@article{HornKellerHildebrandtetal.2016,
  author    = {Horn, Hannes and Keller, Alexander and Hildebrandt, Ulrich and K{\"a}mpfer, Peter and Riederer, Markus and Hentschel, Ute},
  title     = {Draft genome of the \(Arabidopsis\) \(thaliana\) phyllosphere bacterium, \(Williamsia\) sp. ARP1},
  series = {Standards in Genomic Sciences},
  volume    = {11},
  journal   = {Standards in Genomic Sciences},
  number    = {8},
  doi       = {10.1186/s40793-015-0122-x},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146008},
  year      = {2016},
  abstract  = {The Gram-positive actinomycete \(Williamsia\) sp. ARP1 was originally isolated from the \(Arabidopsis\) \(thaliana\) phyllosphere. Here we describe the general physiological features of this microorganism together with the draft genome sequence and annotation. The 4,745,080 bp long genome contains 4434 protein-coding genes and 70 RNA genes. To our knowledge, this is only the second reported genome from the genus \(Williamsia\) and the first sequenced strain from the phyllosphere. The presented genomic information is interpreted in the context of an adaptation to the phyllosphere habitat.},
  language  = {en}
}
@article{PeckSchugZhangetal.2016,
  author    = {Peck, Barrie and Schug, Zachary T. and Zhang, Qifeng and Dankworth, Beatrice and Jones, Dylan T. and Smethurst, Elizabeth and Patel, Rachana and Mason, Susan and Jian, Ming and Saunders, Rebecca and Howell, Michael and Mitter, Richard and Spencer-Dene, Bradley and Stamp, Gordon and McGarry, Lynn and James, Daniel and Shanks, Emma and Aboagye, Eric O. and Critchlow, Susan E. and Leung, Hing Y. and Harris, Adrian L. and Wakelam, Michael J. O. and Gottlieb, Eyal and Schulze, Almut},
  title     = {Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments},
  series = {Cancer \& Metabolism},
  volume    = {4},
  journal   = {Cancer \& Metabolism},
  number    = {6},
  doi       = {10.1186/s40170-016-0146-8},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145905},
  year      = {2016},
  abstract  = {Background Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets. Results Using functional genomics, we identified stearoyl-CoA desaturase (SCD), an enzyme that controls synthesis of unsaturated fatty acids, as essential in breast and prostate cancer cells. SCD inhibition altered cellular lipid composition and impeded cell viability in the absence of exogenous lipids. SCD inhibition also altered cardiolipin composition, leading to the release of cytochrome C and induction of apoptosis. Furthermore, SCD was required for the generation of poly-unsaturated lipids in cancer cells grown in spheroid cultures, which resemble those found in tumour tissue. We also found that SCD mRNA and protein expression is elevated in human breast cancers and predicts poor survival in high-grade tumours. Finally, silencing of SCD in prostate orthografts efficiently blocked tumour growth and significantly increased animal survival. Conclusions Our data implicate lipid desaturation as an essential process for cancer cell survival and suggest that targeting SCD could efficiently limit tumour expansion, especially under the metabolically compromised conditions of the tumour microenvironment.},
  language  = {en}
}
@phdthesis{Sickel2016,
  author    = {Sickel, Wiebke},
  title     = {High-throughput biodiversity assessment - Powers and limitations of meta-barcoding},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144573},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Traditional species identification based on morphological characters is laborious and requires expert knowledge. It is further complicated in the case of species assemblages or degraded and processed material. DNA-barcoding, species identification based on genetic data, has become a suitable alternative, yet species assemblages are still difficult to study. In the past decade meta-barcoding has widely been adopted for the study of species communities, due to technological advances in modern sequencing platforms and because manual separation of individual specimen is not required. Here, meta-barcoding is put into context and applied to the study of bee-collected pollen as well as bacterial communities. These studies provide the basis for a critical evaluation of the powers and limitations of meta-barcoding. Advantages identified include species identification without the need for expert knowledge as well as the high throughput of samples and sequences. In microbiology, meta-barcoding can facilitate directed cultivation of taxa of interest identified with meta-barcoding data. Disadvantages include insufficient species resolution due to short read lengths and incomplete reference databases, as well as limitations in abundance estimation of taxa and functional profiling. Despite these, meta-barcoding is a powerful method for the analysis of species communities and holds high potential especially for automated biomonitoring.},
  subject      = {Biodiversit{\"a}t},
  language  = {en}
}
@phdthesis{Cicova2016,
  author    = {Cicova, Zdenka},
  title     = {Characterization of a novel putative factor involved in host adaptation in Trypanosoma brucei},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level in a systematic way. However, a detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor (Tb927.11.2400) identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38\% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin like (TbFlabarinL) and demonstrate that it is a result of a gene duplication event, which occurred in African trypanosomes. TbFlabarinL is not essential for growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated a TbFlabarinL-specific antibody and showed that it localizes in the flagellum. The co-immunoprecipitation experiment together with a biochemical cell fractionation indicated a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod.},
  subject      = {Trypanosoma brucei},
  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{Costea2016,
  author    = {Costea, Paul Igor},
  title     = {Stratification and variation of the human gut microbiota},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139649},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The microbial communities that live inside the human gastrointestinal tract -the human gut microbiome- are important for host health and wellbeing. Characterizing this new "organ", made up of as many cells as the human body itself, has recently become possible through technological advances. Metagenomics, the high-throughput sequencing of DNA directly from microbial communities, enables us to take genomic snapshots of thousands of microbes living together in this complex ecosystem, without the need for isolating and growing them. Quantifying the composition of the human gut microbiome allows us to investigate its properties and connect it to host physiology and disease. The wealth of such connections was unexpected and is probably still underestimated. Due to the fact that most of our dietary as well as medicinal intake affects the microbiome and that the microbiome itself interacts with our immune system through a multitude of pathways, many mechanisms have been proposed to explain the observed correlations, though most have yet to be understood in depth. An obvious prerequisite to characterizing the microbiome and its interactions with the host is the accurate quantification of its composition, i.e. determining which microbes are present and in what numbers they occur. Historically, standard practices have existed for sample handling, DNA extraction and data analysis for many years. However, these were generally developed for single microbe cultures and it is not always feasible to implement them in large scale metagenomic studies. Partly because of this and partly because of the excitement that new technology brings about, the first metagenomic studies each took the liberty to define their own approach and protocols. From early meta-analysis of these studies it became clear that the differences in sample handling, as well as differences in computational approaches, made comparisons across studies very difficult. This restricts our ability to cross-validate findings of individual studies and to pool samples from larger cohorts. To address the pressing need for standardization, we undertook an extensive comparison of 21 different DNA extraction methods as well as a series of other sample manipulations that affect quantification. We developed a number of criteria for determining the measurement quality in the absence of a mock community and used these to propose best practices for sampling, DNA extraction and library preparation. If these were to be accepted as standards in the field, it would greatly improve comparability across studies, which would dramatically increase the power of our inferences and our ability to draw general conclusions about the microbiome. Most metagenomics studies involve comparisons between microbial communities, for example between fecal samples from cases and controls. A multitude of approaches have been proposed to calculate community dissimilarities (beta diversity) and they are often combined with various preprocessing techniques. Direct metagenomics quantification usually counts sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to technology-inherent differences in sampling depth, normalizing counts is necessary, for instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by subsampling. To derive a single value for community (dis-)similarity, multiple distance measures have been proposed. Although it is theoretically difficult to benchmark these approaches, we developed a biologically motivated framework in which distance measures can be evaluated. This highlights the importance of data transformations and their impact on the measured distances. Building on our experience with accurate abundance estimation and data preprocessing techniques, we can now try and understand some of the basic properties of microbial communities. In 2011, it was proposed that the space of genus level variation of the human gut microbial community is structured into three basic types, termed enterotypes. These were described in a multi-country cohort, so as to be independent of geography, age and other host properties. Operationally defined through a clustering approach, they are "densely populated areas in a multidimensional space of community composition"(source) and were proposed as a general stratifier for the human population. Later studies that applied this concept to other datasets raised concerns about the optimum number of clusters and robustness of the clustering approach. This heralded a long standing debate about the existence of structure and the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in the context of the vastly increased amounts of available data. We propose a refined framework in which the different types should be thought of as weak attractors in compositional space and we try to implement an approach to determining which attractor a sample is closest to. To this end, we train a classifier on a reference dataset to assign membership to new samples. This way, enterotypes assignment is no longer dataset dependent and effects due to biased sampling are minimized. Using a model in which we assume the existence of three enterotypes characterized by the same driver genera, as originally postulated, we show the relevance of this stratification and propose it to be used in a clinical setting as a potential marker for disease development. Moreover, we believe that these attractors underline different rules of community assembly and we recommend they be accounted for when analyzing gut microbiome samples. While enterotypes describe structure in the community at genus level, metagenomic sequencing can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis methodology for this level of resolution has only recently been developed and little exploration has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the landscape of genomic variation seems highly structured even beyond species resolution, indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several cases, these subspecies exhibit geo-stratification, with some subspecies only found in the Chinese population. Generally however, they present only minor dispersion limitations and are seen across most of our study populations. Within one individual, one subspecies is commonly found to dominate and only rarely are several subspecies observed to co-occur in the same ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable over periods of more than three years. When interrogating their functional properties we find many differences, with specific ones appearing relevant to the host. For example, we identify a subspecies of E. rectale that is lacking the flagellum operon and find its presence to be significantly associated with lower body mass index and lower insulin resistance of their hosts; it also correlates with higher microbial community diversity. These associations could not be seen at the species level (where multiple subspecies are convoluted), which illustrates the importance of this increased resolution for a more comprehensive understanding of microbial interactions within the microbiome and with the host. Taken together, our results provide a rigorous basis for performing comparative metagenomics of the human gut, encompassing recommendations for both experimental sample processing and computational analysis. We furthermore refine the concept of community stratification into enterotypes, develop a reference-based approach for enterotype assignment and provide compelling evidence for their relevance. Lastly, by harnessing the full resolution of metagenomics, we discover a highly structured genomic variation landscape below the microbial species level and identify common subspecies of the human gut microbiome. By developing these high-precision metagenomics analysis tools, we thus hope to contribute to a greatly improved understanding of the properties and dynamics of the human gut microbiome.},
  subject      = {Mensch},
  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{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{Koenig2016,
  author    = {K{\"o}nig, Sebastian},
  title     = {Spatially selective visual attention in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134452},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Finding the right behavior at the right time is one of the major tasks of brains. In a natural scenery there is often an abundance of stimuli present and the brain has to separate the relevant from the irrelevant ones. Selective visual attention (SVA) is a property of higher visual systems that achieves this separation, as it allows to '[…] focus on one source of sensory input to the exclusion of others' (Luck and Mangun, 1996). There are probably several forms of SVA depending upon the criteria used for the separation, such as salience, color, location in space, novelty, or motion. Many studies have investigated SVA in humans and non-human primates. However, complex functions like attention were initially not expected to be already implemented in the brains of simple organisms like Drosophila. After a first demonstration of selective attention in the fly (Wolf and Heisenberg, 1980), it took some time until other studies included attentional mechanisms in their argumentation to explain certain behaviors of Drosophila. However, their definition and characterization of attention differed and often was ambiguous. Here, one particular form, spatially selective visual attention in the fly Drosophila is investigated. It has been shown earlier that the fly spontaneously may restrict its behavioral responses in stationary flight to the visual stimuli on one side of the visual field. On the basis of experiments of Sareen et al., (2011) it has been conjectured that the fly has a focus of attention (FoA) and that the fly responds to the visual stimuli within this area of the visual field. Whether the FoA is the adequate concept for this spatial property of SVA in the fly needs to be further discussed and is a subject also of the present study. At this stage, the concept will be used in the description of the new results expanding the characterization of SVA. This study continued the investigation of SVA during tethered flight with variable but controlled visual input and an automated primary data evaluation. This standardized paradigm allowed for analysis of wild-type behavior as well as for a comparison of several mutant and pharmacologically manipulated strains to the wild-type. Some properties of human SVA like the occurrence of externally as well as internally caused shifts of attention were found in Drosophila and it could be shown, that SVA in the fly can be externally guided and has an attention span. Additionally, a neurotransmitter and proteins, which play a significant role in SVA were discovered. Based on this, the genetic tools available for Drosophila provided the means to a first examination of cells and circuits involved in SVA. Finally, the free walk behavior of flies that had been shown to have compromised SVA was characterized. The results suggested that the observed phenotypes of SVA were not behavior specific. Covert shifts of the FoA were investigated. The FoA can be externally guided by visual cues to one or the other side of the visual field and even after the cue has disappeared it remains there for <4s. An intriguing finding of this study is the fact, that the quality of the cue determines whether it is attractive or repellent. For example a cue can be changed from being repellent (negative) to being attractive (positive) by changing its oscillation amplitude from 4° to 2°. Testing the effectiveness of cues in the upper and lower visual field separately, revealed that the perception of a cue by the fly is not exclusively based on a sum of its specifications. Because positive cueing did not have an after-effect in each of the two half-fields alone, but did so if the cue was shown in both, the fly seems to evaluate the cue for each combination of parameters specifically. Whether this evaluation of the cue changed on a trial-to-trial basis or if the cue in some cases failed to shift the FoA can at this point not be determined. Looking at the responses of the fly to the displacement of a black vertical stripe showed that they can be categorized as no responses, syn-directional responses (following the direction of motion of the stripe) and anti-directional responses (in the opposite direction of the motion of the stripe). The yaw-torque patterns of the latter bared similarities with spontaneous body saccades and they most likely represented escape attempts of the fly. Syn-directional responses, however, were genuine object responses, distinguishable by a longer latency until they were elicited and a larger amplitude. These properties as well as the distribution of response polarities were not influenced by the presence or absence of a cue. When two stripes were displaced simultaneously in opposite directions the rate of no responses increased in comparison to the displacement of a single stripe. If one of the stripes was cued, both, the responses towards and away from the side of cue resembled the syn-directional responses. Significant progress was made with the elucidation of the neuronal underpinnings of SVA. Ablation of the mushroom bodies (MB) demonstrated their requirement for SVA. Furthermore, it was shown that dopamine signaling has to be balanced between too much and too little. Either inhibiting the synthesis of dopamine or its re-uptake at the synapse via the dDAT impaired the flies' susceptibility to cueing. Using the Gal4/UAS system, cell specific expression or knockdown of the dDAT was used to scrutinize the role of MB sub-compartments in SVA. The αβ-lobes turned out to be necessary and sufficient to maintain SVA. The Gal4-line c708a labels only a subset of Kenyon cells (KC) within the αβ-lobes, αβposterior. These cells stand out, because of (A) the mesh-like arrangement of their fibers within the lobes and (B) the fact that unlike the other KCs they bypass the calyx and thereby the main source of olfactory input to the MBs, forming connections only in the posterior accessory calyx (Tanaka et al., 2008). This structure receives no or only marginal olfactory input, suggesting for it a role in tasks other than olfaction. This study shows their requirement in a visual task by demonstrating that they are necessary to uphold SVA. Restoring dDAT function in these approximately only 90 cells was probably insufficient to lower the dopamine concentration at the relevant synapses and hence a rescue failed. Alternatively, the processes mediating SVA at the αβ-lobes might require an interplay between all of their KCs. In conclusion, the results provide an initial point for future research to fully understand the localization of and circuitry required for SVA in the brain. In the experiments described so far, attention has been externally guided. However, flies are also able to internally shift their FoA without any cues from the outside world. In a set of 60 consecutive simultaneous displacements of two stripes, they were more likely to produce a response with the same polarity as the preceding one than a random polarity selection predicted. This suggested a dwelling of the FoA on one side of the visual field. Assuming that each response was influenced by the previous one in a way that the probability to repeat the response polarity was increased by a certain factor (dwelling factor, df), a random selection of response type including a df was computed. Implementation of the df removed the difference between observed probability of polarity repetition and the one suggested by random selection. When the interval between displacements was iteratively increased to 5s, no significant df could be detected anymore for pauses longer than 4s. In conclusion, Drosophila has an attention span of approximately 4s. Flies with a mutation in the radish gene expressed no after-effect of cueing and had a shortened attention span of about 1s. The dDAT inhibitor methylphenidate is able to rescue the first, but does not affect the latter phenotype. Probably, radish is differently involved in the two mechanisms. This study showed, that endogenous (covert) shifts of spatially selective visual attention in the fly Drosophila can be internally and externally guided. The variables determining the quality of a cue turned out to be multifaceted and a more systematic approach is needed for a better understanding of what property or feature of the cue changes the way it is evaluated by the fly. A first step has been made to demonstrate that SVA is a fundamental process and compromising it can influence the characteristics of other behaviors like walking. The existence of an attention span, the dependence of SVA on dopamine as well as the susceptibility to pharmacological manipulations, which in humans are used to treat respective diseases, point towards striking similarities between SVA in humans and Drosophila.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Hackl2016,
  author    = {Hackl, Thomas},
  title     = {A draft genome for the Venus flytrap, Dionaea muscipula : Evaluation of assembly strategies for a complex Genome - Development of novel approaches and bioinformatics solutions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133149},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The Venus flytrap, \textit{Dionaea muscipula}, with its carnivorous life-style and its highly specialized snap-traps has fascinated biologist since the days of Charles Darwin. The goal of the \textit{D. muscipula} genome project is to gain comprehensive insights into the genomic landscape of this remarkable plant. The genome of the diploid Venus flytrap with an estimated size between 2.6 Gbp to 3.0 Gbp is comparatively large and comprises more than 70 \% of repetitive regions. Sequencing and assembly of genomes of this scale are even with state-of-the-art technology and software challenging. Initial sequencing and assembly of the genome was performed by the BGI (Beijing Genomics Institute) in 2011 resulting in a 3.7 Gbp draft assembly. I started my work with thorough assessment of the delivered assembly and data. My analysis showed that the BGI assembly is highly fragmented and at the same time artificially inflated due to overassembly of repetitive sequences. Furthermore, it only comprises about on third of the expected genes in full-length, rendering it inadequate for downstream analysis. In the following I sought to optimize the sequencing and assembly strategy to obtain an assembly of higher completeness and contiguity by improving data quality and assembly procedure and by developing tailored bioinformatics tools. Issues with technical biases and high levels of heterogeneity in the original data set were solved by sequencing additional short read libraries from high quality non-polymorphic DNA samples. To address contiguity and heterozygosity I examined numerous alternative assembly software packages and strategies and eventually identified ALLPATHS-LG as the most suited program for assembling the data at hand. Moreover, by utilizing digital normalization to reduce repetitive reads, I was able to substantially reduce computational demands while at the same time significantly increasing contiguity of the assembly. To improve repeat resolution and scaffolding, I started to explore the novel PacBio long read sequencing technology. Raw PacBio reads exhibit high error rates of 15 \% impeding their use for assembly. To overcome this issue, I developed the PacBio hybrid correction pipeline proovread (Hackl et al., 2014). proovread uses high coverage Illumina read data in an iterative mapping-based consensus procedure to identify and remove errors present in raw PacBio reads. In terms of sensitivity and accuracy, proovread outperforms existing software. In contrast to other correction programs, which are incapable of handling data sets of the size of D. muscipula project, proovread's flexible design allows for the efficient distribution of work load on high-performance computing clusters, thus enabling the correction of the Venus flytrap PacBio data set. Next to the assembly process itself, also the assessment of the large de novo draft assemblies, particularly with respect to coverage by available sequencing data, is difficult. While typical evaluation procedures rely on computationally extensive mapping approaches, I developed and implemented a set of tools that utilize k-mer coverage and derived values to efficiently compute coverage landscapes of large-scale assemblies and in addition allow for automated visualization of the of the obtained information in comprehensive plots. Using the developed tools to analyze preliminary assemblies and by combining my findings regarding optimizations of the assembly process, I was ultimately able to generate a high quality draft assembly for D. muscipula. I further refined the assembly by removal of redundant contigs resulting from separate assembly of heterozygous regions and additional scaffolding and gapclosing using corrected PacBio data. The final draft assembly comprises 86 × 10 3 scaffolds and has a total size of 1.45 Gbp. The difference to the estimated genomes size is well explained by collapsed repeats. At the same time, the assembly exhibits high fractions full-length gene models, corroborating the interpretation that the obtained draft assembly provides a complete and comprehensive reference for further exploration of the fascinating biology of the Venus flytrap.},
  subject      = {Venusfliegenfalle},
  language  = {en}
}