@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{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{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{Chouhan2017,
  author    = {Chouhan, Nitin Singh},
  title     = {Time-odor learning in \(Drosophila\) \(melanogaster\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145675},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Endogenous clocks help animals to anticipate the daily environmental changes. These internal clocks rely on environmental cues, called Zeitgeber, for synchronization. The molecular clock consists of transcription-translation feedback loops and is located in about 150 neurons (Helfrich-F{\"o}rster and Homberg, 1993; Helfrich-F{\"o}rster, 2005). The core clock has the proteins Clock (CLK) and Cycle (CYC) that together act as a transcription activator for period (per) and timeless (tim) which then, via PER and TIM block their own transcription by inhibiting CLK/CYC activity (Darlington et al., 1998; Hardin, 2005; Dubruille and Emery, 2008). Light signals trigger the degradation of TIM through a blue-light sensing protein Cryptochrome (CRY) and thus, allows CLK/CYC to resume per and tim transcription (Emery et al., 1998; Stanewsky et al., 1998). Therefore, light acts as an important Zeitgeber for the clock entrainment. The mammalian clock consists of similarly intertwined feedback loops. Endogenous clocks facilitate appropriate alterations in a variety of behaviors according to the time of day. Also, these clocks can provide the phase information to the memory centers of the brain to form the time of day related associations (TOD). TOD memories promote appropriate usage of resources and concurrently better the survival success of an animal. For instance, animals can form time-place associations related to the availability of a biologically significant stimulus like food or mate. Such memories will help the animal to obtain resources at different locations at the appropriate time of day. The significance of these memories is supported by the fact that many organisms including bees, ants, rats and mice demonstrate time-place learning (Biebach et al. 1991; Mistlberger et al. 1997; Van der Zee et al. 2008; Wenger et al. 1991). Previous studies have shown that TOD related memories rely on an internal clock, but the identity of the clock and the underlying mechanism remain less well understood. The present study demonstrates that flies can also form TOD associated odor memories and further seeks to identify the appropriate mechanism. Hungry flies were trained in the morning to associate odor A with the sucrose reward and subsequently were exposed to odor B without reward. The same flies were exposed in the afternoon to odor B with and odor A without reward. Two cycles of the 65 reversal training on two subsequent days resulted in the significant retrieval of specific odor memories in the morning and afternoon tests. Therefore, flies were able to modulate their odor preference according to the time of day. In contrast, flies trained in a non-reversal manner were unable to form TOD related memories. The study also demonstrates that flies are only able to form time-odor memories when the two reciprocal training cycles occur at a minimum 6 h interval. This work also highlights the role of the internal state of flies in establishing timeodor memories. Prolonged starvation motivates flies to appropriate their search for the food. It increases the cost associated with a wrong choice in the T-maze test as it precludes the food discovery. Accordingly, an extended starvation promotes the TOD related changes in the odor preference in flies already with a single cycle of reversal training. Intriguingly, prolonged starvation is required for the time-odor memory acquisition but is dispensable during the memory retrieval. Endogenous oscillators promote time-odor associations in flies. Flies in constant darkness have functional rhythms and can form time-odor memories. In contrast, flies kept in constant light become arrhythmic and demonstrated no change in their odor preference through the day. Also, clock mutant flies per01 and clkAR, show compromised performance compared to CS flies when trained in the time-odor conditioning assay. These results suggest that flies need a per and clk dependent oscillator for establishing TOD related memories. Also, the clock governed rhythms are necessary for the timeodor memory acquisition but not for the retrieval. Pigment-Dispersing Factor (PDF) neuropeptide is a clock output factor (Park and Hall, 1998; Park et al., 2000; Helfrich-F{\"o}rster, 2009). pdf01 mutant flies are unable to form significant time-odor memories. PDF is released by 8 neurons per hemisphere in the fly brain. This cluster includes the small (s-LNvs) and large (l-LNvs) ventral lateral neurons. Restoring PDF in these 16 neurons in the pdf01 mutant background rescues the time-odor learning defect. The PDF neuropeptide activates a seven transmembrane G-protein coupled receptor (PDFR) which is broadly expressed in the fly brain (Hyun et al., 2005). The present study shows that the expression of PDFR in about 10 dorsal neurons (DN1p) is sufficient for robust time-odor associations in flies. 66 In conclusion, flies use distinct endogenous oscillators to acquire and retrieve time-odor memories. The first oscillator is light dependent and likely signals through the PDF neuropeptide to promote the usage of the time as an associative cue during appetitive conditioning. In contrast, the second clock is light independent and specifically signals the time information for the memory retrieval. The identity of this clock and the underlying mechanism are open to investigation.},
  subject      = {Taufliege},
  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{Dusik2015,
  author    = {Dusik, Verena},
  title     = {Immunhistochemische und funktionelle Charakterisierung der Mitogen-aktivierten Proteinkinase p38 in der inneren Uhr von Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124636},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Circadianes und Stress-System sind zwei physiologische Systeme, die dem Organismus helfen sich an Ver{\"a}nderungen ihrer Umwelt anzupassen. W{\"a}hrend letzteres spontane und schnelle Antworten auf akute, unvorhersehbare Umweltreize liefert, sagt das circadiane System t{\"a}glich wiederkehrende Ereignisse vorher and bereitet den Organismus so vorzeitig auf diese nahende Umweltver{\"a}nderung vor. Dennoch, trotz dieser unterschiedlichen Reaktionsmechanismen agieren beide Systeme nicht komplett autonom. Studien der vergangen Jahre belegen vielmehr eine Interaktion beider Systeme. So postulieren sie zum einem Unterschiede in der Stressantwort in Abh{\"a}ngigkeit von der Tageszeit zu der der Reiz auftritt und weisen zugleich auf eine Zunahme von gest{\"o}rten biologischen Tagesrhythmen, wie zum Beispiel Schlafst{\"o}rungen, in Folge von unkontrollierten oder exzessiven Stress hin. Ebenso liefern k{\"u}rzlich durchgef{\"u}hrte Studien an Vertebraten und Pilzen Hinweise, dass mit p38, eine Stress-aktivierte Kinase, an der Signalweiterleitung zur inneren Uhr beteiligt ist (Hayashi et al., 2003), sogar durch dieses endogene Zeitmesssystem reguliert wird (Vitalini et al., 2007; Lamb et al., 2011) und deuten damit erstmals eine m{\"o}gliche Verbindung zwischen Stress-induzierten und regul{\"a}ren rhythmischen Anpassungen des Organismus an Umweltver{\"a}nderungen an. Molekulare und zellul{\"a}re Mechanismen dieser Verkn{\"u}pfung sind bisher noch nicht bekannt. W{\"a}hrend die Rolle von p38 MAPK bei der Stress- und Immunantwort in Drosophila melanogaster gut charakterisiert ist, wurden Expression und Funktion von p38 in der inneren Uhr hingegen bislang nicht untersucht. Die hier vorliegende Arbeit hatte daher zum Ziel mittels immunhistochemischer, verhaltensphysiologischer und molekularer Methoden eine m{\"o}gliche Rolle der Stress-aktivierten Kinase im circadianen System der Fliege aufzudecken. Antik{\"o}rperf{\"a}rbungen sowie Studien mit Reporterlinien zeigen deutliche F{\"a}rbesignale in den s-LNv, l-LNv und DN1a und erbringen erstmals einen Nachweis f{\"u}r p38 Expression in den Uhrneuronen der Fliege. Ebenso scheint die Aktivit{\"a}t von p38 MAPK in den DN1a uhrgesteuert zu sein. So liegt p38 vermehrt in seiner aktiven Form in der Dunkelphase vor und zeigt, neben seiner circadian regulierten Aktivierung, zus{\"a}tzlich auch eine Inaktivierung durch Licht. 15-Minuten-Lichtpulse in der subjektiven Nacht f{\"u}hren zu einer signifikanten Reduktion von aktivierter, phosphorylierter p38 MAPK in den DN1a von Canton S Wildtypfliegen im Vergleich zu Fliegen ohne Lichtpuls-Behandlung. Aufzeichnungen der Lokomotoraktivit{\"a}t offenbaren zus{\"a}tzlich die Notwendigkeit von p38 MAPK f{\"u}r wildtypisches Timing der Abendaktivit{\"a}t sowie zum Erhalt von 24-Stunden-Verhaltensrhythmen unter konstanten Dauerdunkel-Bedindungen. So zeigen Fliegen mit reduzierten p38 Level in Uhrneuronen einen verz{\"o}gerten Beginn der Abendaktivit{\"a}t und stark verl{\"a}ngerte Freilaufperioden. In {\"U}bereinstimmung mit Effekten auf das Laufverhalten scheint dar{\"u}ber hinaus die Expression einer dominant-negativen Form von p38b in Drosophila's wichtigsten Uhrneuronen eine versp{\"a}tete nukle{\"a}re Translokation von Period zur Folge zu haben. Westernblots legen zus{\"a}tzlich einen Einfluss von p38 auf den Phosphorylierungsgrad von Period nahe und liefern damit einen m{\"o}gliche Erkl{\"a}rung f{\"u}r den versp{\"a}teten Kerneintritt des Uhrproteins. Abschließende St{\"u}tzung der Westernblotergebnisse bringen in vitro Kinasenassays und deuten auf p38 als eine potentielle „Uhrkinase" hin, welche auch in vivo Period an Serin 661 sowie weiteren potentiellen Phosphorylierungsstellen phosphorylieren k{\"o}nnte. Zusammengenommen deuten die Ergebnisse der hier vorliegenden Arbeit eindeutig auf eine bedeutende Rolle von p38, neben dessen Funkion im Stress-System, auch im circadianen System der Fliege hin und offenbaren damit die M{\"o}glichkeit, dass p38 als Schnittstelle zwischen beider Systeme fungiert.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Schlichting2015,
  author    = {Schlichting, Matthias},
  title     = {Light entrainment of the circadian clock: the importance of the visual system for adjusting Drosophila melanogaster´s activity pattern},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114457},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The change of day and night is one of the challenges all organisms are exposed to, as they have to adjust their physiology and behavior in an appropriate way. Therefore so called circadian clocks have evolved, which allow the organism to predict these cyclic changes of day and night. The underlying molecular mechanism is oscillating with its endogenous period of approximately 24 hours in constant conditions, but as soon as external stimuli, so called Zeitgebers, are present, the clocks adjust their period to exactly 24h, which is called entrainment. Studies in several species, including humans, animals and plants, showed that light is the most important Zeitgeber synchronizing physiology and behavior to the changes of day and night. Nevertheless also other stimuli, like changes in temperature, humidity or social interactions, are powerful Zeitgebers for entraining the clock. This thesis will focus on the question, how light influences the locomotor behavior of the fly in general, including a particular interest on the entrainment of the circadian clock. As a model organism Drosophila melanogaster was used. During the last years several research groups investigated the effect of light on the circadian clock and their results showed that several light input pathways to the clock contribute to wild-type behavior. Most of the studies focused on the photopigment Cryptochrome (CRY) which is expressed in about half of the 150 clock neurons in the fly. CRY is activated by light, degrades the clock protein Timeless (TIM) and hence entrains the clock to the light-dark (LD)-cycle resulting from changes of day and night. However, also flies lacking CRY are still able to entrain their clock mechanism as well as their activity-rest-rhythm to LD-cycles, clearly showing that the visual system of the fly also contributes to clock synchronization. The mechanism how light information from the visual system is transferred to the clock is so far still unknown. This is also true for so-called masking-effects which are changes in the behavior of the animal that are directly initiated by external stimuli and therefore independent of the circadian clock. These effects complement the behavior of the animals as they enable the fly to react quickly to changes in the environment even during the clock-controlled rest state. Both of these behavioral features were analyzed in more detail in this study. On the one hand, we investigated the influence of the compound eyes on the entrainment of the clock neurons and on the other hand, we tried to separate clock-controlled behavior from masking. To do so "nature-like" light conditions were simulated allowing the investigation of masking and entrainment within one experiment. The simulation of moonlight and twilight conditions caused significant changes in the locomotor behavior. Moonlit nights increased nocturnal activity levels and shifted the morning (M) and evening (E) activity bouts into the night. The opposite was true for the investigation of twilight, as the activity bouts were shifted into the day. The simulation of twilight and moonlight within the same experiment further showed that twilight appears to dominate over moonlight, which is in accordance to the assumption that twilight in nature is one of the key signals to synchronize the clock as the light intensity during early dawn rises similarly in every season. By investigating different mutants with impaired visual system we showed that the compound eyes are essential for the observed behavioral adaptations. The inner receptor cells (R7 and R8) are important for synchronizing the endogenous clock mechanism to the changes of day and night. In terms of masking, a complex interaction of all receptor cells seems to adjust the behavioral pattern, as only flies lacking photopigments in inner and outer receptor cells lacked all masking effects. However, not only the compound eyes seem to contribute to rhythmic activity in moonlit nights. CRY-mutant flies shift their E activity bout even more into the night than wild-type flies do. By applying Drosophila genetics we were able to narrow down this effect to only four CRY expressing clock neurons per hemisphere. This implies that the compound eyes and CRY in the clock neurons have antagonistic effects on the timing of the E activity bout. CRY advances activity into the day, whereas the compound eyes delay it. Therefore, wild-type behavior combines both effects and the two light inputs might enable the fly to time its activity to the appropriate time of day. But CRY expression is not restricted to the clock neurons as a previous study showed a rather broad distribution within the compound eyes. In order to investigate its function in the eyes we collaborated with Prof. Rodolfo Costa (University of Padova). In our first study we were able to show that CRY interacts with the phototransduction cascade and thereby influences visual behavior like phototaxis and optomotor response. Our second study showed that CRY in the eyes affects locomotor activity rhythms. It appears to contribute to light sensation without being a photopigment per se. Our results rather indicate that CRY keeps the components of the phototransduction cascade close to the cytoskeleton, as we identified a CRY-Actin interaction in vitro. It might therefore facilitate the transformation of light energy into electric signals. In a further collaboration with Prof. Orie Shafer (University of Michigan) we were able to shed light on the significance of the extraretinal Hofbauer-Buchner eyelet for clock synchronization. Excitation of the eyelet leads to Ca2+ and cAMP increases in specific clock neurons, consequently resulting in a shift of the flies´ rhythmic activity. Taken together, the experiments conducted in this thesis revealed new functions of different eye structures and CRY for fly behavior. We were furthermore able to show that masking complements the rhythmic behavior of the fly, which might help to adapt to natural conditions.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Grebler2015,
  author    = {Grebler, Rudi},
  title     = {Untersuchung der Rolle von Rhodopsin 7 und Cryptochrom im Sehprozess von Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114466},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Ausgangspunkt f{\"u}r die Detektion von Licht ist im gesamten Tierreich die Absorption von Photonen durch photorezeptive Proteine, die sogenannten Opsine und in geringerem Ausmaß die Typ 1 Cryptochrome. Die Taufliege Drosophila melanogaster besitzt sechs eingehend charakterisierte, auch als Rhodopsine bezeichnete Opsine (Rh1-Rh6) und ein Cryptochrom (CRY). Neben den Ocellen und den Hofbauer-Buchner {\"A}uglein werden die Rhodopsine in erster Linie in den Photorezeptorzellen der Komplexaugen, den Hauptorganen der Lichtperzeption exprimiert, wo sie der Vermittlung der visuellen Wahrnehmung dienen. Basierend auf Sequenzvergleichen wurde im Jahr 2000 ein neues Protein namens Rh7 zur Gruppe der Drosophila Opsine hinzugef{\"u}gt. Bis heute fehlt allerdings jeglicher experimentelle Beleg f{\"u}r die photorezeptive Funktion dieses Proteins. Im Gegensatz dazu wird Cryptochrom in erster Linie in einigen Uhrneuronen des Drosophila Gehirns exprimiert, wo es diesen Neuronen die F{\"a}higkeit zur Lichtdetektion verleiht und das Photoentrainment der inneren Uhr lenkt. Neueren Untersuchungen zu folge spielt CRY allerdings auch bei der visuellen Wahrnehmung der Augen eine Rolle. Die vorliegende Arbeit zielte nun darauf ab die potentielle Funktion von Rh7 als neuen Photorezeptor in Drosophila sowie die Rolle von CRY bei der visuellen Lichtperzeption zu untersuchen. Die Aufnahmen der Elektroretinogramme (ERGs) von transgenen Fliegen, die Rh7 anstelle von oder zusammen mit dem dominanten Photorezeptor Rh1 in den Komplexaugen exprimieren, zeigen, dass Rh7 die Phototransduktionskaskade bei Belichtung mit Weißlicht nicht aktivieren kann. Die Abwesenheit von Rh7 sorgt allerdings trotzdem f{\"u}r eine Beeintr{\"a}chtigung der lichtinduzierten Antwort der Rezeptorzellen im Komplexauge. So zeigen die Intensit{\"a}ts-Response Kurven der ERG Rezeptorpotentialamplitude von rh7 Knockout-Fliegen unter Weißlicht niedriger und mittlerer Intensit{\"a}t nach einer anf{\"a}nglichen Dunkeladaptation von 15min eine insgesamt, im Vergleich zur Kontrolle erh{\"o}hte Rezeptorpotentialamplitude. Der Verlauf dieser Kurven deutet außerdem darauf hin, dass die Zunahme der Rezeptorpotentialamplitude mit steigender Lichtintensit{\"a}t gr{\"o}ßer wird. Zudem zeigt das Aktionsspektrum f{\"u}r die Rezeptorpotentialamplitude der rh7 Knockout-Fliegen, dass diese Empfindlichkeitszunahme im gesamten Bereich von 370-648nm auftritt. Diese Beeintr{\"a}chtigung scheint jedoch zu fehlen, wenn die Fliegen vor Experimentbeginn nur 1min dunkeladaptiert wurden, oder wenn intensives Blaulicht zur Belichtung verwendet wird. Des weiteren ist auch das 4s nach Ende des Lichtpulses im ERG gemessene Nachpotential bei fehlendem Rh7 reduziert. Zusammengenommen deuten diese Ergebnisse darauf hin, dass Rh7, wenn auch nicht als Photorezeptor, bei Belichtung mit Weißlicht niedriger und mittlerer Intensit{\"a}t die Lichtantwort in den Rezeptorzellen des Komplexauges in Abh{\"a}ngigkeit von Intensit{\"a}t und Adaptationszustand beeinflusst und dass dieser Einfluss scheinbar nicht durch Licht eines eng begrenzten Wellenl{\"a}ngenbereichs induziert wird. Des weiteren legt die Untersuchung des ERG Nachpotentials nahe, dass Rh7 m{\"o}glicherweise f{\"u}r eine normale Beendigung der Lichtantwort ben{\"o}tigt wird. Die allgemeine Funktion von Rh7 als Photorezeptor in Drosophila sowie die Eigenschaften der endogenen Funktion von Rh7 werden diskutiert. Unabh{\"a}ngig davon wird in der vorliegenden Arbeit auch gezeigt, dass Fliegen ohne CRY zwar nach 15-min{\"u}tiger, nicht jedoch nach 1-min{\"u}tiger Dunkeladaptation bei Belichtung mit Weißlicht niedriger Intensit{\"a}t eine insgesamt geringere ERG Rezeptorpotentialamplitude aufweisen. Dies k{\"o}nnte auf eine Beeintr{\"a}chtigung der Dunkeladaptationsprozesse bei Abwesenheit von CRY hindeuten.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Gmeiner2014,
  author    = {Gmeiner, Florian},
  title     = {Der Einfluss der Neurotransmitter Dopamin, Serotonin und GABA sowie ihrer Transporter auf das Schlafverhalten von Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-99152},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {In der vorliegenden Arbeit wurde der Einfluss von Dopamin, Serotonin und GABA auf das Schlafverhalten von Drosophila melanogaster genauer untersucht. Mit Hilfe von Mutanten in Wiederaufnahmetransportern f{\"u}r Dopamin und Serotonin konnte gezeigt werden, dass Dopamin und Serotonin entgegengesetzte Wirkungen auf die Schlafmenge der Fliegen haben. Dopamin hat eine schlafhemmende, Serotonin eine schlaff{\"o}rdernde Wirkung. Die Nutzung eines neuronal dopamindefizienten Fliegenstammes erweitert diese Erkenntnisse. Die Nutzung von RNAi zur Hinunterregulierung der Rezeptoren f{\"u}r Dopamin brachte keine weiteren Erkenntnisse, da sie zu keinem messbaren Effekt f{\"u}hren. Jedoch ergab eine parallel dazu durchgef{\"u}hrte Hinunterregulierung des GABABR2 Rezeptors, dass dieser maßgeblich f{\"u}r die Aufrechterhaltung des Schlafes in der zweiten H{\"a}lfte der Nacht verantwortlich ist. Es konnte gezeigt werden, dass f{\"u}r diese Aufgabe vor allem ihre Expression in den l-LNv Neuronen relevant ist. Dabei ist f{\"u}r die GABABR2 Rezeptoren kein Effekt, f{\"u}r Dopamin und Serotonin nur in geringen Ausmaß ein Effekt auf die Innere Uhr in Form von gering ver{\"a}nderter Periode zu beobachten. Durch eine Kombination der Transportermutanten f{\"u}r Dopamin und Serotonin mit dem intakten, als auch mutierten WHITE Transporter zeigte sich eine interessante Interaktion dieser drei Transporter bei der Regulation der Gesamtschlafmenge, wobei die white Mutation zu einer Reduzierung der Gesamtschlafmenge f{\"u}hrt. UPLC Messungen der St{\"a}mme ergaben, dass der Effekt von white vermutlich auf dessen Einfluss auf den beta-Alanyldopamingehalt der Fliegen basiert. beta-Alanyldopamin wird bei dem Transport von Dopamin {\"u}ber die Gliazellen durch das Enzym EBONY gebildet, dessen Mutation in der Kombination mit intaktem WHITE und mutiertem Dopamintransporter zu einer drastischen Reduktion des Schlafes w{\"a}hrend der Nacht f{\"u}hrt. Im Rahmen der Untersuchung konnte zudem gezeigt werden, dass entgegen des bisherigen Wissens aus Zellkulturstudien in Drosophila melanogaster kein beta-Alanylserotonin gebildet wird. M{\"o}glicherweise wird nur Dopamin, nicht jedoch Serotonin {\"u}ber die Gliazellen recycelt. Dies ist ein interessanter Unterschied, der sowohl eine zeitliche, als auch lokale Feinregulation der Gegenspieler Dopamin und Serotonin erm{\"o}glicht. Die Untersuchung der Dimerpartner BROWN und SCARLET zeigte, dass lediglich BROWN zu einer Reduktion des Schlafes f{\"u}hrt. Ein Effekt, der auch in einer Fliegenlinie mit spontaner white Mutation beobachtet werden konnte. Die genaue Funktion dieses Heterodimertransporters und seine neuronale Lokalisation wurden im Rahmen dieser Arbeit noch nicht gekl{\"a}rt. Dennoch liegt eine Funktion als Dopamin- oder beta-Alanyldopamintransporter in Gliazellen auf Grund der ermittelten Ergebnisse nahe. Zus{\"a}tzlich konnte zum ersten Mal in Drosophila melanogaster eine Funktion der Amintransporter bei der Anpassung der Inneren Uhr an extreme kurze bzw. lange Photoperioden gezeigt werden. Eine anatomische Lokalisierung des WHITE Transporters im Gehirn von Drosophila melanogaster, die weitere Charakterisierung der Rolle des WHITE/BROWN Dimers und die Zuordnung bestimmter dopaminerger und serotonerger Neurone bei der Modulation der Aktivit{\"a}tsmaxima stellen spannende Fragen f{\"u}r zuk{\"u}nftige Arbeiten dar.},
  subject      = {Taufliege},
  language  = {de}
}
@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}
}