspanne eines Individuums flexibel bleibt. Erfahrene Fourageure wurden in Dunkelheit zur{\"u}ckgef{\"u}hrt, um zu untersuchen, ob die lichtausgel{\"o}ste synaptische Umstrukturierung reversibel ist, doch ihre PK zeigen nur einige die Zur{\"u}ckf{\"u}hrung widerspiegelnde Plastizit{\"a}tsauspr{\"a}gungen, besonders eine {\"A}nderung der pr{\"a}synaptischen Synapsinexprimierung. Mithilfe immunozytochemischer F{\"a}rbungen, konfokaler Mikroskopie und 3D-Rekonstruktionen wurden die pr{\"a}- und postsynaptischen Strukturen synaptischer Komplexe des LAL in C. fortis analysiert und potentielle strukturelle {\"A}nderungen bei Innendiensttieren und Fourageuren quantifiziert. Die Ergebnisse zeigen, dass diese Komplexe aus postsynaptischen, in einer zentralen Region angeordneten Forts{\"a}tzen bestehen, welche umringt sind von einem pr{\"a}synaptischen kelchartigen Profil. Eingehende und ausgehende Trakte wurden durch Farbstoffinjektionen identifiziert: Projektionsneurone des Anterioren Optischen Tuberkels kontaktieren Neurone, welche in den Zentralkomplex ziehen. Der Verhaltens{\"u}bergang wird von einer Zunahme an synaptischen Komplexen um ~13\% begleitet. Dieser Zuwachs suggeriert eine Art Kalibrierungsprozess in diesen potentiell kr{\"a}ftigen synaptischen Kontakten, welche vermutlich eine schnelle und belastbare Signal{\"u}bertragung in der Polarisationsbahn liefern. Die Analyse von im Freiland aufgenommener Verhaltenweisen von C. fortis enth{\"u}llen, dass die Ameisen, bevor sie mit ihrer Fouragiert{\"a}tigkeit anfangen, bis zu zwei Tage lang in unmittelbarer N{\"a}he des Nestes Entdeckungsl{\"a}ufe unternehmen, welche Pirouetten {\"a}hnliche Drehungen beinhalten. W{\"a}hrend dieser Entdeckungsl{\"a}ufe sammeln die Ameisen Lichterfahrung und assoziieren m{\"o}glicherweise den Nesteingang mit spezifischen Landmarken oder werden anderen visuellen Informationen, wie denen des Polarisationsmusters, ausgesetzt und adaptieren begleitend ihre neuronalen Netzwerke an die bevorstehende Herausforderung. Dar{\"u}ber hinaus k{\"o}nnten die Pirouetten einer Stimulation der an der Polarisationsbahn beteiligten neuronalen Netzwerke dienen. Videoanalysen legen dar, dass Lichtaussetzung nach drei Tagen die Bewegungsaktivit{\"a}t der Ameisen heraufsetzt. Die Tatsache, dass die neuronale Umstrukturierung in visuellen Zentren wie auch die Ver{\"a}nderungen im Verhalten im selben Zeitrahmen ablaufen, deutet darauf hin, dass ein Zusammenhang zwischen struktureller synaptischer Plastizit{\"a}t und dem Verhaltens{\"u}bergang von der Innendienst- zur Fouragierphase bestehen k{\"o}nnte. Cataglyphis besitzen hervorragende visuelle Navigationsf{\"a}higkeiten, doch sie nutzen zudem olfaktorische Signale, um das Nest oder die Futterquelle aufzusp{\"u}ren. Mithilfe konfokaler Mikroskopie und 3D-Rekonstruktionen wurden potentielle Anpassungen der prim{\"a}ren olfaktorischen Gehirnzentren untersucht, indem die Anzahl, Gr{\"o}ße und r{\"a}umliche Anordnung olfaktorischer Glomeruli im Antennallobus von C. fortis, C. albicans, C. bicolor, C. rubra, und C. noda verglichen wurde. Arbeiterinnen aller Cataglyphis-Arten haben eine geringere Glomeruli-Anzahl im Vergleich zu denen der mehr olfaktorisch-orientierten Formica Arten - einer Gattung nah verwandt mit Cataglyphis - und denen schon bekannter olfaktorisch-orientierter Ameisenarten. C. fortis hat die geringste Anzahl an Glomeruli im Vergleich zu allen anderen Cataglyphis-Arten und besitzt einen vergr{\"o}ßerten Glomerulus, der nahe dem Eingang des Antennennerves lokalisiert ist. C. fortis M{\"a}nnchen besitzen eine signifikant geringere Glomeruli-Anzahl im Vergleich zu Arbeiterinnen und K{\"o}niginnen und haben einen hervorstechenden M{\"a}nnchen-spezifischen Makroglomerulus, welcher wahrscheinlich an der Pheromon-Kommunikation beteiligt ist. Die Verhaltensrelevanz des vergr{\"o}ßerten Glomerulus der Arbeiterinnen bleibt schwer fassbar. Die Tatsache, dass C. fortis Mikrohabitate bewohnt, welche von allen anderen Cataglyphis Arten gemieden werden, legt nahe, dass extreme {\"o}kologische Bedingungen nicht nur zu Anpassungen der visuellen F{\"a}higkeiten, sondern auch des olfaktorischen Systems gef{\"u}hrt haben. Die vorliegende Arbeit veranschaulicht, dass Cataglyphis ein exzellenter Kandidat ist bei der Erforschung neuronaler Mechanismen, welche Navigationsfunktionalit{\"a}ten zugrundeliegen, und bei der Erforschung neuronaler Plastizit{\"a}t, welche verkn{\"u}pft ist mit der lebenslangen Flexibilit{\"a}t eines individuellen Verhaltensrepertoires.}, subject = {Neuroethologie}, language = {en} } @phdthesis{EngelhardtgebChristiansen2013, author = {Engelhardt [geb. Christiansen], Frauke}, title = {Synaptic Connectivity in the Mushroom Body Calyx of Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85058}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {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.}, subject = {Taufliege}, language = {en} } @phdthesis{Hieke2019, author = {Hieke, Marie}, title = {Synaptic arrangements and potential communication partners of \(Drosophila's\) PDF-containing clock neurons within the accessory medulla}, doi = {10.25972/OPUS-17598}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175988}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla's edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors' are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons' conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs' terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations}, subject = {Taufliege}, language = {en} } @phdthesis{Knapek2010, author = {Knapek, Stephan}, title = {Synapsin and Bruchpilot, two synaptic proteins underlying specific phases of olfactory aversive memory in Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-49726}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Memory is dynamic: shortly after acquisition it is susceptible to amnesic treatments, gets gradually consolidated, and becomes resistant to retrograde amnesia (McGaugh, 2000). Associative olfactory memory of the fruit fly Drosophila melanogaster also shows these features. After a single associative training where an odor is paired with electric shock (Quinn et al., 1974; Tully and Quinn, 1985), flies form an aversive odor memory that lasts for several hours, consisting of qualitatively different components. These components can be dissociated by mutations, their underlying neuronal circuitry and susceptibility to amnesic treatments (Dubnau and Tully, 1998; Isabel et al., 2004; Keene and Waddell, 2007; Masek and Heisenberg, 2008; Xia and Tully, 2007). A component that is susceptible to an amnesic treatment, i.e. anesthesia-sensitive memory (ASM), dominates early memory, but decays rapidly (Margulies et al., 2005; Quinn and Dudai, 1976). A consolidated anesthesia-resistant memory component (ARM) is built gradually within the following hours and lasts significantly longer (Margulies et al., 2005; Quinn and Dudai, 1976). I showed here that the establishment of ARM requires less intensity of shock reinforcement than ASM. ARM and ASM rely on different molecular and/or neuronal processes: ARM is selectively impaired in the radish mutant, whereas for example the amnesiac and rutabaga genes are specifically required for ASM (Dudai et al., 1988; Folkers et al., 1993; Isabel et al., 2004; Quinn and Dudai, 1976; Schwaerzel et al., 2007; Tully et al., 1994). The latter comprise the cAMP signaling pathway in the fly, with the PKA being its supposed major target (Levin et al., 1992). Here I showed that a synapsin null-mutant encoding the evolutionary conserved phosphoprotein Synapsin is selectively impaired in the labile ASM. Further experiments suggested Synapsin as a potential downstream effector of the cAMP/PKA cascade. Similar to my results, Synapsin plays a role for different learning tasks in vertebrates (Gitler et al., 2004; Silva et al., 1996). Also in Aplysia, PKA-dependent phosphorylation of Synapsin has been proposed to be involved in regulation of neurotransmitter release and short-term plasticity (Angers et al., 2002; Fiumara et al., 2004). Synapsin is associated with a reserve pool of vesicles at the presynapse and is required to maintain vesicle release specifically under sustained high frequency nerve stimulation (Akbergenova and Bykhovskaia, 2007; Li et al., 1995; Pieribone et al., 1995; Sun et al., 2006). In contrast, the requirement of Bruchpilot, which is homologous to the mammalian active zone proteins ELKS/CAST (Wagh et al., 2006), is most pronounced in immediate vesicle release (Kittel et al., 2006). Under repeated stimulation of a bruchpilot mutant motor neuron, immediate vesicle release is severely impaired whereas the following steady-state release is still possible (Kittel et al., 2006). In line with that, knockdown of the Bruchpilot protein causes impairment in clustering of Ca2+ channels to the active zones and a lack of electron-dense projections at presynaptic terminals (T-bars). Thus, less synaptic vesicles of the readily-releasable pool are accumulated to the release sites and their release probability is severely impaired (Kittel et al., 2006; Wagh et al., 2006). First, I showed that Bruchpilot is required for aversive olfactory memory and localized the requirement of Bruchpilot to the Kenyon cells of the mushroom body, the second-order olfactory interneurons in Drosophila. Furthermore, I demonstrated that Bruchpilot selectively functions for the consolidated anesthesia-resistant memory. Since Synapsin is specifically required for the labile anesthesia sensitive memory, different synaptic proteins can dissociate consolidated and labile components of olfactory memory and two different modes of neurotransmission (high- vs. low frequency dependent) might differentiate ASM and ARM.}, subject = {Taufliege}, language = {en} } @phdthesis{Subota2011, author = {Subota, Ines}, title = {Switches in trypanosome differentiation: ALBA proteins acting on post-transcriptional mRNA control}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85707}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Trypanosoma brucei is a digenetic eukaryotic parasite that develops in different tissues of a mammalian host and a tsetse fly. It is responsible for sleeping sickness in sub-saharan Africa. The parasite cycle involves more than nine developmental stages that can be clearly distinguished by their general morphology, their metabolism and the relative positioning of their DNA-containing organelles. During their development, trypanosomes remain exclusively extracellular and encounter changing environments with different physico-chemical properties (nutritional availability, viscosity, temperature, etc.). It has been proposed that trypanosomes use their flagellum as a sensing organelle, in agreement with the established role of structurally-related cilia in metazoa and ciliates. Recognition of environmental triggers is presumed to be at the initiation of differentiation events, leading to the parasite stage that is the best suited to the new environment. These changes are achieved by the modification of gene expression programmes, mostly underlying post-transcriptional control of mRNA transcripts. We first demonstrate that the RNA-binding proteins ALBA3/4 are involved in specific differentiation processes during the parasite development in the fly. They are cytosolic and expressed throughout the parasite cycle with the exception of the stages found in the tsetse fly proventriculus, as shown by both immunofluorescence and live cell analysis upon endogenous tagging with YFP. Knock-down of both proteins in the developmental stage preceding these forms leads to striking modifications: cell elongation, cell cycle arrest and relocalization of the nucleus in a posterior position, all typical of processes acting in parasites found in the proventriculus region. When ALBA3 is over-expressed from an exogenous copy during infection, it interferes with the relocalization of the nucleus in proventricular parasites. This is not observed for ALBA4 over-expression that does not visibly impede differentiation. Both ALBA3/4 proteins react to starvation conditions by accumulating in cytoplasmic stress granules together with DHH1, a recognized RNA-binding protein. ALBA3/4 proteins also partially colocalize with granules formed by polyA+ RNA in these conditions. We propose that ALBA are involved in trypanosome differentiation processes where they control a subset of developmentally regulated transcripts. These processes involving ALBA3/4 are likely to result from the specific activation of sensing pathways. In the second part of the thesis, we identify novel flagellar proteins that could act in sensing mechanisms. Several protein candidates were selected from a proteomic analysis of intact flagella performed in the host laboratory. This work validates their flagellar localization with high success (85\% of the proteins examined) and defines multiple different patterns of protein distribution in the flagellum. Two proteins are analyzed during development, one of them showing down-regulation in proventricular stages. The functional analysis of one novel flagellar membrane protein reveals its rapid dynamics within the flagellum but does not yield a visible phenotype in culture. This is coherent with sensory function that might not be needed in stable culture conditions, but could be required in natural conditions during development. In conclusion, this work adds new pieces to the puzzle of identifying molecular switches involved in developmental mRNA control and environmental sensing in trypanosome stages in the tsetse fly.}, subject = {Trypanosoma brucei}, language = {en} } @phdthesis{Georgiev2021, author = {Georgiev, Kostadin}, title = {Sustainable management of naturally disturbed forests}, doi = {10.25972/OPUS-24285}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242854}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Owing to climate change, natural forest disturbances and consecutive salvage logging are drastically increasing worldwide, consequently increasing the importance of understanding how these disturbances would affect biodiversity conservation and provision of ecosystem services. In chapter II, I used long-term water monitoring data and mid-term data on α-diversity of twelve species groups to quantify the effects of natural disturbances (windthrow and bark beetle) and salvage logging on concentrations of nitrate and dissolved organic carbon (DOC) in streamwater and α-diversity. I found that natural disturbances led to a temporal increase of nitrate concentrations in streamwater, but these concentrations remained within the health limits recommended by the World Health Organization for drinking water. Salvage logging did not exert any additional impact on nitrate and DOC concentrations, and hence did not affect streamwater quality. Thus, neither natural forest disturbances in watersheds nor associated salvage logging have a harmful effect on the quality of the streamwater used for drinking water. Natural disturbances increased the α-diversity in eight out of twelve species groups. Salvage logging additionally increased the α-diversity of five species groups related to open habitats, but decreased the biodiversity of three deadwood-dependent species groups. In chapter III, I investigated whether salvage logging following natural disturbances (wildfire and windthrow) altered the natural successional trajectories of bird communities. I compiled data on breeding bird assemblages from nine study areas in North America, Europe and Asia, over a period of 17 years and tested whether bird community dissimilarities changed over time for taxonomic, functional and phylogenetic diversity when rare, common and dominant species were weighted differently. I found that salvage logging led to significantly larger dissimilarities than expected by chance and that these dissimilarities persisted over time for rare, common and dominant species, evolutionary lineages, and for rare functional groups. Dissimilarities were highest for rare, followed by common and dominant species. In chapter IV, I investigated how β-diversity of 13 taxonomic groups would differ in intact, undisturbed forests, disturbed, unlogged forests and salvage-logged forests 11 years after a windthrow and salvage logging. The study suggests that both windthrow and salvage logging drive changes in between-treatment β-diversity, whereas windthrow alone seems to drive changes in within-treatment β-diversity. Over a decade after the windthrow at the studied site, the effect of subsequent salvage logging on within-treatment β-diversity was no longer detectable but the effect on between-treatment β-diversity persisted, with more prominent changes in saproxylic groups and rare species than in non-saproxylic groups or common and dominant species. Based on these results, I suggest that salvage logging needs to be carefully weighed against its long-lasting impact on communities of rare species. Also, setting aside patches of naturally disturbed areas is a valuable management alternative as these patches would enable post-disturbance succession of bird communities in unmanaged patches and would promote the conservation of deadwood-dependent species, without posing health risks to drinking water sources.}, subject = {species richness}, language = {en} } @phdthesis{Aufmkolk2018, author = {Aufmkolk, Sarah}, title = {Super-Resolution Microscopy of Synaptic Proteins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151976}, school = {Universit{\"a}t W{\"u}rzburg}, pages = {X, 97}, year = {2018}, abstract = {The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit. The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples. In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity. The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm. These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data. Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM. In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes.}, subject = {Hochaufl{\"o}sende Mikroskopie}, language = {en} } @phdthesis{Goetz2020, author = {G{\"o}tz, Ralph}, title = {Super-resolution microscopy of plasma membrane receptors and intracellular pathogens}, doi = {10.25972/OPUS-20716}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207165}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Humans tend to believe in what they can see with their own eyes. Hence, visualization methods like microscopy have always been extremely popular since their invention in the 17th century. With the advent of super-resolution microscopy, the diffraction limit of ~200 - 250 nm could be overcome to enable more detailed insights into biological samples. Especially the single molecule localization microscopy method dSTORM offers the possibility of quantitative bioimaging. Hereby, the repetitive photoswitching of organic dyes in the presence of thiols is exploited to enable a lateral resolution of 20 nm. Another, recently introduced super-resolution method is expansion microscopy (ExM) which physically expands the sample to increase the resolution by the expansion factor from four to even twenty. To enable this, the sample is embedded into a hydrogel, homogenized using an unspecific proteinase and expanded in distilled water. Within this thesis, both methods were used to shed light on plasma membrane receptor distributions and different bacterial and fungal pathogens. In the first part of this thesis dSTORM was used to elucidate the "Receptome", the entirety of all membrane receptors, of the cell line Jurkat T-cells and primary T-cells. Within this project we could successfully visualize and quantify the distribution of the plasma membrane receptors CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 and CD105 with receptor densities ranging from 0.8 cluster/µm² in case of CD20 and 81.4 cluster/µm² for the highly abundant CD45 in activated primary T-cells at the basal membrane. Hereby, we could also demonstrate a homogeneous distribution of most receptors, while only few were clustered. In the case of CD3-clusters were detected in Jurkat T-cells and in primary activated T-cells, but not in na{\"i}ve ones, demonstrating the activation of this receptor. This was followed by the application of dSTORM to three different clinical projects involving the receptors CD38, BCMA and CD20 which are immunotherapeutic targets by monoclonal antibodies and CAR T-cells. In the first two projects dSTORM was applied to determine the receptor upregulation upon exposure of various drugs to MM1.S cells or primary multiple myeloma patient cells. This increase in membrane receptor expression can subsequently enhance the efficacy of therapies directed against these receptors. Within the CD20-project, the superior sensitivity of dSTORM compared to flow cytometry could be demonstrated. Hereby, a substantially higher fraction of CD20-positive patient cells was detected by dSTORM than by flow cytometry. In addition, we could show that by dSTORM CD20-positive evaluated cells were eradicated by immunotherapeutic CAR T-cell treatment. These studies were followed by whole cell super-resolution imaging using both LLS-3D dSTORM and 10x ExM to exclude any artifacts caused by interactions with the glass surface. In 10x ExM signal amplification via biotinylated primary antibodies and streptavidin ATTO 643 was essential to detect even single antibodies directed against the heterodimer CD11a with standard confocal microscopes. Albeit probably not quantitative due to the process of gelation, digestion and expansion during the ExM protocol, even some putative dimers of the receptor CD2 could be visualized using 10x ExM-SIM, similar to dSTORM experiments. Within the second part of this thesis, expansion microscopy was established in bacterial and fungal pathogens. ExM enabled not only an isotropic fourfold expansion of Chlamydia trachomatis, but also allowed the discrimination between the two developmental forms by the chlamydial size after expansion into reticulate and elementary bodies. Hereafter, a new α-NH2-ω-N3-C6-ceramide was introduced enabling an efficient fixation and for the first time the use of lipids in both, 4x and 10x ExM, termed sphingolipid ExM. This compound was used to investigate the ceramide uptake and incorporation into the cell membrane of Chlamydia trachomatis and Simkania negevensis. For Chlamydia trachomatis the combined resolution power of 10x ExM and SIM even allowed the visualization of both bacterial membranes within a distance of ~30 nm. Finally, ExM was applied to the three different fungi Ustilago maydis, Fusarium oxysporum and Aspergillus fumigatus after enzymatic removal of the fungal cell wall. In case of Ustilago maydis sporidia this digestion could be applied to both, living cells resulting in protoplasts and to fixed cells, preserving the fungal morphology. This new protocol could be demonstrated for immunostainings and fluorescent proteins of the three different fungi.}, subject = {Mikroskopie}, language = {en} } @phdthesis{Eiring2021, author = {Eiring, Patrick}, title = {Super-resolution microscopy of plasma membrane receptors}, doi = {10.25972/OPUS-25004}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250048}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Plasma membrane receptors are the most crucial and most commonly studied components of cells, since they not only ensure communication between the extracellular space and cells, but are also responsible for the regulation of cell cycle and cell division. The composition of the surface receptors, the so-called "Receptome", differs and is characteristic for certain cell types. Due to their significance, receptors have been important target structures for diagnostic and therapy in cancer medicine and often show aberrant expression patterns in various cancers compared to healthy cells. However, these aberrations can also be exploited and targeted by different medical approaches, as in the case of personalized immunotherapy. In addition, advances in modern fluorescence microscopy by so-called single molecule techniques allow for unprecedented sensitive visualization and quantification of molecules with an attainable spatial resolution of 10-20 nm, allowing for the detection of both stoichiometric and expression density differences. In this work, the single molecule sensitive method dSTORM was applied to quantify the receptor composition of various cell lines as well as in primary samples obtained from patients with hematologic malignancies. The focus of this work lies on artefact free quantification, stoichiometric analyses of oligomerization states and co localization analyses of membrane receptors. Basic requirements for the quantification of receptors are dyes with good photoswitching properties and labels that specifically mark the target structure without generating background through non-specific binding. To ensure this, antibodies with a predefined DOL (degree of labeling) were used, which are also standard in flow cytometry. First background reduction protocols were established on cell lines prior analyses in primary patient samples. Quantitative analyses showed clear expression differences between the cell lines and the patient cells, but also between individual patients. An important component of this work is the ability to detect the oligomerization states of receptors, which enables a more accurate quantification of membrane receptor densities compared to standard flow cytometry. It also provides information about the activation of a certain receptor, for example of FLT3, a tyrosine kinase, dimerizing upon activation. For this purpose, different well-known monomers and dimers were compared to distinguish the typical localization statistics of single bound antibodies from two or more antibodies that are in proximity. Further experiments as well as co localization analyses proved that antibodies can bind to closely adjacent epitopes despite their size. These analytical methods were subsequently applied for quantification and visualization of receptors in two clinically relevant examples. Firstly, various therapeutically relevant receptors such as CD38, BCMA and SLAMF7 for multiple myeloma, a malignant disease of plasma cells, were analyzed and quantified on patient cells. Furthermore, the influence of TP53 and KRAS mutations on receptor expression levels was investigated using the multiple myeloma cell lines OPM2 and AMO1, showing clear differences in certain receptor quantities. Secondly, FLT3 which is a therapeutic target receptor for acute myeloid leukemia, was quantified and stoichiometrically analyzed on both cell lines and patient cells. In addition, cells that have developed resistance against midostaurin were compared with cells that still respond to this type I tyrosine-kinase-inhibitor for their FLT3 receptor expression and oligomerization state.}, subject = {Fluoreszenzmikroskopie}, language = {en} } @phdthesis{Nayak2007, author = {Nayak, Arnab}, title = {Sumoylation Modulates NFATc1-mediated Lymphokine Gene Expression}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-24722}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Die Aktivit{\"a}t von Transkriptionsfaktoren kann durch die Modifikation mit SUMO positiv oder negativ beeinflusst werden, indem Protein-Protein-Interaktionen als auch die subzellul{\"a}re bzw. subnukle{\"a}re Lokalisation ver{\"a}ndert werden. In T-Zellen spielt die Familie der NFAT (Nuclear Factor of Activated T cells)-Transkriptionsfaktoren eine wichtige Rolle bei der Zytokingenregulation. NFATc1 wird durch die Verwendung zwei verschiedener Promotoren (P1 \& P2) bzw. Polyadenylierungsstellen (pA1 \& pA2) und alternativen Spleißens in sechs Isoformen exprimiert. Sie werden als NFATc1/alphaA, betaA, alphaB, betaB, alphaC und betaC bezeichnet, wobei alpha und beta sich auf die beiden unterschiedlichen 1. Exons und A, B, C sich auf die differentiell gespleißten und unterschiedlich langen C-Termini beziehen. Die NFATc1/A-Isoformen umfassen einen relativ kurzen C-Terminus, w{\"a}hrend die langen Isoformen B und C extra-C-terminale Peptide von 128 bzw. 246 Aminos{\"a}uren aufweisen. Um die spezifischen, biologischen Effekte der NFATc1-Isoformen zu untersuchen, wurde ein sog. ‚Yeast two Hybrid screen' mit einer humanen Milz-cDNA-Bibliothek und dem NFATc1/C-spezifischen C-Terminus durchgef{\"u}hrt. Am Ende wurden Ubc9 und PIAS1, Proteine, die an der Sumoylierung beteiligt sind, am h{\"a}ufigsten dedektiert. Anschließend konnte gezeigt werden, dass NFATc1 tats{\"a}chlich sumoyliert wird. Das Ausmaß an Sumoylierung ist Isoformen abh{\"a}ngig. W{\"a}hrend NFATc1/A, das eine einzige Sumoylierungsstelle besitzt, nur eine geringe Sumoylierung aufweist, f{\"u}hren die beiden zus{\"a}tzlichen Stellen in NFATc1/C zu einer effizienten Modifikation mit SUMO. Diese C-terminale Modifikation dirigiert NFATc1/C in SUMO-1-K{\"o}rperchen, die mit PML-nbs kolokalisieren. Dar{\"u}ber hinaus rekrutiert sumoyliertes NFATc1/C die transkriptionellen Korepressoren HDAC (sowohl Klasse I wie Klasse II HDACs), was zu einer signifikanten Verringerung der Histonazetylierung am IL-2-Promotor, eines wichtigen NFATc1-Zielgens, f{\"u}hrt. Konsequenterweise wurde eine Verminderung der IL-2-Produktion beobachtet, w{\"a}hrend NFATc1/C, das wegen Mutation der entscheidenden Lysine nicht mehr sumoyliert werden kann, ein dramatisch erh{\"o}htes Transaktivierungspotential am IL-2-Promotor aufwies. Das unterst{\"u}tzt unsere Daten, die mit einem IL-2-Promotor getriebenen Reporterassay gewonnen wurden und zeigen, dass das Transaktivierungspotential von NFATc1/C durch Sumoylierung herabgesetzt wird. Demzufolge {\"u}bt Sumoylierung einen negativen Effekt auf die transkriptionelle NFATc1-Aktivit{\"a}t aus. Immunfluoreszenzversuche zeigten, dass die Modifikation mit SUMO außerdem zur Relokalisation von NFATc1/C in transkriptionell inaktive, heterochromatische Regionen f{\"u}hrt, was durch die F{\"a}rbung von trimethyliertem Histon mit anti-H3K9 m3 nachgewiesen wurde. Interessanterweise war in Abwesenheit von Sumoylierung NFATc1 teilweise mit transkriptionellen Hotspots im Kern lokalisiert. Das mag zu dem h{\"o}heren Transkriptionspotential des nicht-sumoylierten NFATc1 beitragen. Es ist wichtig zu erw{\"a}hnen, dass die transkriptionelle Aktivit{\"a}t auf andere NFATc1-Zielgene durch die Sumoylierung von NFATc1 positiv verst{\"a}rkt war. Dies deutet auf einen nicht-universalen Effekt der Sumoylierung auf die NFATc1/C-Funktion hin. Demzufolge dirigiert Sumoylierung NFATc1 in Kernk{\"o}rperchen, wo es mit transkriptionellen Korepressoren interagiert und selbst ans Heterochromatin relokalisiert, was zu einer Repression der NFATc1/C vermittelten Transkription f{\"u}hrt. Als sehr wichtig erscheint, dass der Effekt der NFATc1/C-Sumoylierung Promotor spezifisch ist. Zusammengenommen ver{\"a}ndet die Modifikation mit SUMO die NFATc1-Funktion von einem Transaktivator zu einem DNA-Bindungsstellen spezifischen Repressor. Daher wird hier ein neuer regulatorischer Mechanismus aufgezeigt, der die Isoform spezifische NFAT-Funktion kontrolliert.}, language = {en} } @phdthesis{Thum2006, author = {Thum, Andreas Stephan}, title = {Sugar reward learning in Drosophila : neuronal circuits in Drosophila associative olfactory learning}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-17930}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Genetic intervention in the fly Drosophila melanogaster has provided strong evidence that the mushroom bodies of the insect brain act as the seat of memory traces for aversive and appetitive olfactory learning (reviewed in Heisenberg, 2003). In flies, electroshock is mainly used as negative reinforcer. Unfortunately this fact complicates a comparative consideration with other inscets as most studies use sugar as positive reinforcer. For example, several lines of evidence from honeybee and moth have suggested another site, the antennal lobe, to house neuronal plasticity underlying appetitive olfactory memory (reviewed in Menzel, 2001; Daly et al., 2004). Because of this I focused my work mainly on appetitive olfactory learning. In the first part of my thesis, I used a novel genetic tool, the TARGET system (McGuire et al., 2003), which allows the temporally controlled expression of a given effector gene in a defined set of cells. Comparing effector genes which either block neurotransmission or ablate cells showed important differences, revealing that selection of the appropriate effector gene is critical for evaluating the function of neural circuits. In the second part, a new engram of olfactory memory in the Drosophila projection neurons is described by restoring Rutabaga adenlylate cyclase (rut-AC) activity specifically in these cells. Expression of wild-type rutabaga in the projection neurons fully rescued the defect in sugar reward memory, but not in aversive electric shock memory. No difference was found in the stability of the appetitive memories rescued either in projection neurons or Kenyon cells. In the third part of the thesis I tried to understand how the reinforcing signals for sugar reward are internally represented. In the bee Hammer (1993) described a single octopaminergic neuron - called VUMmx1 - that mediates the sugar stimulus in associative olfactory reward learning. Analysis of single VUM neurons in the fly (Selcho, 2006) identified a neuron with a similar morphology as the VUMmx1 neuron. As there is a mutant in Drosophila lacking the last enzymatic step in octopamine synthesis (Monastirioti et al., 1996), Tyramine beta Hydroxylase, I was able to show that local Tyramine beta Hydroxylase expression successfully rescued sugar reward learning. This allows to conclude that about 250 cells including the VUM cluster are sufficient for mediating the sugar reinforcement signal in the fly. The description of a VUMmx1 similar neuron and the involvement of the VUM cluster in mediating the octopaminergic sugar stimulus are the first steps in establishing a neuronal map for US processing in Drosophila. Based on this work several experiments are contrivable to reach this ultimate goal in the fly. Taken together, the described similiarities between Drosophila and honeybee regarding the memory organisation in MBs and PNs and the proposed internal representation of the sugar reward suggest an evolutionarily conserved mechanism for appetitive olfactory learning in insects.}, subject = {Taufliege}, language = {en} } @phdthesis{DeğirmencineePoelloth2023, author = {Değirmenci [n{\´e}e P{\"o}lloth], Laura}, title = {Sugar perception and sugar receptor function in the honeybee (\(Apis\) \(mellifera\))}, doi = {10.25972/OPUS-32187}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321873}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {In the eusocial insect honeybee (Apis mellifera), many sterile worker bees live together with a reproductive queen in a colony. All tasks of the colony are performed by the workers, undergoing age-dependent division of labor. Beginning as hive bees, they take on tasks inside the hive such as cleaning or the producing of larval food, later developing into foragers. With that, the perception of sweetness plays a crucial role for all honeybees whether they are sitting on the honey stores in the hive or foraging for food. Their ability to sense sweetness is undoubtedly necessary to develop and evaluate food sources. Many of the behavioral decisions in honeybees are based on sugar perception, either on an individual level for ingestion, or for social behavior such as the impulse to collect or process nectar. In this context, honeybees show a complex spectrum of abilities to perceive sweetness on many levels. They are able to perceive at least seven types of sugars and decide to collect them for the colony. Further, they seem to distinguish between these sugars or at least show clear preferences when collecting them. Additionally, the perception of sugar is not rigid in honeybees. For instance, their responsiveness towards sugar changes during the transition from in-hive bees (e.g. nurses) to foraging and is linked to the division of labor. Other direct or immediate factors changing responsiveness to sugars are stress, starvation or underlying factors, such as genotype. Interestingly, the complexity in their sugar perception is in stark contrast to the fact that honeybees seem to have only three predicted sugar receptors. In this work, we were able to characterize the three known sugar receptors (AmGr1, AmGr2 and AmGr3) of the honeybee fully and comprehensively in oocytes (Manuscript II, Chapter 3 and Manuscript III, Chapter 4). We could show that AmGr1 is a broad sugar receptor reacting to sucrose, glucose, maltose, melezitose and trehalose (which is the honeybees' main blood sugar), but not fructose. AmGr2 acts as its co-receptor altering AmGr1's specificity, AmGr3 is a specific fructose receptor and we proved the heterodimerization of all receptors. With my studies, I was able to reproduce and compare the ligand specificity of the sugar receptors in vivo by generating receptor mutants with CRISPR/Cas9. With this thesis, I was able to define AmGr1 and AmGr3 as the honeybees' basis receptors already capable to detect all sugars of its known taste spectrum. In the expression analysis of my doctoral thesis (Manuscript I, Chapter 2) I demonstrated that both basis receptors are expressed in the antennae and the brain of nurse bees and foragers. This thesis assumes that AmGr3 (like the Drosophila homologue) functions as a sensor for fructose, which might be the satiety signal, while AmGr1 can sense trehalose as the main blood sugar in the brain. Both receptors show a reduced expression in the brain of foragers when compared with nurse bees. These results may reflect the higher concentrated diet of nurse bees in the hive. The higher number of receptors in the brain may allow nurse bees to perceive hunger earlier and to consume the food their sitting on. Forager bees have to be more persistent to hunger, when they are foraging, and food is not so accessible. The findings of reduced expression of the fructose receptor AmGr3 in the antennae of nurse bees are congruent with my other result that nurse bees are also less responsive to fructose at the antennae when compared to foragers (Manuscript I, Chapter 2). This is possible, since nurse bees sit more likely on ripe honey which contains not only higher levels of sugars but also monosaccharides (such as fructose), while foragers have to evaluate less-concentrated nectar. My investigations of the expression of AmGr1 in the antennae of honeybees found no differences between nurse bees and foragers, although foragers are more responsive to the respective sugar sucrose (Manuscript I, Chapter 2). Considering my finding that AmGr2 is the co-receptor of AmGr1, it can be assumed that AmGr1 and the mediated sucrose taste might not be directly controlled by its expression, but indirectly by its co-receptor. My thesis therefore clearly shows that sugar perception is associated with division of labor in honeybees and appears to be directly or indirectly regulated via expression. The comparison with a characterization study using other bee breeds and thus an alternative protein sequence of AmGr1 shows that co-expression of different AmGr1 versions with AmGr2 alters the sugar response differently. Therefore, this thesis provides first important indications that alternative splicing could also represent an important regulatory mechanism for sugar perception in honeybees. Further, I found out that the bitter compound quinine lowers the reward quality in learning experiments for honeybees (Manuscript IV, Chapter 5). So far, no bitter receptor has been found in the genome of honeybees and this thesis strongly assumes that bitter substances such as quinine inhibit sugar receptors in honeybees. With this finding, my work includes other molecules as possible regulatory mechanism in the honeybee sugar perception as well. We showed that the inhibitory effect is lower for fructose compared to sucrose. Considering that sugar signals might be processed as differently attractive in honeybees, this thesis concludes that the sugar receptor inhibition via quinine in honeybees might depend on the receptor (or its co-receptor), is concentration-dependent and based on the salience or attractiveness and concentration of the sugar present. With my thesis, I was able to expand the knowledge on honeybee's sugar perception and formulate a complex, comprehensive overview. Thereby, I demonstrated the multidimensional mechanism that regulates the sugar receptors and thus the sugar perception of honeybees. With this work, I defined AmGr1 and AmGr3 as the basis of sugar perception and enlarged these components to the co-receptor AmGr2 and the possible splice variants of AmGr1. I further demonstrated how those sugar receptor components function, interact and that they are clearly involved in the division of labor in honeybees. In summary, my thesis describes the mechanisms that enable honeybees to perceive sugar in a complex way, even though they inhere a limited number of sugar receptors. My data strongly suggest that honeybees overall might not only differentiate sugars and their diet by their general sweetness (as expected with only one main sugar receptor). The found sugar receptor mechanisms and their interplay further suggest that honeybees might be able to discriminate directly between monosaccharides and disaccharides or sugar molecules and with that their diet (honey and nectar).}, subject = {Biene}, language = {en} } @phdthesis{Barth2008, author = {Barth, Enrico}, title = {Study of the properties of channel-forming proteins of the cell walls of different Corynebacteriae}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-36325}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Die Gattung Corynebacterium geh{\"o}rt, neben Mycobacterium, Nocardia, Rhodococcus und weiteren nahverwandten Gattungen, dem unverwechselbaren, gattungs{\"u}bergreifenden Taxon Mycolata an. Viele Spezies aus dieser heterogenen Gruppe Mycols{\"a}ure-haltiger Actinomyceten sind entweder aufgrund ihrer medizinischen oder ihrer biotechnologischen Bedeutung bekannt. Beispielsweise z{\"a}hlen Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae und Nocardia farcinica, welche weltweit Verursacher besonders gef{\"a}hrlicher bakterieller Infektionskrankheiten sind, zu dieser ungew{\"o}hnlichen Gruppe Gram-positiver Bakterien. Ebenso bedeutsam sind einige apathogene Mycolata-Arten, die industrielle Anwendung finden. Corynebacterium glutamicum und Corynebacterium efficiens sind leistungsf{\"a}hige Bakterien, die zum Beispiel in der Produktion des Geschmacksverst{\"a}rkers Glutamat und des Tierfuttermittelzusatzes Lysin eingesetzt werden, w{\"a}hrend verschiedene Rhodococcus Spezies Anwendung bei der Herstellung von Acryls{\"a}uren finden. Die Zellwand der Mycolata zeigt, verglichen mit der klassischer Gram-positiver Bakterien, eine außergew{\"o}hnliche Zusammensetzung und Struktur auf. Abgesehen von einem Arabinogalactan-Peptidoglycan-Komplex enth{\"a}lt die Zellwand der meisten Actinomyceten einen hohen Anteil an Mycols{\"a}uren. Diese langkettigen, verzweigten Fetts{\"a}uren formen eine, mit der {\"a}ußeren Membran Gram-negativer Bakterien vergleichbare, stark undurchl{\"a}ssige, hydrophobe {\"a}ußere H{\"u}lle, welche die Grundlage der außergew{\"o}hnlichen Medikamentenresistenz bei den Mycolata bildet. Wie die {\"a}ußere Membran Gram-negativer Bakterien enth{\"a}lt die Zellwand der Mycolata porenformende Proteine, die den Durchlass hydrophiler Substanzen gestatten. Indem sie eine Verbindung zwischen dem Zellinneren und der Umwelt, in der das Bakterium lebt, schaffen und einen kontrollierten Austausch zwischen beiden erm{\"o}glichen, tragen die Kanalproteine entscheidend zur Funktion der bakteriellen Zellh{\"u}lle bei. Das Ziel dieser Arbeit war das Wissen {\"u}ber Zellwandkan{\"a}le in Corynebakterien zu erweitern. Deshalb untersuchten wir PorA und PorH Proteine, die basierend auf fr{\"u}heren Studien Zellwandkan{\"a}len in C. glutamicum, C. efficiens und Corynebacterium callunae zugeordnet werden, um ungekl{\"a}rten Fragen nachzugehen und um Wissen {\"u}ber deren Struktur zu erlangen. Ferner inspizierten wir Zellw{\"a}nde pathogener Corynebakterien, genauer gesagt von Corynebacterium diphtheriae und Corynebacterium jeikeium, um herauszufinden, ob diese Spezies wie ihre harmlosen Verwandten Kanalproteine besitzen. In dieser Arbeit wiesen wir mit C. diphtheriae und C. jeikeium in zwei weiteren Corynebacterium-Arten offene, mit Wasser gef{\"u}llte Zellwandkan{\"a}le nach. Des Weiteren stellten wir fest, dass sich die Zellwandkan{\"a}le von C. glutamicum, C. efficiens und C. diphtheriae aus zwei Proteinen zusammensetzen, einem zugeh{\"o}rig zu der Gruppe der PorH Proteine und einem weiteren aus der Gruppe der PorA Proteine. Diese heteromere Struktur von Zellwandkan{\"a}len bei Corynebakterien stellt ein Novum f{\"u}r Zellwandkan{\"a}le bei den Mycolata dar. Indessen besteht der Zellwandkanal von C. jeikeium aus nur einem Protein, CjPorA, angeordnet zu einem Oligomer. Obgleich das Molekulargewicht dieses Proteins (4 kDa) mit dem von PorH und PorA Proteinen vergleichbar ist (5-7 kDa), weißt seine Prim{\"a}rsequenz keine eindeutige Homologie zu diesen auf. Dennoch deutet vieles auf eine Verwandtschaft zwischen CjPorA und PorH/PorA Proteinen hin, da das Gen jk0268, welches f{\"u}r CjPorA kodiert, sich in einer Region des C. jeikeium Chromosoms befindet, die der Genomregion entspricht in welcher die porH/porA Gene der anderen Corynebakterien lokalisiert sind. Dies l{\"a}sst vermuten, dass jk0268 (welches f{\"u}r den homomeren Zellwandkanal in C. jeikeium kodiert) und die porH/porA Gene von C. glutamicum, C. efficiens und C. diphtheriae (die einen heteromeren Zellwandkanal kodieren) wahrscheinlich Nachkommen eines gemeinsamen Vorl{\"a}ufergens sind. Phylogenetische Analysen der Gattung Corynebacterium unterst{\"u}tzen diese Annahme. Desweitern legen sie nahe, dass die hier untersuchten Zellwandkan{\"a}le innerhalb dieser Gattung wahrscheinlich weit verbreitet sind. Ein umfassendes Wissen {\"u}ber Zellwandkan{\"a}le, denen beim Transport gel{\"o}ster Stoffe {\"u}ber die {\"a}ußere Membran in Corynebakterien und anderen Mitgliedern der Mycolata eine entscheidende Rolle zukommt, k{\"o}nnte von großem wirtschaftlichem und medizinischem Nutzen sein.}, subject = {Zellwand}, language = {en} } @phdthesis{Stegmeier2006, author = {Stegmeier, Johannes Friedrich}, title = {Study of Omp85 Family Proteins YaeT and YtfM and Multidrug Export Machineries in Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18171}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {In this study the Omp85 family proteins YaeT and YtfM of Escherichia coli were investigated by using biochemical and electrophysiological methods as well as bioinformatical and structural analysis. In addition, knock-out strains were constructed to further study the relevance of these proteins in vivo. The prediction that Omp85 proteins are composed of two domains, a periplasmic amino-terminal POTRA (polypeptide translocation associated) domain and a carboxy-terminal domain anchoring these proteins in the outer membrane, was confirmed by the construction of mutants. It could be shown that the carboxy-terminal part of the proteins is able to insert into the outer bacterial membrane, even if the POTRA domain is removed. Furthermore, pore-forming activity in the black-lipid bilayer was observed for both full-length proteins as well as their carboxy-terminal membrane located parts. The channels formed by both proteins in the black lipid bilayer showed variable single channel conductance states rather than a defined value for conductance. In 1M KCl, e.g. YaeT forms pores with a channel conductance of 100 to 600 pS containing a most abundant value at 400 pS. This variability is at least reasonable for YaeT due to a prerequisite flexibility of its channel for OMP insertion. YaeT was identified to form a cation selective, YtfM an anion selective channel, which is less pH dependent than YaeT. Another feature of the YaeT channel is that its selectivity and conductance is influenced by charged detergent molecules indicating an accumulation of these molecules in hydrophobic pockets inside the compact channel. YaeT revealed heat-modifiable mobility in SDS-PAGE which is characteristic for \&\#946;-barrel OMPs, whereas YtfM did not show this behaviour. This result could be explained by sequence alignment and structural comparison of YaeT and YtfM via CD and FTIR spectra displaying much higher \&\#946;-strand content for the carboxy-terminal part of YaeT compared to YtfM. Since the carboxy-terminal parts were shown to have pore forming ability and are inserted in the OM in vivo, the substitution of the essential protein YaeT by its carboxy-terminal mutant was attempted in a yaeT knock-out strain. The carboxy-terminal half of YaeT was not sufficient to compensate depletion of the full-length protein indicating an important role of the amino-terminus for cell viability. In contrary, YtfM is shown to be a non-essential protein and lack of YtfM had no effects on the composition and integrity of the OM. However, chromosomal deletion of ytfM remarkably reduced the growth rate of cells. This study provides the first detailed investigation of the structure of YaeT and describes its electrophysiological behaviour, which could be a basis for further studies of YaeT and its substrate proteins. Furthermore, YtfM was characterised and its in vivo function was investigated revealing YtfM as the second Omp85 family protein of importance in E. coli. In a second part of this study assembly and function of multidrug efflux pumps were investigated. Drug efflux pumps are tripartite export machineries in the cell envelope of Gram-negative bacteria conferring multidrug resistance and therefore causing severe problems for medical treatment of diseases. Protein structures of all three efflux pump components are solved, but the exact interaction sites are still unknown. Assembly of a hybrid exporter system composed of the Pseudomonas aeruginosa channel tunnel OprM, the E. coli adaptor protein AcrA and its associated transporter AcrB could be shown by chemical cross-linking, even though this efflux pump is not functional. Exchange of the hairpin domain of AcrA by the corresponding hairpin from the adaptor protein MexA of P. aeruginosa restored functionality tested by antibiotic sensitivity assays. This shows the importance of the MexA hairpin domain for functional interaction with the OprM channel tunnel. Interestingly, the hybrid protein was also able to assemble with TolC as outer membrane component to form a functional efflux pump indicating a higher flexibility of TolC compared to OprM concerning interaction partners. Based on these results, an interaction model of the hairpin domain and the channel tunnel on molecular level for AcrA and TolC as well as MexA and OprM, respectively, is presented. This model provides a basis for directed mutagenesis to reveal the exact contact sites of the hairpin of the adapter protein and the outer membrane component}, subject = {Escherichia coli}, language = {en} } @phdthesis{Polleichtner2006, author = {Polleichtner, Johann Georg}, title = {Studies of structure-function relationship of components of multidrug efflux pumps and type I secretion systems}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18671}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {This work deals with channel-tunnel dependent multidrug efflux pumps and type I secretion systems, more concrete with the improved classification of the adaptor protein family, the characterization of the TolC-homologue protein HI1462 of Haemophilus influenzae, and the molecular characterization of the interaction between TolC and AcrA of Escherichia coli.}, subject = {Gram-negative Bakterien}, language = {en} } @phdthesis{Schwebs2024, author = {Schwebs, Marie}, title = {Structure and dynamics of the plasma membrane: a single-molecule study in \(Trypanosoma\) \(brucei\)}, doi = {10.25972/OPUS-27569}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-275699}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The unicellular, flagellated parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in livestock. In the last decades, it has become an established eukaryotic model organism in the field of biology, as well as in the interdisciplinary field of biophysics. For instance, the dense variant surface glycoprotein (VSG) coat offers the possibility to study the dynamics of GPI-anchored proteins in the plasma membrane of living cells. The fluidity of the VSG coat is not only an interesting object of study for its own sake, but is critically important for the survival of the parasite in the mammalian host. In order to maintain the integrity of the coat, the entire VSG coat is recycled within a few minutes. This is surprisingly fast for a purely diffusive process with the flagellar pocket (FP) as the sole site for endo- and exocytosis. Previous studies characterising VSG dynamics using FRAP reported diffusion coefficients that were not sufficient to to enable fast turnover based on passive VSG randomisation on the trypanosome surface. In this thesis, live-cell single-molecule fluorescence microscopy (SMFM) was employed to elucidate whether VSG diffusion coefficients were priorly underestimated or whether directed forces could be involved to bias VSGs towards the entrance of the FP. Embedding the highly motile trypanosomes in thermo-stable hydrogels facilitated the investigation of VSG dynamics on living trypanosomes at the mammalian host's temperature of 37°C. To allow for a spatial correlation of the VSG dynamics to the FP entrance, a cell line was employed harbouring a fluorescently labelled structure as a reference. Sequential two-colour SMFM was then established to allow for recording and registration of the dynamic and static single-molecule information. In order to characterise VSG dynamics, an algorithm to obtain reliable information from short trajectories was adapted (shortTrAn). It allowed for the quantification of the local dynamics in two distinct scenarios: diffusion and directed motion. The adaptation of the algorithm to the VSG data sets required the introduction of an additional projection filter. The algorithm was further extended to take into account the localisation errors inherent to single-particle tracking. The results of the quantification of diffusion and directed motion were presented in maps of the trypanosome surface, including an outline generated from a super-resolved static structure as a reference. Information on diffusion was displayed in one map, an ellipse plot. The colour code represented the local diffusion coefficient, while the shape of the ellipses provided an indication of the diffusion behaviour (aniso- or isotropic diffusion). The eccentricity of the ellipses was used to quantify deviations from isotropic diffusion. Information on directed motion was shown in three maps: A velocity map, representing the amplitude of the local velocities in a colour code. A quiver plot, illustrating the orientation of directed motion, and a third map which indicated the relative standard error of the local velocities colour-coded. Finally, a guideline based on random walk simulations was used to identify which of the two motion scenarios dominated locally. Application of the guideline to the VSG dynamics analysed by shortTrAn yielded supermaps that showed the locally dominant motion mode colour-coded. I found that VSG dynamics are dominated by diffusion, but several times faster than previously determined. The diffusion behaviour was additionally characterised by spatial heterogeneity. Moreover, isolated regions exhibiting the characteristics of round and elongated traps were observed on the cell surface. Additionally, VSG dynamics were studied with respect to the entrance of the FP. VSG dynamics in this region displayed similar characteristics compared to the remainder of the cell surface and forces biasing VSGs into the FP were not found. Furthermore, I investigated a potential interference of the attachment of the cytoskeleton to the plasma membrane with the dynamics of VSGs which are anchored to the outer leaflet of the membrane. Preliminary experiments were conducted on osmotically swollen trypanosomes and trypanosomes depleted for a microtubule-associated protein anchoring the subpellicular microtubule cytoskeleton to the plasma membrane. The measurements revealed a trend that detachment of the cytoskeleton could be associated with a reduction in the VSG diffusion coefficient and a loss of elongated traps. The latter could be an indication that these isolated regions were caused by underlying structures associated with the cytoskeleton. The measurements on cells with an intact cytoskeleton were complemented by random walk simulations of VSG dynamics with the newly determined diffusion coefficient on long time scales not accessible in experiments. Simulations showed that passive VSG randomisation is fast enough to allow for a turnover of the full VSG coat within a few minutes. According to an estimate based on the known rate of endocytosis and the newly determined VSG diffusion coefficient, the majority of exocytosed VSGs could escape from the FP to the cell surface without being immediately re-endocytosed.}, subject = {Trypanosoma brucei}, language = {en} } @phdthesis{Bollmann2013, author = {Bollmann, Stefan}, title = {Structural Dynamics of Oligopeptides determined by Fluorescence Quenching of Organic Dyes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-92191}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {For determination of structures and structural dynamics of proteins organic fluorophores are a standard instrument. Intra- and intermolecular contact of biomolecular structures are determined in time-resolved and stationary fluorescence microscopy experiments by quenching of organic fluorophores due to Photoinduced Electron Transfer (PET) and dimerization interactions. Using PET we show in this work that end-to-end contact dynamics of serine-glycine peptides are slowed down by glycosylation. This slow down is due to a change in reaction enthalpy for end-to-end contact and is partly compensated by entropic effects. In a second step we test how dimerization of MR121 fluorophore pairs reports on end-to-end contact dynamics. We show that in aqueous solutions containing strong denaturants MR121 dimerization reports advantageously on contact dynamics for glycine-serine oligopeptides compared to the previously used MR121/tryptophane PET reporters. Then we analyze dimer interactions and quenching properties of different commercially available fluorophores being standards in F{\"o}rster Resonance Energy Transfer (FRET) measurements. Distances in biomolecules are determinable using FRET, but for very flexible biomolecules the analysis of masurement data can be distorted if contact of the two FRET fluorophores is likely. We quantify how strong the quenching of fluorophore pairs with two different or two identical fluorophores is. Dimer spectra and association constants are quantified to estimate if fluophores are applicable in various applications, e.g. in FRET measurements with unstructured peptides and proteins.}, subject = {Fluorophore}, language = {en} } @phdthesis{Noskov2003, author = {Noskov, Andrey}, title = {Structural and functional studies of the Interleukin-5 receptor system}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-8195}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2003}, abstract = {The aim of current work was contribution to the long-term ongoing project on developing human IL-5 agonists/antagonists that intervene with or inhibit IL-5 numerous functions in cell culture and/or in animal disease models. To facilitate design of an IL-5 antagonist variant or low-molecular weight mimetics only capable of binding to the specific receptor alpha chain, but would lack the ability to attract the receptor common \&\#946;-chain and thus initiate receptor complex activation it is necessary to gain the information on minimal structural and functional epitopes. Such a strategy was successfully adopted in our group on example of Interleukin 4. To precisely localize minimal structural epitope it is essential to have structure of the ligand in its bound form and especially informative would be structure of complex of the ligand and its specific receptor alpha chain. For this purpose large quantities (tens of milligrams), retaining full biological activity IL-5 and extracellular domain of IL-5 specific receptor \&\#945;-chain were expressed in a bacterial expression system (E.coli). After successful refolding proteins were purified to 95-99\% Stable and soluble receptor:ligand complex was prepared. Each established purification and refolding procedures were subjected to optimization targeting maximal yields and purity. Produced receptor:ligand complex was applied to crystallization experiments. Microcrystals were initially obtained with a flexible sparse matrix screening methodology. Crystal quality was subsequently improved by fine-tuning of the crystallization conditions. At this stage crystals of about 800x150x30µm in size can be obtained. They possess desirable visible characteristics of crystals including optical clarity, smooth facecs and sharp edges. Crystals rotate plane polarized light reflecting their well internal organization. Unfortunately relative slimness and sometimes cluster nature of the produced crystals complicates acquisition of high-resolution dataset and resolution of the structure. With some of obtained crystals diffraction to a resolution up to 4{\AA} was observed.}, subject = {Interleukin 5}, language = {en} } @phdthesis{Bartossek2018, author = {Bartossek, Thomas}, title = {Structural and functional analysis of the trypanosomal variant surface glycoprotein using x-ray scattering techniques and fluorescence microscopy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144775}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Trypanosoma brucei is an obligate parasite and causative agent of severe diseases affecting humans and livestock. The protist lives extracellularly in the bloodstream of the mammalian host, where it is prone to attacks by the host immune system. As a sophisticated means of defence against the immune response, the parasite's surface is coated in a dense layer of the variant surface glycoprotein (VSG), that reduces identification of invariant epitopes on the cell surface by the immune system to levels that prevent host immunity. The VSG has to form a coat that is both dense and mobile, to shield invariant surface proteins from detection and to allow quick recycling of the protective coat during immune evasion. This coat effectively protects the parasite from the harsh environment that is the mammalian bloodstream and leads to a persistent parasitemia if the infection remains untreated. The available treatment against African Trypanosomiasis involves the use of drugs that are themselves severely toxic and that can lead to the death of the patient. Most of the drugs used as treatment were developed in the early-to-mid 20th century, and while developments continue, they still represent the best medical means to fight the parasite. The discovery of a fluorescent VSG gave rise to speculations about a potential interaction between the VSG coat and components of the surrounding medium, that could also lead to a new approach in the treatment of African Trypanosomiasis that involves the VSG coat. The initially observed fluorescence signal was specific for a combination of a VSG called VSG'Y' and the triphenylmethane (TPM) dye phenol red. Exchanging this TPM to a bromo-derivative led to the observation of another fluorescence effect termed trypanicidal effect which killed the parasite independent of the expressed VSG and suggests a structurally conserved feature between VSGs that could function as a specific drug target against T. b. brucei. The work of this thesis aims to identify the mechanisms that govern the unique VSG'Y' fluorescence and the trypanocidal effect. Fluorescence experiments and protein mutagenesis of VSG'Y' as well as crystallographic trials with a range of different VSGs were utilized in the endeavour to identify the binding mechanisms between TPM compounds and VSGs, to find potentially conserved structural features between VSGs and to identify the working mechanisms of VSG fluorescence and the trypanocidal effect. These trials have the potential to lead to the formulation of highly specific drugs that target the parasites VSG coat. During the crystallographic trials of this thesis, the complete structure of a VSG was solved experimentally for the first time. This complete structure is a key component in furthering the understanding of the mechanisms governing VSG coat formation. X-ray scattering techniques, involving x-ray crystallography and small angle x-ray scattering were applied to elucidate the first complete VSG structures, which reveal high flexibility of the protein and supplies insight into the importance of this flexibility in the formation of a densely packed but highly mobile surface coat.}, subject = {Trypanosoma brucei brucei}, language = {en} } @phdthesis{Milenkovic2006, author = {Milenkovic, Vladimir M.}, title = {Structural and functional analysis of bestrophin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19372}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Morbus Best (OMIM 153700), auch als vitelliforme Makuladystrophie Typ 2 (VMD2) bezeichnet, ist eine autosomal dominant vererbte Makuladystrophie mit juvenilem Beginn. Die Erkrankung geht einher mit einer Ansammlung von Lipofuscin-{\"a}hnlichem Material im sowie unterhalb des retinalen Pigmentepithels (RPE). Das bei Morbus Best mutierte VMD2- Gen kodiert f{\"u}r ein 585 Aminos{\"a}uren langes Transmembranprotein, genannt Bestrophin, und wird vorwiegend im RPE exprimiert. Das Protein hat eine komplexe Membrantopologie mit 4-6 putativen Transmembrandom{\"a}nen (TMD) und ist vermutlich in den Ca2+-abh{\"a}ngigen Transport von Chloridionen durch die Plasmamembran involviert. Die {\"u}berwiegende Mehrheit der krankheitsassoziierten Ver{\"a}nderungen bei M. Best Patienten sind Missense-Mutationen, die innerhalb der hochkonservierten N-terminalen H{\"a}lfte des Proteins nahe der mutmaßlichen Transmembrandom{\"a}nen akkumulieren. Der Zusammenhang zwischen Pathologie und identifizierter Mutationen bzw. der Chloridkanal- Funktion von Bestrophin-1 ist noch unklar. Um die biologische Funktion von Bestrohin-1 weiter aufzukl{\"a}ren und die zugrunde liegenden molekularen Mechanismen der BMD besser zu verstehen, wurde mit Hilfe des GAL4-basierenden Hefe-Zwei-Hybridsystems (Y2H) nach interagierenden Partnern von Bestrophin-1 gesucht. Ein Screen in einer bovinen RPE cDNABank mit verschiedenen verk{\"u}rzten Fragmenten von Bestrophin-1 ergab 53 m{\"o}gliche interagierende Partner. Allerdings schlossen anschließende Verifikationsexperimente die Kandidatengene aus. Somit deuten die Resultate dieser umfangreichen YH2-Studie daraufhin, dass Bestrophin f{\"u}r das herk{\"o}mmliche Zwei-Hybrid-System nicht geeignet ist. Zum einen k{\"o}nnte dies daran liegen, dass das Protein ein integraler Bestandteil der Membran ist und zum anderen, dass m{\"o}glicherweise der Transport der gew{\"a}hlten Bestrophin-Fragmente zum Nukleus nicht stattfindet. Dies gilt jedoch als Grundvoraussetzung f{\"u}r eine Proteininteraktion im Hefe-2-Hybridsystem. Bestrophin geh{\"o}rt zu einer großen Familie von integralen Membranproteinen, von der bis heute bereits {\"u}ber 100 Mitglieder bei verschiedenen Organismengruppen wie denS{\"a}ugern, Insekten und W{\"u}rmern identifiziert werden konnten. Als auff{\"a}lligste Besonderheit in der Familie der Bestrophine zeigt sich neben einer nicht-variablen RFP-Dom{\"a}ne (Arginin- Phenylalanin-Prolin) eine evolution{\"a}r hochkonservierte N-terminale Region. Um die phylogenetische Beziehung der Bestrophine zu untersuchen sowie den Aufbau und die Funktion von konservierten Motiven innerhalb der Familienmitglieder zu identifizieren, wurde diese konservierte N-terminale Region sowohl bioinformatisch wie auch Chapter Two: Zusammenfassung 4 phylogenetisch weiter untersucht. Die phylogenetische Analyse der Bestrophin Homologen brachte vier evolution{\"a}r konservierte Familienmitglieder in S{\"a}ugern hervor, die jeweils eine starke Homologie zu den Proteinen VMD2, VMD2-L1 bis VMD2-L3 des Menschen zeigen. Die signifikante {\"A}hnlichkeit der Proteinsequenz innerhalb der vier Familienmitglieder l{\"a}sst die Schlussfolgerungen zu, dass zum einen jedes einzelne Familienmitglied ihre eigene evolution{\"a}r konservierte Funktion hat und zum anderen dass die Divergenz des Bestrophins in verschiedene Familienmitglieder zeitlich vor der Divergenz der verschiedenen S{\"a}ugerspezien erfolgt sein muss.}, subject = {Makuladegeneration}, language = {en} }