Refine
Has Fulltext
- yes (363) (remove)
Year of publication
- 2014 (363) (remove)
Document Type
- Doctoral Thesis (363) (remove)
Keywords
- Maus (11)
- T-Lymphozyt (10)
- Genexpression (6)
- Thrombozyt (6)
- Transkriptionsfaktor (6)
- Apoptosis (5)
- Ackerschmalwand (4)
- Angiogenese (4)
- Dendritische Zelle (4)
- Epigenetik (4)
Institute
- Graduate School of Life Sciences (62)
- Theodor-Boveri-Institut für Biowissenschaften (48)
- Physikalisches Institut (20)
- Medizinische Klinik und Poliklinik I (15)
- Medizinische Fakultät (14)
- Institut für Informatik (12)
- Fakultät für Chemie und Pharmazie (10)
- Institut für Pharmazie und Lebensmittelchemie (10)
- Institut für Anorganische Chemie (9)
- Julius-von-Sachs-Institut für Biowissenschaften (9)
Schriftenreihe
Sonstige beteiligte Institutionen
- Universitätsklinikum Münster (2)
- CBIO, University of Cape Town, South Africa (1)
- DLR (1)
- Department of Biochemistry (1)
- Fachhochschule Kaiserslautern, Campus Zweibrücken (1)
- Fraunhofer-Institut für Silicatforschung ISC, Würzburg (1)
- IBMP - Institut für Biomedizinische und Pharmazeutische Forschung in Nürnberg-Heroldsberg (1)
- Institut für Tierökologie und Tropenbiologie (1)
- Institut für klinische Neurobiologie (1)
- Instituto de Hygiene Montevideo, Uruguay (1)
ResearcherID
- I-5818-2014 (1)
In der vorliegenden Studie wurden 220 Patientinnen und Patienten prospektiv 1 Jahr lang nach der Anlage einer PEG-Sonde verfolgt. Die Studie sollte der Frage nachgehen, welche Patienten bzw. welche Indikationsgruppen in welchem Umfang von einer PEG-Anlage kurz- oder langfristig profitieren.
Außerdem wurde die Zufriedenheit der Patienten, ihrer Angehörigen, Betreuer und Hausärzte sowie auf den Einfluss einer PEG-Anlage und der Sondenernährung auf die Lebensqualität der Betroffenen untersucht. Die Befragung wurde über strukturierte Telefoninterviews nach 7 Tagen, 4 Wochen und dann im Abstand von jeweils 3 Monaten bis abschließend ein Jahr nach Sondenanlage durchgeführt.
Die Gesamtheit der Patienten setzte sich zu 41% aus Frauen und 59% aus Männern zusammen. Das Durchschnittsalter lag bei 72 Jahren, die Altersspanne bewegte sich zwischen 39 und 97 Jahren.
Es zeigte sich eine außergewöhnlich hohe Zufriedenheit aller Befragten mit der PEG-Sonde und deren Auswirkung auf die Lebensqualität. 95,7% der befragten Patienten beurteilten ihre Zufriedenheit mit der PEG als sehr gut oder gut. Der Grad der Zufriedenheit war auch bei den Angehörigen, Pflegekräften und Ärzten sehr hoch: 94%, 95,2% und 92% bewerteten die PEG mit sehr gut/ gut. Die wenigen negativen Beurteilungen basierten im Wesentlichen auf Komplikationen, die in Verbindung mit der Sondenernährung auftraten, und auf dem Hinterfragen der Notwendigkeit der PEG-Anlagen. Unterschiede zwischen den einzelnen Indikationsgruppen gaben die Befragten mit einer Ausnahme nicht an. Während Angehörige und Pflegekräfte ihre hohe Zustimmung zur PEG auch bei den Demenzkranken zum Ausdruck brachten (100%, bzw. 95% sehr gute/gute Bewertung), lassen die Hausärzte in dieser Indikation gewisse Vorbehalte erkennen. 9,1% vergaben die Noten mangelhaft/ungenügend und weitere 13,6% die Bewertung befriedigend/ausreichend. Sie begründen ihre Kritik mit ethischen und medizinischen Überlegungen.
Zusammenfassend bestätigt sich die PEG in dieser prospektiven Studie als sichere und effektive Maßnahme zur enteralen Ernährung. Die Indikationsstellung darf nicht nur den Erhalt oder die Steigerung des Körpergewichtes berücksichtigen, sondern sollte auch die Aspekte wie Komorbiditäten, Prognose und nicht zuletzt Allgemeinzustand und Lebensqualität der Patienten beachten. Nach einer PEG-Anlage ist die allgemeine Zufriedenheit bei Patienten Angehörigen, Pflegekräften und Ärzten fast ausnahmslos sehr hoch.
In einer ständig älter werdenden Bevölkerung, in der auch die Senioren einen unverändert hohen Anspruch an ihre Lebensqualität aufrechterhalten, kommt dem Ersatz verschlissener Gelenke und hier besonders der Hüftendoprothetik eine ständig wachsende Bedeutung zu. Ausgehend von den Erfolgen der minimal-invasiven Chirurgie in anderen chirurgischen Disziplinen entwickelte sich dieser Zweig seit Beginn des Jahrtausendes auch in der Hüftchirurgie: 2005 stellte Rachbauer eine minimal-invasive Variante des seit 1887 bekannten anterioren Zugangs zur Hüfttotalendoprothesenimplantation vor. Zum kurzfristigen postoperativen Intervall existieren bereits zahlreiche Studien. Hier zeigt der anteriore Zugang entscheidende Vorteile im Vergleich zu anderen Zugängen. Hervorzuheben sind ein geringeres postoperatives Schmerzausmaß, eine kürzere stationäre Liegedauer und eine schnellere Rehabiliation. Ein gravierender Nachteil des anterioren Zugangs besteht darin, dass er aufgrund des schmalen Operationskanals eine hohe Qualifikation des Operateurs erfordert und mit einer anfänglich erhöhten Komplikationsrate vergesellschaftet sein kann.
Ziel der vorliegenden Arbeit war es, die Resultate des minimal-invasiven anterioren Zugangs in einem mittelfristigen postoperativen Intervall mit den Resultaten des lateralen Zugangs, den man als eine weithin etablierte konventionelle Methode betrachten kann, zu vergleichen. Untersucht wurden 85 über einen minimal-invasiven anterioren Zugang implantierte primäre Hüfttotalendoprothesen nach durchschnittlich 3,7 Jahren post operationem, denen 86 Fälle mit einem lateralen Zugang nach durchschnittlich 5,5 Jahren entgegengestellt wurden. Beide Gruppen unterschieden sich nicht signifikant in Alter und Geschlechterverteilung. Der signifikante Unterschied der Nachuntersuchungsintervalle ist ohne Relevanz, da sich sich beide Gruppen in einer komplikationsarmen postoperativen Phase befinden, die in etwa vom 2. bis zum 10. Postoperativen Jahr geht.
Die Gruppen wurden in Funktion (HHS), Aktivität (UCLA activity score), Schmerzen (abgeleitet aus dem HHS), Komplikationsraten und Gesundheitsempfinden (SF-36) miteinander verglichen.
Die vorliegende Arbeit kam zu den folgenden Ergebnissen über den mittelfristigen Zeitraum: Die Unterschiede in der Funktion waren marginal, die HHS-Durchschnittswerte in beiden Gruppen waren exzellent und nicht signifikant unterschiedlich. Ebenso zeigten beide Gruppen gleichwertige Ergebnisse im subjektiven Gesundheitsempfinden (SF-36). Ein weiteres wichtiges Erfolgskriterium war die Frage nach der durch die Operation erreichten Aktivität. Hier fand sich eine Diskrepanz zwischen UCLA und TWB. Der UCLA ist zur Aktivitätsmessung weit verbreitet. Im UCLA zeigten sich die Patienten der lateralen Gruppe aktiver. Der TWB ergab gleiche Aktivität in beiden Gruppen. Nach dem eingehenden Vergleich beider Fragebögen, kamen wir in dieser Arbeit jedoch zu der Auffassung, dass der UCLA zwar als ein schnell erhebbarer Test für den klinischen Alltag praktisch ist, der wesentlich aufwendigere TWB aber zuverlässigere Ergebnisse liefert.
Die Komplikationsraten waren in beiden Gruppen gering. Die Wahrscheinlichkeit, eine der erfassten Komplikationen zu erleiden, betrug 10,7% für den minimal-invasiven und 10,5% für den klassischen Zugang. Um aber nicht nur die Quantität, sondern auch die Qualität der aufgetretenen Komplikationen erfassen zu können, wurde in dieser Arbeit der Komplikationsindex eingeführt, der die registrierten Komplikationen in Gruppen verschiedener Schweregrade unterteilt. Eine schwere Komplikation floss mit 10 Punkten, eine mittlere mit 5 und eine leichte Komplikation mit einem Punkt in den Komplikationsindex ein. Der anteriore Zugang zeigte so mit insgesamt 26 Punkten einen deutlich geringeren Komplikationsindex als der laterale mit insgesamt 60 Punkten. Dem entsprach eine Reoperationsrate von 1,2% unter den anterioren Patienten im Vergleich zu 3,3% bei den lateralen. Die Schaft- und die Pfannenüberlebensrate lag in beiden Gruppen bei 100%.
Zusammenfassend zeigt die vorliegende Arbeit, dass der anteriore Zugang viele Vorteile gegenüber dem lateralen Zugang aufweist. Die entscheidenden Unterschiede finden sich im kurzfristigen postoperativen Zeitraum. Zu einem mittelfristigen Zeitpunkt erzielen beide Zugänge gleichwertige Ergebnisse.
Welche langfristigen Resultate das noch relativ junge Verfahren des minimal-invasiven anterioren Zugangs hervorbringen wird, muss durch künftige prospektiv randomisierte Studien großer Fallzahlen belegt werden.
SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype.
The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1.
Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage.
Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis.
To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.
Galectin-1 (hGal-1) is overexpressed by numerous cancer types and previously conducted studies confirmed that the β-galactoside-binding protein mediates various molecular interactions associated with tumor growth, spread and survival. Upon interaction with carbohydrate-based binding epitopes of glycan structures on human cell surfaces galectin-1 induces proliferative, angiogenetic and migratory signals and modulates negative T cell regulation which essentially helps the tumor to evade the immune response. These findings attributed galectin-1 a pivotal role in tumor physiology and strongly suggest the protein as target for diagnostic and therapeutic applications.
Within the scope of this work a strategy was elaborated for designing tailor-made galectin-1 ligands by functionalizing selected hydroxyl groups of the natural binding partner N-acetyllactosamine (LacNAc) that are not involved in the sophisticated interplay between the disaccharide and the protein. Synthetic modifications intended to introduce chemical groups i) to address a potential binding site adjacent to the carbohydrate recognition domain (CRD) with extended hGal-1-ligand interactions, ii) to implement a tracer isotope for diagnostic detection and iii) to install a linker unit for immobilization on microarrays.
Resulting structures were investigated regarding their targeting ability towards galectin-1 by cocrystallization experiments, SPR and ITC studies. Potent binders were further probed for their diagnostic potential to trace elevated galectin-1 levels in microarray experiments and for an application in positron emission tomography (PET).
Abstract
Tobacco addiction is considered as a chronic relapsing disorder, characterized by compul-sive drug seeking and intake. Learning processes are stressed to account for the situational-specific expression of core features of the disorder, e.g., craving for drug, tolerance and ex-cessive consumption. According to incentive theories, smoke conditioned stimuli are hy-pothesized to be appetitive in nature, promoting craving, approach and consummatory be-havior. Commonly, smoking cues are treated as simple excitatory conditioned stimuli formed by a close and reliable overlap with the drug effect. However, the smoking ritual comprises a multitude of stimuli which may give rise to different forms of learning and con-ditioned responses partially opposing each other. Previous research suggests the predictive content and the temporal proximity of smoking stimuli to the drug effect as important de-terminants of cue reactivity. In contrast to stimuli related to the preparatory stage of smok-ing and the start of consumption (BEGIN stimuli), stimuli from the terminal stage of smok-ing (END stimuli) apparently lack high cue reactivity. Several lines of evidence suggest the poor cue properties of terminal stimuli to be related to their signaling of poor smoke availa-bility. Indeed, cue reactivity is commonly decreased when smoking appears to be unavaila-ble. Moreover, the learning literature suggests that stimuli predictive for the non-availability of reward may acquire the capacity to modulate or oppose the responses of ex-citatory conditioned stimuli. Therefore, the aim of the present thesis was to enhance our knowledge of stimulus control in human drug addiction and incentive motivation by running a series of conditioning studies with smoke intake and monetary reward as reinforcer. Sub-jective report and physiological measures of motivational valence and consummatory re-sponse tendencies were used as dependent variables.
The first experiment of this thesis used a differential conditioning paradigm to reveal evi-dence for the conditioning of preparatory and consummatory responses to a CS+ for smok-ing. Neutral pictograms served as CSs and single puffs on a cigarette as US. In line with the predictions of incentive theories, the excitatory CS+ for smoking acquired the ability to evoke an appetitive conditioned response, as indicated by enhanced activity of the M. zy-gomaticus major. Moreover, anticipation of puffing on the cigarette increased the activity of the M. orbicularis oris (lip muscle), indicating the activation of consummatory response tendencies. Finally, the CS+ evoked stronger skin conductance responses, indicative of in-creased autonomic arousal and orienting in preparation for action. In contrast, the rating data were apparently unaffected by the experimental contingency. In sum, the physiological data provide support for the notion that excitatory smoke conditioning gives rise to appeti-tive and consummatory conditioned responses, which may at least partially contribute to the maintenance of tobacco addiction.
The second experiment of this thesis adapted the conditioning protocol of the first study to probe the functional significance of terminal stimuli in the control of addictive behavior. This study manipulated the predictive relationship of BEGIN and END stimuli to smoke rein-forcement to provide further support for the differential reactivity to both stimuli and the retarded (i.e., delayed) conditioning of END stimuli. Overall, the results of the first study of this thesis were conceptually replicated as the association of a BEGIN stimulus with smoke intake resulted in the acquisition of appetitive and consummatory physiological responses. Importantly, the results revealed evidence for a retarded excitatory conditioning of END stimuli. Thus, pairing of an END stimulus with smoke intake failed to produce a conditioned discrimination in terms of motivational valence and autonomic arousal, as indicated by the activity of the M. corrugator supercilii and the skin conductance data. These results provide further support for the notion that END stimuli may be weak cues for smoking. Moreover, in light of the results of the first study of this thesis, the retarded excitatory conditioning of terminal stimuli may be suggestive of an inhibitory response component, which may be re-lated to their signaling of poor smoke availability. In sum, these results add to a growing body of data, which suggest that the expression of cue reactivity may be modulated by the temporal proximity and the availability of the drug effect.
The aim of the third study of this thesis was to provide “proof of concept” for an inhibi-tory conditioning notion of terminal stimuli. In this analog study BEGIN and END stimuli were emulated as discriminative SD and S for monetary reward. During an acquisition phase conditioned inhibition was established to the S predictive of the non-availability of re-ward. Subsequently a retardation test was used to substantiate conditioned inhibition. In this test, excitatory conditioning of the previous S was compared to the excitatory condi-tioning of a novel control stimulus. Importantly, the results revealed evidence for reward conditioned inhibition as indicated by the retarded acquisition of subjective (pleasure and reward expectancy) and physiological (skin conductance and activity of the M. orbicularis oculi) responses. In sum, these results provide support for the notion that stimuli predictive for the non-availability of reward may acquire the capacity to oppose the responses of ex-citatory conditioned stimuli. Thus, future research may benefit from the consideration of inhibitory conditioning processes in drug addiction, which may be of theoretical, methodo-logical and clinical importance.
In sum, the present thesis revealed evidence for 1) an appetitive nature of excitatory condi-tioned smoking cues, 2) the dependency of this learning process on the temporal position of the conditioned stimuli in the intake ritual and 3) the acquisition of conditioned inhibition by a stimulus predictive for the non-availability of reward, as evident in retarded excitatory conditioning. Overall, these studies made a novel contribution to the field of human drug addiction and incentive motivation and provided valuable suggestions for further research.
Structural and biochemical characterization of gephyrin and various gephyrin-ligand complexes
(2014)
Efficient synaptic neurotransmission requires the exact apposition of presynaptic terminals and matching neurotransmitter receptor clusters on the postsynaptic side. The receptors are embedded in the postsynaptic density, which also contains scaffolding and regulatory proteins that ensure high local receptor concentrations. At inhibitory synapses the cytosolic scaffolding protein gephyrin assumes an essential organizing role within the postsynaptic density by the formation of self-oligomers which provide a high density of binding sites for certain -amino butyric acid type A (GABAA) and the large majority of glycine receptors (GlyR). Gephyrin contains two oligomerization domains: In isolation, the 20 kDa N-terminal G domain (GephG) and the 46 kDa E domain (GephE) trimerize and dimerize, respectively. In the full-length protein the domains are interconnected by a central ~150 amino acid linker, and only GephG trimerization is utilized, whereas GephE dimerization is prevented, thus suggesting the need for a trigger to release GephE autoinhibition, which would pave the way for the formation of higher oligomers and for efficient receptor clustering. The structural basis for this GephE autoinhibition has remained elusive so far, but the linker was reported to be sufficient for autoinhibition. This work dealt with the biochemical and structural characterization of apo-gephyrin and gephyrin in complexes with ligands which are known to promote the formation of synaptic gephyrin clusters (collybistin and neuroligin 2) and reorganize them (dynein light chain 1).
For full-length gephyrin no structural information has been available so far. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) analyses described in this thesis disclosed that the gephyrin trimer forms a highly flexible assembly, which, due to the long linker, can switch between compact and extended conformational states in solution, with a preference for compact states. This partial compaction and potentially GephE autoinhibition are achieved by interactions of parts of the linker with the G and E domains, as suggested by circular dichroism spectroscopy. However, the linker on its own cannot account for GephE blockage, as size exclusion chromatography experiments coupled with multi angle light scattering detection (SEC-MALS) and SAXS analyses revealed that a gephyrin variant only encompassing the linker and GephE (GephLE) forms dimers and not monomers as suggested by an earlier study. The oligomeric state of GephLE and the observation that several gephyrin variants, in which linker segments of varying length were deleted, predominantly formed trimers, suggested the presence of a linker independent mechanism of GephE dimerization blockade. Taken together, the data indicated that linker-dependent and linker-independent mechanisms mediate gephyrin autoinhibition.
In the second project gephyrin’s interaction with DYNLL1 (Dynein LC8 Light Chain 1) was characterized. DYNLL1 is a 25 kDa dimer incorporated into the dynein motor and provides two binding sites, each of which can accommodate an octapeptide derived from gephyrin’s linker region (referred to as GephDB). Originally, DYNLL1 was regarded as a cargo adaptor, linking gephyrin-GlyR complexes to the dynein motor, thus driving their retrograde transport and leading to a decrease of synaptic gephyrin-GlyR complexes.
Building on these studies, this thesis assessed the cargo hypothesis as well as the so far unclear stoichiometry of the gephyrin-DYNLL1 complex. The cargo scenario would require ternary complex formation between gephyrin, DYNLL1 and the dynein intermediate chain (DIC) of the dynein motor. However, such a complex could not be detected by analytical size exclusion chromatography (aSEC) experiments – presumably because gephyrin and DIC competed for a common binding site in DYNLL1. This finding was consistent with a single DYNLL1 dimer capturing two linker segments of a single gephyrin trimer as suggested by a 26 kDa mass increase of the gephyrin species in the presence of DYNLL1 in SEC-MALS experiments. aSEC experiments at even higher concentrations (~20 µM gephyrin and ~80 µM DYNLL1) indicated that the affinity of GephDB was significantly impaired in the context of full-length gephyrin but also in a variant that bears only GephG and the first 39 residues of the linker (GephGL220). Presumably due to avidity effects two linkers stably associated with a single DYNLL1 dimer, whereas the third DYNLL1 binding motif remained predominantly unoccupied unless high concentrations of GephGL220 (50 µM) and DYNLL1 (200 µM) were used. These findings indicate that an interplay between GephG and the N-terminal linker segment mediates the attenuation of GephDB affinity towards DYNLL1 and that preventing DYNLL1 from the induction of higher gephyrin oligomers is either advantageous for DYNLL1-mediated reorganization of gephyrin-GlyR clusters or that DYNLL1 exerts possibly two (concentration-dependent) actions on gephyrin.
The gephyrin-collybistin-neuroligin 2 complex was the subject of the third project. Previously, collybistin and gephyrin were observed to mutually trigger their translocation to the postsynaptic membrane, where the disordered cytoplasmic tail of the postsynaptic cell adhesion molecule NL2 (NL2cyt) causes the anchoring of collybistin 2 (CB2) by binding to its SH3 domain, thereby releasing SH3 domain mediated autoinhibiton of CB2 binding to the membrane phospholipid phosphatidylinositol-3-phosphate. Critical for this event is the binding of gephyrin to both CB2 and NL2, presumably via GephE.
Following up on these previous studies biochemical data presented in this thesis confirm the formation of the ternary complex. Unexpectedly, analyses by means of native polyacrylamide gel electrophoresis pointed to: (1) The existence of a complex containing NL2cyt and CB2 lacking the SH3 domain and consequently an additional NL2 binding site in CB2. (2) Attenuated gephyrin-collybistin complex formation in the presence of the SH3 domain. (3) A requirement for high NL2cyt concentrations (> 30 µM) during the formation of the ternary complex. This might allow for the regulation by other factors such as additional binding partners or posttranslational modifications. Although of preliminary character, these results provide a starting point for future studies, which will hopefully elucidate the interplay between gephyrin, collybistin, NL2 and certain GABAA receptors.
Die zunehmende Versalzung des Bodens führt weltweit zu starken Ernteeinbußen. Ob- wohl die Wurzeln der Pflanzen als erstes mit dem Salzstress in Berührung kommen, ist noch nicht viel über Signaltransduktionswege in Wurzeln zur Anpassung der Pflanze an Salzstress bekannt. Die bZIP-Transkriptionsfaktoren der Gruppe S1, bZIP1 und bZIP53, werden gewebespezifisch in der Wurzel nach Salzstress aktiviert. In dieser Arbeit werden diese bZIPs in ein Netzwerk eingeordnet, von der Aktivierung der Tran- skriptionsfaktoren bis zur Funktion in der Regulation des Stoffwechsels in der salzgest- ressten Pflanze.
Die Aktivierung von bZIP1 kann über verschiedene sowohl ionische als auch osmotische Stimuli erfolgen und ist abhängig von Calcium, der HEXOKINASE 1 und SnRK1- Kinasen (Snf1 RELATED PROTEIN KINASE 1). Die dunkelinduzierte Expression von bZIP1 wird HXK1-abhängig durch Glucose inhibiert, bei Energiemangelbedingungen ist die Aktivierung von bZIP1 SnRK1-abhängig. Beide Enzyme spielen auch in der salzinduzierten Expression von bZIP1 eine Rolle. Über Transkriptom- und Me- tabolomanalysen kann gezeigt werden, dass bZIP1 und bZIP53 an der Umprogram- mierung des Kohlenhydrat- und Aminosäuremetabolismus teilhaben. Besonders Gene der Glukoneogenese (PYRUVAT ORTHOPHOSPHAT DIKINASE und FRUCTOSE- 1,6-BISPHOS- PHATASE) bzw. des Aminosäurekatabolismus (BRANCHED- CHAIN AMINO ACID TRANSAMINASE 2, METHYLCROTONYL- COA-CARBOXYLASE A und HOMOGENTISATE 1,2-DIOXYGENASE ) werden von den Transkriptionsfaktoren reguliert. Das spricht für eine Umprogrammierung des Metabolismus und der Mobilisierung von Energie aus Aminosäuren zur Anpassung an die Stressbedingungen. Die Transkriptionsfaktoren der Gruppe S1 bilden vorzugsweise Heterodimere mit der Gruppe C. Mit Mutantenanalysen, die zum einen die Transkriptionsfaktoren des C/S1-Netzwerks und zum anderen Komponenten der Abscisinsäure (ABA) abhängigen Signaltransduktion beinhalten, konnte ein Signaltransduktionsnetzwerk aufgestellt werden, das die Antwort auf abiotischen Stress mittels des Signalwegs über ABA, SnRK2 und AREB (ABA RESPONSIVE ELEMENTS-BINDING PROTEIN) mit der SnRK1-vermittelten Antwort auf Energiemangelbedingungen in der Pflanze verknüpft. Die gefundenen stress- bzw. energieresponsiven Gene konnten nach den Mutantenana- lysen auf Grund ihrer unterschiedlichen Regulation in vier Klassen eingeteilt werden, wovon nur eine, die Klasse 4, von dem C/S1 Netzwerk reguliert wird. Die Klassen 1- 3 sind unabhängig von den bZIP-Transkriptionsfaktoren der Gruppe C. Die Klasse 1 bilden typische ABA-responsive Gene, die von den Gruppe A-bZIPs reguliert werden. Faktoren der Gruppe A sind auch an der Expression der Gene der Klasse 2 beteiligt, diese werden aber auch durch bZIP1 und bZIP53 induziert. Dieser Klasse konnten Gene zugeordnet werden, die im Abbau verzweigtkettiger Aminosäuren eine Rolle spielen. Am Aminosäureabbau sind außerdem die Gene der Klasse 2 beteiligt. Für diese Gene konnte eine Expressionsregulation durch bZIP1 und bZIP53 gezeigt werden. Für die Bestimmung möglicher Heterodimerisierungspartner bedarf es noch weiterer Analysen. Dieses Model, das den abitoschen Stress abhängigen ABA-Signalweg mit dem ener- gieabhängigen SnRK1-Signaltransduktionsweg verknüpft, zeigt die präzise Regulation von mindestens 4 Gen-Klassen, deren Expression durch die Kombination verschiedener bZIP-Transkriptionsfaktoren aktiviert wird.
Invasive Zygomykosen verzeichnen in den letzten Jahren eine steigende Inzidenz, insbesondere im Risikokollektiv immunsupprimierter Patienten. Aufgrund des häufig letalen Verlaufs dieser Infektionen ist eine rasche, korrekte Diagnosestellung essentiell, um rechtzeitig eine adäquate Therapie einzuleiten. Jedoch sieht sich die konventionelle, mikrobiologische Diagnostik mit vielen Problemen konfrontiert, so dass molekularbiologische Nachweisverfahren zunehmend in den Fokus der Aufmerksamkeit rücken. Eine zuverlässige, mit relativ geringem Zeit- und Kostenaufwand praktizierbare Methode stellt in diesem Zusammenhang die Real-time-PCR dar, deren Aussagekraft durch anschließende Speziesidentifizierung mittels Sequenzierung noch verstärkt werden kann.
Aus diesem Grund wurden im Rahmen dieser Arbeit 3 PCR-Assays entwickelt und deren Sensitivität, Spezifität und klinische Anwendbarkeit evaluiert. Alle 3 Systeme nutzten Multi-copy-Gene des ribosomalen Operons der Zygomyzeten als Target und erwiesen sich als zuverlässige Werkzeuge zur Amplifikation fungaler DNA. Sie wurde sowohl an Pilzkulturen, als auch an klinischen Proben und einem Quasi-Tiermodell mit Erfolg ausgetestet und werden möglicherweise in Zukunft der klinischen Routinediagnostik zur Verfügung stehen.
Bedingt durch die Seltenheit invasiver Zygomykosen besteht in diesem Bereich noch ein großer Forschungsbedarf, auch, um die noch nicht optimale Therapie dieser Erkrankungen zu verbessern. Es bleibt daher zu hoffen, dass sich in absehbarer Zeit mehr Forschungsgruppen mit diesen Erregern beschäftigen, damit den schwer kranken Patienten eine echte Heilungschance geboten werden kann.
Organisms use different resources in different habitat types during their life cycle. Thereby, they connect habitats and provide ecosystem services or disservices in several habitat types. In agricultural landscapes, the spillover of organisms, i.e. movement of an organism and its function from one habitat to another, especially from semi-natural to managed habitats, is one of the most important processes that influence population dynamics and community composition. Importantly, spillover connects habitats not only spatially, but also on different temporal scales, because availability of resources changes over time in agricultural landscapes, e.g. by mass-flowering events of crops, harvesting or crop rotation. Most often, semi-natural habitats are seen as beneficial source of organisms, but also managed habitats can provide valuable resources, and thereby initiate spillover to other habitats. Mass-flowering crops, like oil-seed rape, are such valuable feeding resources for pollinators, and pollinators might spillover from oil-seed rape to other habitats which provide alternative foraging resources. The focus of this dissertation was to evaluate the influence of oil-seed rape on pollinators in agricultural landscapes by studying effects (1) on different temporal scales (from effects during the flowering period of oil-seed rape, Chapter II & IV, to intermediate effects on a second mass-flowering crop, Chapter III, to spillover effects to the flowering period in the next year, Chapter IV), (2) semi-natural (Chapter II) and crop (Chapter III, IV) habitats, and (3) on various pollinator groups which differ in their life cycle (Chapter II, III, IV).
In this dissertation effects from oil-seed rape on all temporal scales – in the short term during mass-flowering and in the long term on a late-flowering crop and even in the next year on oil-seed rape fields ─ were found. These effects might be important for crop and wild plant pollination, and pollinator conservation. Importantly, the effects on different temporal scales depend on the considered habitat (managed or different semi-natural habitats) and on the investigated pollinator group. The more pollinators match the flowering period of oil-seed rape in their activity period and the more dependent they are on flowering resources in their life cycle, the more pronounced are their responses. Effects were found for wild bees, but not for hoverflies and honey bees. Moreover, the availability of semi-natural habitats in the landscape is important and may modulate effects from oil-seed rape. The longevity of effects of oil-seed rape shows the importance of including several temporal scales into ecosystem-service studies, not only for pollinators, but also for other ecosystem-service providing species groups.
The Fanconi anemia (FA) pathway is a replication-dependent DNA repair mechanism which is essential for the removal of interstrand crosslink (ICL) DNA damages in higher eukaryotes (Moldovan and D’Andrea, 2009). Malfunctions in this highly regulated repair network lead to genome instability (Deans and West, 2011). Pathological phenotypes of the disease FA which is caused by mutations in the eponymous pathway are very heterogeneous, involving congenital abnormalities, bone-marrow failure, cancer predisposition and infertility (Auerbach, 2009). The FA pathway comprises a complex interaction network and to date 16 FA complementation groups and associated factors have been identified (Kottemann and Smogorzewska, 2013). Additionally, components of nucleotide excision repair (NER), homologous recombination repair (HRR), and translesion synthesis (TLS) are involved and coordinated by the FA proteins (Niedzwiedz et al., 2004; Knipscheer et al., 2009). One of the FA proteins is the DEAH helicase FANCM. In complex with its binding partners FAAP24 and MHF1/2 it binds the stalled replication fork and activates the FA damage response (Wang et al., 2013). However, the exact steps towards removal of the ICL damage still remain elusive.
To decipher the underlying process of FA initiation by FANCM, this thesis mainly focuses on the archaeal FANCM homolog helicase-associated endonuclease for fork-structured DNA (Hef). Hef from the archaeal organism Thermoplasma acidophilum (taHef) differs from other archaeal Hef proteins and exclusively comprises an N-terminal helicase entity with two RecA and a thumb-like domain while others additionally contain a nuclease portion at the C-terminus. I solved the crystal structure of full-length taHef at a resolution of 2.43 Å. In contrast to the crystal structure of the helicase domain of Hef from Pyrococcus furiosus (pfHef), taHef exhibits an extremely open conformation (Nishino et al., 2005b) which implies that a domain movement of the RecA-like helicase motor domains of 61° is possible thus highlighting the flexibility of helicases which is required to translocate along the DNA. However, small-angle x-ray scattering (SAXS) measurements confirm an intermediate conformation of taHef in solution indicating that both crystal structures represent rather edge states. Most
importantly, proliferating cell nuclear antigen (PCNA) was identified as an interaction partner of Hef. This interaction is mediated by a highly conserved canonical PCNA interacting peptide (PIP) motif. Intriguingly, the presence of PCNA does not alter the ATPase nor the helicase activity of taHef, thus suggesting that the interaction is entirely dedicated to recruit taHef to the replication fork to fulfill its function. Due to a high level of flexibility the taHef-taPCNA complex could not be crystallized and therefore SAXS was utilized to determine a low-resolution model of this quaternary structure.
This newly discovered PCNA interaction could also be validated for the eukaryotic FANCM homolog Mph1 from the thermophilic fungus Chaetomium thermophilum (ctMph1). As the first step towards the characterization of this interaction I solved the crystal structure of PCNA from Chaetomium thermophilum (ctPCNA).
Furthermore, it was possible to achieve preliminary results on the putative interaction between the human proteins FANCM and PCNA (hsFANCM, hsPCNA). In collaboration with Detlev Schindler (Human Genetics, Würzburg) and Weidong Wang (National Institute on Aging, Baltimore, USA) co-immunoprecipitation (CoIP) experiments were performed using hsFANCM and hsPCNA expressed in HEK293 cells. Although an interaction was reproducibly observed in hydroxyurea stimulated cells
further experiments and optimization procedures are required and ongoing.