@phdthesis{Schubert2010,
  author    = {Schubert, Alice},
  title     = {Immunhistochemische und funktionelle Charakterisierung der Serin/Arginin-Proteinkinase SRPK79D mit Identifizierung von Interaktionspartnern in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-53841},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Auf der Suche nach Mutanten mit einer vom Wildtyp abweichenden Verteilung des Aktive Zone-Proteins Bruchpilot wurde die Serin/Arginin-Proteinkinase SRPK79D identifiziert. Hier zeigte sich, dass die Mutation im Srpk79D-Gen zu einer Agglomeration von Bruchpilot in den larvalen segmentalen und intersegmentalen Nerven f{\"u}hrt. In der vorliegenden Arbeit sollte die SRPK79D genauer charakterisiert werden. Nach Pr{\"a}adsorptionen und Affinit{\"a}tsreinigungen von in einer fr{\"u}heren Arbeit erzeugten Antiseren, gelang es die Lokalisation der {\"u}berexprimierten SRPK79D-GFP-Isoformen zu bestimmen. Dabei zeigte sich, dass keines der Antiseren die endogene Kinase im Western Blot oder immunhistocheimisch detektieren konnte. Dies legt den Schluss nahe, dass die Expression der SRPK79D in einer geringen Konzentration erfolgt. Es war jedoch m{\"o}glich die endogene SRPK79D-PC-Isoform mittels einer Immunpr{\"a}zipitation soweit anzureichern, dass sie im Western Blot nachweisbar war. F{\"u}r die SRPK79D-PB-Isoform gelang dies allerdings nicht. Anhand von larvalen Nerv-Muskel-Pr{\"a}paraten konnte gezeigt werden, dass die panneural {\"u}berexprimierte SRPK79D-PC-GFP-Isoform an die Aktiven Zone transportiert wird und dort mit Bruchpilot, sowie den Interaktionspartnern von Bruchpilot Liprin-α und Rab3 kolokalisiert. Außerdem liegt sie diffus im Zytoplasma von neuronalen Zellk{\"o}rpern vor. In adulten Gehirnen lokalisiert die transgen {\"u}berexprimierte SRPK79D-PC-GFP im Fanshaped body, Ringkomplex und in neuronalen Zellk{\"o}rpern. Die panneural {\"u}berexprimierte SRPK79D-PB-GFP-Isoform liegt im larvalen und adulten Gehirn lokal im Zytoplasma der Perikaryen akkumuliert vor und wird nicht an die Aktive Zone transportiert. Das PB-Antiserum erkennt im adulten Gehirn neuronale Zellk{\"o}rper und das Neuropil in der Calyxregion der Pilzk{\"o}rper. Immunhistochemische F{\"a}rbungen von larvalen Nerv-Muskel-Pr{\"a}paraten mit verschiedenen Antik{\"o}rpern gegen neuronale Proteine belegen, dass die Agglomerate in der Srpk79D-Mutante f{\"u}r Bruchpilot spezifisch sind. Es konnten bisher keine weiteren Komponenten der Agglomerate detektiert werden. Auch ein genereller axonaler Defekt konnte durch F{\"a}rbungen gegen CSP, Synaptotagmin und Experimenten mit dem Mitochondrienfarbstoff MitoTracker® FM Green ausgeschlossen werden. Die quantitative Auswertung der Pr{\"a}parate zeigte, dass die Morphologie der synaptischen Boutons und die Zahl der Aktiven Zonen durch die Mutation im Srpk79D-Gen nicht beeinflusst werden. Um gesicherte Kenntnis dar{\"u}ber zu erlangen, ob die Mutation im Srpk79D-Gen die beobachteten Ph{\"a}notypen verursacht, wurden Rettungsexperimente durchgef{\"u}hrt. Es konnte sowohl f{\"u}r das hypomorphe Srpk79DP1-Allel, als auch f{\"u}r die Nullmutante Srpk79DVN eine nahezu vollst{\"a}ndige Rettung des Agglomerat-Ph{\"a}notyps mit der panneural exprimierten SRPK79D-PF- oder der SRPK79D-PB-Isoform erreicht werden. Aus diesen Ergebnissen folgt, dass beide Isoformen der SRPK79D in der Lage sind den Bruchpilot-Agglomerat-Ph{\"a}notyp zu retten, die Rettung der Verhaltensdefizite jedoch alle Isoformgruppen ben{\"o}tigen. Um zu untersuchen, ob der Agglomerations-Ph{\"a}notyp der Srpk79D-Mutanten auf einer {\"U}berexpression des Bruchpilotgens oder auf Fehlspleißen seiner pr{\"a}-mRNA beruht, wurden Immunpr{\"a}zipitationen, semiquantitative RT-PCRs und Real Time-PCRs durchgef{\"u}hrt. Ausgehend von den Ergebnissen kann eine m{\"o}gliche {\"U}berexpression bzw. Spleißdefekte von Bruchpilot weitgehend ausgeschlossen werden. Die simultane {\"U}berexpression von SRPK79D und Bruchpilot konnte den Ph{\"a}notyp der Bruchpilot-{\"U}berexpression nicht retten. Anhand der stimulated emission depletion-Mikroskopie konnte gezeigt werden, dass die gebildeten Agglomerate das charakteristische Donut-f{\"o}rmige Muster der T-bars zeigen und wahrscheinlich als fusionierte Ketten von T-bars in den larvalen Nerven vorliegen. Beim in vivo Imaging Versuch konnte demonstriert werden, dass das verk{\"u}rzte Bruchpilot-D3-Strawberry in die Bruchpilot-Agglomerate der Srpk79D-Nullmutante eingebaut wird und dass gr{\"o}ßere Agglomerate unbewegt im Nerv verharren. Der anterograde und retrograde Transport kleinerer Agglomerate konnte verzeichnet werden. Bei CytoTrap-Yeast-two-hybrid-Experimenten konnten f{\"u}r die SRPK79D-PB Isoform vier potentielle Interaktionspartner identifiziert werden: das Hitzeschockprotein Hsp70Bbb, die mitochondriale NADH-Dehydrogenase mt:ND5, das large ribosomal RNA Gen in Mitochondrien und das am Spleißen beteiligte Protein 1.3CC/Caper. Die Sequenzierung zeigte, dass nur das letzte Exon von Caper im pMyr-Vektor vorliegt. Der f{\"u}r die PC-Isoform durchgef{\"u}hrte CytoTrap-Versuch ergab nur Temperatur-Revertanten. SR-Proteinkinasen phosphorylieren die RS-Dom{\"a}ne von SR-Proteinen und sind dadurch an der Regulation des konstitutiven und alternativen Spleißens beteiligt. Somit stellen die acht identifizierten SR-Proteine in Drosophila potentielle Interaktionspartner der SRPK79D dar. Die durch RNAi-vermittelte Reduktion von sieben SR-Proteinen f{\"u}hrte zu keiner Agglomeration von Bruchpilot. Jedoch f{\"u}hrte die RNAi-vermittelte Reduktion des SR-Proteins Spleißfaktor 2 (SF2) zu kleineren Bruchpilot-Agglomeraten in den axonalen Nerven. SF2 ist selbst kein Bestandteil der Agglomerate der Srpk79D-Nullmutante. Die {\"U}berexpression von SF2 f{\"u}hrt wahrscheinlich zu einem axonalen Transportdefekt, wie die F{\"a}rbung gegen das Cysteine string protein zeigte. Weiterhin f{\"u}hrt die {\"U}berexpression zu einer Akkumulation von SF2 in larvalen Axonen und im adulten Gehirn der Fliegen. SF2 ist nicht nur in Zellkernen s{\"a}mtlicher Zellen nachweisbar, sondern es konnte auch ein spezifisches Signal im subsynaptischen Retikulum der Postsynapse detektiert werden, wie die F{\"a}rbungen gegen Disc large best{\"a}tigten.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Schmid2010,
  author    = {Schmid, Benjamin},
  title     = {Computational tools for the segmentation and registration of confocal brain images of Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51490},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Neuroanatomical data in fly brain research are mostly available as spatial gene expression patterns of genetically distinct fly strains. The Drosophila standard brain, which was developed in the past to provide a reference coordinate system, can be used to integrate these data. Working with the standard brain requires advanced image processing methods, including visualisation, segmentation and registration. The previously published VIB Protocol addressed the problem of image registration. Unfortunately, its usage was severely limited by the necessity of manually labelling a predefined set of neuropils in the brain images at hand. In this work I present novel tools to facilitate the work with the Drosophila standard brain. These tools are integrated in a well-known open-source image processing framework which can potentially serve as a common platform for image analysis in the neuroanatomical research community: ImageJ. In particular, a hardware-accelerated 3D visualisation framework was developed for ImageJ which extends its limited 3D visualisation capabilities. It is used for the development of a novel semi-automatic segmentation method, which implements automatic surface growing based on user-provided seed points. Template surfaces, incorporated with a modified variant of an active surface model, complement the segmentation. An automatic nonrigid warping algorithm is applied, based on point correspondences established through the extracted surfaces. Finally, I show how the individual steps can be fully automated, and demonstrate its application for the successful registration of fly brain images. The new tools are freely available as ImageJ plugins. I compare the results obtained by the introduced methods with the output of the VIB Protocol and conclude that our methods reduce the required effort five to ten fold. Furthermore, reproducibility and accuracy are enhanced using the proposed tools.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Nuwal2010,
  author    = {Nuwal, Tulip},
  title     = {Characterization of Synapsin, Tubulin-Binding Chaperone E-like, And Their Putative Interactions With Synapse Associated Protein Of 47 kDa In Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51683},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {In this thesis we have used Drosophila melanogaster as a model organism to investigate proteins and their putative interacting partners that are directly or indirectly involved in the release of neurotransmitters at the synapse. We have used molecular techniques to investigate conserved synaptic proteins, synapsin and synapse associated protein of 47 kD (SAP47), and a putative interaction partner of SAP47, tubulin binding chaperone E-like (TBCEL). SAP47 and synapsins are highly conserved synaptic vesicle associated proteins in Drosophila melanogaster. To further investigate the role and function of Sap47 and Syn genes, we had earlier generated the null mutants by P-element mutagenesis (Funk et al., 2004; Godenschwege et al., 2004). Western blots and ELISA of brain homogenates from Sap47156 null mutants showed the presence of up-regulated phospho-synapsin in comparison to wild-type (CS) and the presence of up-regulated phospho-synapsin was partially abolished when a pan-neuronal rescue of SAP47 was performed by the Gal4- UAS technique. Thus, the results suggest a qualitative and quantitative modulation of synapsin by SAP47. At the transcript level, we did not observe any difference in content of Syn transcript in Sap47156 and wild-type CS flies. The question of a direct molecular interaction between SAP47 and synapsin was investigated by co-immunoprecipitation (Co-IP) experiments and we did not find any stable interactions under the several IP conditions we tested. The possibility of Sap47 as a modifier of Syn at the genetic level was investigated by generating and testing homozygous double null mutants of Sap47 and Syn. The Syn97, Sap47156 double mutants are viable but have a reduced life span and decreased locomotion when compared to CS. In 2D-PAGE analysis of synapsins we identified trains of spots corresponding to synapsins, suggesting that synapsin has several isoforms and each one of them is posttranslationally modified. In an analysis by Blue native-SDS-PAGE (BN-SDS-2D- PAGE) and Western blot we observed synapsin and SAP47 signals to be present at 700-900 kDa and 200-250 kDa, respectively, suggesting that they are part of large but different complexes. We also report the possibility of Drosophila synapsin forming homo- and heteromultimers, which has also been reported for synapsins of vertebrates. In parallel to the above experiments, phosphorylation of synapsins in Drosophila was studied by IP techniques followed by 1D-SDS gel electrophoresis and mass spectrometry (in collaboration with S. Heo and G. Lubec). We identified and verified 5 unique phosphorylation sites in Drosophila synapsin from our MS analysis. Apart from phosphorylation modifications we identified several other PTMs which have not been verified. The significance of these phosphorylations and other identified PTMs needs to be investigated further and their implications for synapsin function and Drosophila behavior has to be elucidated by further experiments. In a collaborative project with S. Kneitz and N. Nuwal, we investigated the effects of Sap156 and Syn97 mutations by performing a whole Drosophila transcriptome microarray analysis of the individual null mutants and the double mutants (V2 and V3). We obtained several candidates which were significantly altered in the mutants. These genes need to be investigated further to elucidate their interactions with Sap47 and Syn. In another project, we investigated the role and function of Drosophila tubulin- binding chaperone E-like (Tbcel, CG12214). The TBCEL protein has high homology to vertebrate TBCE-like (or E-like) which has high sequence similarity to tubulin-binding chaperone E (TBCE) (hence the name TBCE-Like). We generated an anti-TBCEL polyclonal antiserum (in collaboration with G. Krohne). According to flybase, the Tbcel gene has only one exon and codes for two different transcripts by alternative transcription start sites. The longer transcript RB is present only in males whereas the shorter transcript RA is present only in females. In order to study the gene function we performed P- element jump-out mutagenesis to generate deletion mutants. We used the NP4786 (NP) stock which has a P(GawB) insertion in the 5' UTR of the Tbcel gene. NP4786 flies are homozygous lethal due to a second-site lethality as the flies are viable over a deficiency (Df) chromosome (a deletion of genomic region spanning the Tbcel gene and other upstream and downstream genes). We performed the P-element mutagenesis twice. In the first trial we obtained only revertants and the second experiment is still in progress. In the second attempt, jump-out was performed over the deficiency chromosome to prevent homologous chromosome mediated double stranded DNA repair. During the second mutagenesis an insertion stock G18151 became available. These flies had a P-element insertion in the open reading frame (ORF) of the Tbcel gene but was homozygous viable. Western blots of fresh tissue homogenates of NP/Df and G18151 flies probed with anti-TBCEL antiserum showed no TBCEL signal, suggesting that these flies are Tbcel null mutants. We used these flies for further immunohistochemical analyses and found that TBCEL is specifically expressed in the cytoplasm of cyst cells of the testes and is associated with the tubulin of spermatid tails in wild-type CS, whereas in NP/Df and G18151 flies the TBCEL staining in the cyst cells was absent and there was a disruption of actin investment cones. We also found enrichment of TBCEL staining around the actin investment cone. These results are also supported by the observation that the enhancer trap expression of the NP4786 line is localised to the cyst cells, similar to TBCEL expression. Also, male fertility of NP/Df and G18151 flies was tested and they were found to be sterile with few escapers. Thus, these results suggest that TBCEL is involved in Drosophila spermatogenesis with a possible role in the spermatid elongation and individualisation process.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Nuwal2010,
  author    = {Nuwal, Nidhi},
  title     = {Optogenetic investigation of nervous system functions using walking behavior and genome wide transcript analysis of Synapsin and Sap47 mutants of Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51694},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {PART I Animals need to constantly evaluate their external environment in order to survive. In some cases the internal state of the animal changes to cope with it's surrounding. In our study we wanted to investigate the role of amines in modulating internal states of Drosophila. We have designed a behavioral paradigm where the flies are fixed in space but can walk on a small styrofoam ball suspended by a gentle stream of air. The walking activity of flies was used as behavioral readout. PART I Animals need to constantly evaluate their external environment in order to survive. In some cases the internal state of the animal changes to cope with it's surrounding. In our study we wanted to investigate the role of amines in modulating internal states of Drosophila. We have designed a behavioral paradigm where the flies are fixed in space but can walk on a small styrofoam ball suspended by a gentle stream of air. The walking activity of flies was used as behavioral readout. An operant training paradigm was established by coupling one of the walking directions to incidence of heat punishment. We observed that animals quickly realized the contingency of punishment with walking direction and avoided walking in the punished direction in the presence of punishment, but did not continue walking in the unpunished direction in the absence of the punishment. This would indicate that the flies do not form a memory for the punished direction or rapidly erase it under new conditions. On having established the paradigm with heat punishment we have attempted to activate selected subsets of neuronal populations of Drosophila while they were walking on the ball. The selective activation of neurons was achieved by expressing the light-activated ion channel channelrhodopsin-2 (ChR2) using the Gal4-UAS system and coupling the unidirectional walking of the animals on the ball with the incidence of blue light required to activate the channels and depolarize the neurons. The feasibility of this approach was tested by light-activating sugar sensitive gustatory receptor neurons expressing ChR2, we found that when the light was actuated the flies preferred to turn in one direction the optically "rewarded" direction. Next we similarly activated different subsets of aminergic neurons. We observed that in our setup animals avoided to turn in the direction which was coupled to activation of dopaminergic neurons indicating that release of dopamine is disliked by the animals. This is in accordance with associative learning experiments where dopamine is believed to underlie the formation of an association between a neutral conditioned stimulus with the aversive unconditioned stimulus. However, when we activated tyraminergic/octopaminergic neurons we did not observe any directional preference. The activation of dopaminergic and tyraminergic/octopaminergic neurons led to arousal of the animals indicating that we were indeed successful in activating those neurons. Also, the activation of serotonergic neurons did not have any effect on directional preference of the animals. With this newly established paradigm it will be interesting to find out if in insects like in mammals a reward mediating system exists and to test subsets of aminergic or peptidergic neurons that could possibly be involved in a reward signaling system which has not been detected in our study. Also, it would be interesting to localize neuropile regions that would be involved in mediating choice behavior in our paradigm. PART II In collaboration with S. Kneitz (IZKF Wuerzburg) and T. Nuwal we performed genome-wide expression analysis of two pre-synaptic mutants - Synapsin (Syn97) and Synapse associated protein of 47 kDa (Sap47156). The rationale behind these experiments was to identify genes that were up- or down-regulated due to these mutations. The microarray experiments provided us with several candidate genes some of which we have verified by qPCR. From our qPCR analysis we can conclude that out of the verified genes only Cirl transcripts seem to be reproducibly down regulated in Synapsin mutants. The Cirl gene codes for a calcium independent receptor for latrotoxin. Further qPCR experiments need to be performed to verify other candidate genes. The molecular interactions between CIRL and SYN or their genes should now be investigated in detail.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Niewalda2010,
  author    = {Niewalda, Thomas},
  title     = {Neurogenetic analyses of pain-relief learning in the fruit fly},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65035},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {All animals learn in order to cope with challenges imposed on them by their environment. This is true also for both larval and adult fruit flies as exemplified in pavlovian conditioning. The focus of this Thesis is on various aspects of the fruit flies learning ability. My main project deals with two types of learning which we call punishment-learning and pain-relief learning. Punishment learning happens when fruit flies are exposed to an odour which is followed by electric shock. After such training, flies have learned that that odour signals pain and consequently will avoid it in the future. If the sequence of the two stimuli is reversed such that odour follows shock, flies learn the odour as a signal for relief and will later on approach it. I first report a series of experiments investigating qualitative and parametric features of relief-learning; I find that (i) relief learning does result from true associative conditioning, (ii) it requires a relatively high number of training trials, (iii) context-shock training is ineffective for subsequent shock-odour learning. A further question is whether punishment-learning and pain-relief learning share genetic determinants. In terms of genetics, I test a synapsin mutant strain, which lacks all Synapsin protein, in punishment and relief-learning. Punishment learning is significantly reduced, and relief-learning is abolished. Pan-neuronal RNAi-mediated knock-down of Synapsin results in mutant-like phenotypes, confirming the attribution of the phenotype to lack of Synapsin. Also, a rescue of Synapsin in the mushroom body of syn97 mutants restores both punishment- and relief-learning fully, suggesting the sufficiency of Synapsin in the mushroom body for both these kinds of learning. I also elucidate the relationship between perception and physiology in adult fruit flies. I use odour-shock conditioning experiments to identify degrees of similarity between odours; I find that those similarity measures are consistent across generalization and discrimination tasks of diverse difficulty. Then, as collaborator of T. V{\"o}ller and A. Fiala, I investigate how such behavioural similarity/dissimilarity is reflected at the physiological level. I combine the behaviour data with calcium imaging data obtained by measuring the activity patterns of those odours in either the sensory neurons or the projection neurons at the antennal lobe. Our interpretation of the results is that the odours perceptual similarity is organized by antennal lobe interneurons. In another project I investigate the effect of gustatory stimuli on reflexive behaviour as well as their role as reinforcer in larval learning. Drosophila larvae greatly alter their behaviour in presence of sodium chloride. Increasing salt concentration modulates choice behaviour from weakly appetitive to strongly aversive. A similar concentration-behaviour function is also found for feeding: larval feeding is slightly enhanced in presence of low salt concentrations, and strongly decreased in the presence of high salt concentrations. Regarding learning, relatively weak salt concentrations function as appetitive reinforcer, whereas high salt concentrations function as aversive reinforcer. Interestingly, the behaviour-concentration curves are shifted towards higher concentrations from reflexive behaviour (choice behaviour, feeding) as compared to associative learning. This dissociation may reflect a different sensitivity in the respective sensory-motor circuitry.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Knapek2010,
  author    = {Knapek, Stephan},
  title     = {Synapsin and Bruchpilot, two synaptic proteins underlying specific phases of olfactory aversive memory in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-49726},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Memory is dynamic: shortly after acquisition it is susceptible to amnesic treatments, gets gradually consolidated, and becomes resistant to retrograde amnesia (McGaugh, 2000). Associative olfactory memory of the fruit fly Drosophila melanogaster also shows these features. After a single associative training where an odor is paired with electric shock (Quinn et al., 1974; Tully and Quinn, 1985), flies form an aversive odor memory that lasts for several hours, consisting of qualitatively different components. These components can be dissociated by mutations, their underlying neuronal circuitry and susceptibility to amnesic treatments (Dubnau and Tully, 1998; Isabel et al., 2004; Keene and Waddell, 2007; Masek and Heisenberg, 2008; Xia and Tully, 2007). A component that is susceptible to an amnesic treatment, i.e. anesthesia-sensitive memory (ASM), dominates early memory, but decays rapidly (Margulies et al., 2005; Quinn and Dudai, 1976). A consolidated anesthesia-resistant memory component (ARM) is built gradually within the following hours and lasts significantly longer (Margulies et al., 2005; Quinn and Dudai, 1976). I showed here that the establishment of ARM requires less intensity of shock reinforcement than ASM. ARM and ASM rely on different molecular and/or neuronal processes: ARM is selectively impaired in the radish mutant, whereas for example the amnesiac and rutabaga genes are specifically required for ASM (Dudai et al., 1988; Folkers et al., 1993; Isabel et al., 2004; Quinn and Dudai, 1976; Schwaerzel et al., 2007; Tully et al., 1994). The latter comprise the cAMP signaling pathway in the fly, with the PKA being its supposed major target (Levin et al., 1992). Here I showed that a synapsin null-mutant encoding the evolutionary conserved phosphoprotein Synapsin is selectively impaired in the labile ASM. Further experiments suggested Synapsin as a potential downstream effector of the cAMP/PKA cascade. Similar to my results, Synapsin plays a role for different learning tasks in vertebrates (Gitler et al., 2004; Silva et al., 1996). Also in Aplysia, PKA-dependent phosphorylation of Synapsin has been proposed to be involved in regulation of neurotransmitter release and short-term plasticity (Angers et al., 2002; Fiumara et al., 2004). Synapsin is associated with a reserve pool of vesicles at the presynapse and is required to maintain vesicle release specifically under sustained high frequency nerve stimulation (Akbergenova and Bykhovskaia, 2007; Li et al., 1995; Pieribone et al., 1995; Sun et al., 2006). In contrast, the requirement of Bruchpilot, which is homologous to the mammalian active zone proteins ELKS/CAST (Wagh et al., 2006), is most pronounced in immediate vesicle release (Kittel et al., 2006). Under repeated stimulation of a bruchpilot mutant motor neuron, immediate vesicle release is severely impaired whereas the following steady-state release is still possible (Kittel et al., 2006). In line with that, knockdown of the Bruchpilot protein causes impairment in clustering of Ca2+ channels to the active zones and a lack of electron-dense projections at presynaptic terminals (T-bars). Thus, less synaptic vesicles of the readily-releasable pool are accumulated to the release sites and their release probability is severely impaired (Kittel et al., 2006; Wagh et al., 2006). First, I showed that Bruchpilot is required for aversive olfactory memory and localized the requirement of Bruchpilot to the Kenyon cells of the mushroom body, the second-order olfactory interneurons in Drosophila. Furthermore, I demonstrated that Bruchpilot selectively functions for the consolidated anesthesia-resistant memory. Since Synapsin is specifically required for the labile anesthesia sensitive memory, different synaptic proteins can dissociate consolidated and labile components of olfactory memory and two different modes of neurotransmission (high- vs. low frequency dependent) might differentiate ASM and ARM.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Jauch2010,
  author    = {Jauch, Mandy},
  title     = {Die Serin/Arginin Proteinkinase 79D (SRPK79D) von Drosophila melanogaster und ihre Rolle bei der Bildung Aktiver Zonen von Synapsen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-53974},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Synapsen als Stellen der Kommunikation zwischen Neuronen besitzen spezialisierte Bereiche - Aktive Zonen (AZs) genannt -, die aus einem hoch komplexen Netzwerk von Proteinen aufgebaut sind und die Maschinerie f{\"u}r den Prozess der Neurotransmitter-Aussch{\"u}ttung und das Vesikel-Recycling beinhalten. In Drosophila ist das Protein Bruchpilot (BRP) ein wichtiger Baustein f{\"u}r die T-f{\"o}rmigen B{\"a}nder („T-Bars") der pr{\"a}synaptischen Aktiven Zonen. BRP ist notwendig f{\"u}r eine intakte Struktur der Aktiven Zone und eine normale Exocytose von Neurotransmitter-Vesikeln. Auf der Suche nach Mutationen, welche die Verteilung von Bruchpilot im Gewebe beeintr{\"a}chtigen, wurde eine P-Element-Insertion im Gen CG11489 an der Position 79D identifiziert, welches eine Kinase kodiert, die einen hohen Grad an Homologie zur Familie der SR Proteinkinasen (SRPKs) von S{\"a}ugern aufweist. Die Mitglieder dieser Familie zeichnen sich durch eine evolution{\"a}r hoch konservierte zweigeteilte Kinasedom{\"a}ne aus, die durch eine nicht konservierte Spacer-Sequenz unterbrochen ist. SRPKs phosphorylieren SR-Proteine, die zu einer evolution{\"a}r hoch konservierten Familie Serin/Arginin-reicher Spleißfaktoren geh{\"o}ren und konstitutive sowie alternative Spleißprozesse steuern und damit auf post-transkriptioneller Ebene die Genexpression regulieren. Mutation des Srpk79D-Gens durch die P-Element-Insertion (Srpk79DP1) oder eine Deletion im Gen (Srpk79DVN Nullmutante) f{\"u}hrt zu auff{\"a}lligen BRP-Akkumulationen in larvalen und adulten Nerven. In der vorliegenden Arbeit wird gezeigt, dass diese BRP-Akkumulationen auf Ultrastruktur-Ebene ausgedehnten axonalen Agglomeraten elektronendichter B{\"a}nder entsprechen und von klaren Vesikeln umgeben sind. Charakterisierung durch Immuno-Elektronenmikroskopie ergab, dass diese Strukturen BRP-immunoreaktiv sind. Um die Bildung BRP-enthaltender Agglomerate in Axonen zu verhindern und damit eine intakte Gehirnfunktion zu gew{\"a}hrleisten, scheint die SRPK79D nur auf niedrigem Niveau exprimiert zu werden, da die endogene Kinase mit verschiedenen Antik{\"o}rpern nicht nachweisbar war. Wie in anderen Arbeiten gezeigt werden konnte, ist die Expression der PB-, PC- oder PF-Isoform der vier m{\"o}glichen SRPK79D-Varianten, die durch alternativen Transkriptionsstart in Exon eins beziehungsweise drei und alternatives Spleißen von Exon sieben zustande kommen, zur Rettung des Ph{\"a}notyps der BRP-Akkumulation im Srpk79DVN Nullmutanten-Hintergrund ausreichend. Zur Charakterisierung der Rescue-Eigenschaften der SRPK79D-PE-Isoform wurde mit der Klonierung der cDNA in einen UAS-Vektor begonnen. Offenbar beruht die Bildung der axonalen BRP-Agglomerate nicht auf einer {\"U}berexpression von BRP in den betroffenen Neuronen, denn auch bei reduzierter Expression des BRP-Proteins im Srpk79DVN Nullmutanten-Hintergrund entstehen die BRP-Agglomerate. In K{\"o}pfen der Srpk79DVN Nullmutante ist die Gesamtmenge an Bruchpilot-Protein im Vergleich zum Wildtyp nicht deutlich ver{\"a}ndert. Auch die auf Protein-Ebene untersuchte Expression der verschiedenen Isoformen der pr{\"a}synaptischen Proteine Synapsin, Sap47 und CSP weicht in der Srpk79DVN Nullmutante nicht wesentlich von der Wildtyp-Situation ab, sodass sich keine Hinweise auf ver{\"a}ndertes Spleißen der entsprechenden pr{\"a}-mRNAs ergeben. Jedes der sieben bekannten SR-Proteine von Drosophila ist ein potentielles Zielprotein der SRPK79D. Knock-down-Experimente f{\"u}r die drei hier untersuchten SR-Proteine SC35, X16/9G8 und B52/SRp55 im gesamten Nervensystem durch RNA-Interferenz zeigten allerdings keinen Effekt auf die Verteilung von BRP im Gewebe. Hinsichtlich der Flugf{\"a}higkeit der Tiere hat die Srpk79DVN Nullmutation keinen additiven Effekt zum Knock-down des BRP-Proteins, denn die Doppelmutanten zeigten bei der Bestimmung des Anteils an flugunf{\"a}higen Tieren vergleichbare Werte wie die Einzelmutanten, die entweder die Nullmutation im Srpk79D-Gen trugen, oder BRP reduziert exprimierten. Vermutlich sind Bruchpilot und die SR Proteinkinase 79D somit Teil desselben Signalwegs. Durch Doppelf{\"a}rbungen mit Antik{\"o}rpern gegen BRP und CAPA-Peptide wurde abschließend entdeckt, dass Bruchpilot auch im Median- und Transvers-Nervensystem (MeN/TVN) von Drosophila zu finden ist, welche die Neuroh{\"a}mal-Organe beherbergen. Aufgabe dieser Organe ist die Speicherung und Aussch{\"u}ttung von Neuropeptid-Hormonen. Daher ist zu vermuten, dass das BRP-Protein neben Funktionen bei der Neurotransmitter-Exocytose m{\"o}glicherweise eine Rolle bei der Aussch{\"u}ttung von Neuropeptiden spielt. Anders als in den Axonen der larvalen Segmental- und Intersegmentalnerven der Srpk79DVN Nullmutante, die charakteristische BRP-Agglomerate aufweisen, hat die Mutation des Srpk79D-Gens in den Axonen der Va-Neurone, die das MeN/TVN-System bilden, keinen sichtbaren Effekt auf die Verteilung von Brp, denn das Muster bei F{\"a}rbung gegen BRP weist keine deutlichen Ver{\"a}nderungen zum Wildtyp auf.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Gruber2010,
  author    = {Gruber, Franz Andreas},
  title     = {Untersuchung zur Regulation der Expression des zuckerkonditionierten Verhaltens bei Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48802},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {In dieser Doktorarbeit habe ich die Regulation der Expression des zuckerbelohnten Verhaltens durch den F{\"u}tterungszustand bei Drosophila melanogaster untersucht. Die Fliegen k{\"o}nnen w{\"a}hrend einer Trainingsphase mit Hilfe einer Zuckerbelohnung auf einen bestimmten Duft konditioniert werden. Nach dem Training k{\"o}nnen die Fliegen dann auf das olfaktorische Ged{\"a}chtnis getestet werden. Die Bereitschaft das zuckerkonditionierte Ged{\"a}chtnis im Test zu zeigen wird vom F{\"u}tterungszustand kontrolliert, wie ich in {\"U}bereinstimmung mit den Ergebnissen fr{\"u}herer Arbeiten demonstrierte (Tempel et al. 1983; Gruber 2006; Krashes et al. 2008). Nur nicht gef{\"u}tterte Fliegen exprimieren das Ged{\"a}chtnis, w{\"a}hrend F{\"u}tterungen bis kurz vor dem Test eine reversibel supprimierende Wirkung haben. Einen {\"a}hnlichen regulatorischen Einfluss {\"u}bt der Futterentzug auch auf die Expression anderer futterbezogener Verhaltensweisen, wie z.B. die naive Zuckerpr{\"a}ferenz, aus. Nachdem ich den drastischen Einfluss des F{\"u}tterungszustands auf die Auspr{\"a}gung des zuckerkonditionierten Verhaltens gezeigt bzw. best{\"a}tigt hatte, habe ich nach verhaltensregulierenden Faktoren gesucht, die bei einer F{\"u}tterung die Ged{\"a}chtnisexpression unterdr{\"u}cken. Als m{\"o}gliche Kandidaten untersuchte ich Parameter, die zum Teil bereits bei verschiedenen futterbezogenen Verhaltensweisen unterschiedlicher Tierarten als „S{\"a}ttigungssignale" identifiziert worden waren (Marty et al. 2007; Powley and Phillips 2004; Havel 2001; Bernays and Chapman 1974; Simpson and Bernays 1983; Gelperin 1971a). Dabei stellte sich heraus, dass weder die „ern{\"a}hrende" Eigenschaft des Futters, noch ein durch Futteraufnahme bedingter Anstieg der internen Glukosekonzentration f{\"u}r die Suppression des zuckerkonditionierten Ged{\"a}chtnisses notwendig sind. Die Unterdr{\"u}ckung der Ged{\"a}chtnisexpression kann auch nicht durch Unterschiede in den aufgenommenen Futtermengen, die als verhaltensinhibitorische Dehnungssignale des Verdauungstrakts wirken k{\"o}nnten, oder mit der St{\"a}rke des s{\"u}ßen Geschmacks erkl{\"a}rt werden. Die Suppression des zuckerbelohnten Verhaltens folgte den Konzentrationen der gef{\"u}tterten Substanzen und war unabh{\"a}ngig von deren chemischen Spezifit{\"a}t. Deshalb wird die Osmolarit{\"a}t des aufgenommenen Futters als ein entscheidender Faktor f{\"u}r die Unterdr{\"u}ckung der zuckerkonditionierten Ged{\"a}chtnisexpression angenommen. Weil nur inkorporierte Substanzen einen Unterdr{\"u}ckungseffekt hatten, wird ein osmolarit{\"a}tsdetektierender Mechanismus im K{\"o}rper 67 postuliert, wahrscheinlich im Verdauungstrakt und/oder der H{\"a}molymphe. Die H{\"a}molymphosmolarit{\"a}t ist als „S{\"a}ttigungssignal" bei einigen wirbellosen Tieren bereits nachgewiesen worden (Bernays and Chapman 1974; Simpson and Raubenheimer 1993; Gelperin 1971a; Phifer and Prior 1985). Deshalb habe ich mit Hilfe genetischer Methoden und ohne die Fliegen zu f{\"u}ttern, versucht {\"u}ber einen k{\"u}nstlich induzierten Anstieg der Trehaloseund Lipidkonzentrationen die Osmolarit{\"a}t der H{\"a}molymphe in Drosophila zu erh{\"o}hen. Eine solche konzentrationserh{\"o}hende Wirkung f{\"u}r Lipide und die Trehalose, dem Hauptblutzucker der Insekten, ist bereits f{\"u}r das adipokinetische Hormon (AKH), das von Zellen der Corpora cardiaca exprimiert wird, nachgewiesen worden (Kim and Rulifson 2004; Lee and Park 2004; Isabel et al. 2005). Es stellte sich heraus, dass die k{\"u}nstliche Stimulierung AKH-produzierender Neurone das zuckerkonditionierten Verhalten tempor{\"a}r, reversible und selektiv unterdr{\"u}ckt. Gleiche Behandlungen hatten keinen Effekt auf ein aversiv konditioniertes olfaktorisches Ged{\"a}chtnis oder ein naives Zuckerpr{\"a}ferenzverhalten. Wie aus dieser Arbeit hervorgeht, stellt wahrscheinlich die Osmolarit{\"a}t des Verdauungstrakts und der H{\"a}molymphe oder nur der H{\"a}molymphe ein physiologisches Korrelat zum F{\"u}tterungszustand dar und wirkt als unterdr{\"u}ckendes Signal. Dass F{\"u}tterungen das zuckerkonditionierte Verhalten und die Zuckerpr{\"a}ferenz supprimieren, die k{\"u}nstliche Stimulation AKH-produzierender Zellen aber selektiv nur die zuckerbelohnte Ged{\"a}chtnisexpression unterdr{\"u}ckt, deutet auf mindestens zwei unterschiedliche „S{\"a}ttigungssignalwege" hin. Außerdem macht es deutlich wie uneinheitlich futterbezogene Verhaltensweisen, wie das zuckerbelohnte Verhalten und die naive Zuckerpr{\"a}ferenz, reguliert werden.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Aso2010,
  author    = {Aso, Yoshinori},
  title     = {Dissecting the neuronal circuit for olfactory learning in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-55483},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, "General introduction and discussion" section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila.},
  subject      = {Taufliege},
  language  = {en}
}