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Pavlovian fear conditioning describes a form of associative learning in which a previously neutral stimulus elicits a conditioned fear response after it has been temporally paired with an aversive consequence. Once acquired, the fear response can be extinguished by repeatedly presenting the former neutral stimulus in the absence of the aversive consequence. Although most patients suffering from anxiety disorders cannot recall a specific conditioned association between a formerly neutral stimulus and the feeling of anxiety, the produced behavioral symptoms, such as avoidance or safety behavior to prevent the anticipated aversive consequence are commonly exhibited in all anxiety disorders. Moreover, there is considerable similarity between the neural structures involved in fear and extinction in the rodent and in the human. Translational research thus contributes to the understanding of neural circuitries involved in the development and maintenance of anxiety disorders, and further provides hypotheses for improvements in treatment strategies aiming at inhibiting the fear response.
Since the failure to appropriately inhibit or extinguish a fear response is a key feature of pathological anxiety, the present preclinical research focuses on the interplay between the amygdala and the medial prefrontal cortex (mPFC) during fear learning with particular regard to the prefrontal recruitment during fear extinction and its recall. By firstly demonstrating an increased mPFC activity over the time course of extinction learning with functional near-infrared spectroscopy, the main study of this dissertation focused on repetitive transcranial magnetic stimulation (rTMS) as brain stimulation technique suitable to enhance extinction learning. Since hypofrontality is assumed to underlie the maintenance of pathological anxiety, rTMS application revealed an increased mPFC activity, which resulted in a decreased fear response on the behavioral level both during extinction learning as well as during the recall of extinction 24 hours later and in the absence of another stimulation. The following attempt to improve the generalization of extinction with rTMS from an extinguished stimulus to a second stimulus which was reinforced but not extinguished was at least partially evidenced. By revealing an increased prefrontal activity to the non-extinguished stimulus, the active and the placebo rTMS condition, however, did not differ on behavioral parameters. These preclinical findings were discussed in the light of genetic and environmental risk factors with special regard to the combination of a risk variant of the neuropeptide S receptor 1 gene polymorphism (NPSR1 rs324981) and anxiety sensitivity. While the protective homozygous AA genotype group showed no correlation with anxiety sensitivity, the NPSR1 T genotype group exhibited an inverse correlation with anxiety sensitivity in the presence of emotionally negative stimuli. In light of other findings assuming a role of the NPSR1 T allele in panic disorder, the revealed hypofrontality was discussed to define a risk group of patients who might particularly benefit from an augmentation of exposure therapy with rTMS.
Taken together, the presented studies support the central role of the prefrontal cortex in fear extinction and suggest the usefulness of rTMS as an augmentation strategy to exposure therapy in order to decrease therapy relapse rates. The combination of rTMS and extinction has been herein evidenced to modulate fear processes in a preclinical approach thereby establishing important implications for the design of future clinical studies.
Anxiety is an affective state characterized by a sustained, long-lasting defensive response, induced by unpredictable, diffuse threat. In comparison, fear is a phasic response to predictable threat. Fear can be experimentally modeled with the help of cue conditioning. Context conditioning, in which the context serves as the best predictor of a threat due to the absence of any conditioned cues, is seen as an operationalization of sustained anxiety.
This thesis used a differential context conditioning paradigm to examine sustained attention processes in a threat context compared to a safety context for the first time. In three studies, the attention mechanisms during the processing of contextual anxiety were examined by measuring heart rate responses and steady-state-visually evoked potentials (ssVEPs). An additional focus was set on the processing of social cues (i.e. faces) and the influence of contextual information on these cues. In a last step, the correlates of sustained anxiety were compared to evoked responses by phasic fear, which was realized in a previously established paradigm combining predictable and unpredictable threat.
In the first study, a contextual stimulus was associated with an aversive loud noise, while a second context remained unpaired. This conditioning paradigm created an anxiety context (CTX+) and a safety context (CTX-). After acquisition, a social agent vs. an object was presented as a distractor in both contexts. Heart rate and cortical responses, with ssVEPs by using frequency tagging, to the contexts and the distractors were assessed. Results revealed enhanced ssVEP amplitudes for the CTX+ compared to the CTX− during acquisition and during presentation of distractor stimuli. Additionally, the heart rate was accelerated in the acquisition phase, followed by a heart rate deceleration as a psychophysiological marker of contextual anxiety.
Study 2 used the same context conditioning paradigm as Study 1. In contrast to the first study, persons with different emotional facial expressions were presented in the anxiety and safety contexts in order to compare the differential processing of these cues within periods of threat and safety. A similar anxiety response was found in the second study, although only participants who
Abstract
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were aware of the contingency between contexts and aversive event showed a sensory amplification of the threat context, indicated by heart rate response and ssVEP activation. All faces irrespective of their emotional expression received increased attentional resources when presented within the anxiety context, which suggests a general hypervigilance in anxiety contexts.
In the third study, the differentiation of predictable and unpredictable threat as an operationalization of fear and anxiety was examined on a cortical and physiological level. In the predictable condition, a social cue was paired with an aversive event, while in the unpredictable condition the aversive event remained unpaired with the respective cue. A fear response to the predictable cue was found, indicated by increased oscillatory response and accelerated heart rate. Both predictable and unpredictable threat yielded increased ssVEP amplitudes evoked by the context stimuli, while the response in the unpredictable context showed longer-lasting ssVEP activation to the threat context.
To sum up, all three studies endorsed anxiety as a long-lasting defensive response. Due to the unpredictability of the aversive events, the individuals reacted with hypervigilance in the anxiety context, reflected in a facilitated processing of sensory information and an orienting response. This hypervigilance had an impact on the processing of novel cues, which appeared in the anxiety context. Considering the compared stimuli categories, the stimuli perceived in a state of anxiety received increased attentional resources, irrespective of the emotional arousal conveyed by the facial expression. Both predictable and unpredictable threat elicited sensory amplification of the contexts, while the response in the unpredictable context showed longer-lasting sensory facilitation of the threat context.
Die invasive Aspergillose (IA) z¨ ahlt zu den seltenen, bei immunsupprimierten Patienten
jedoch mit einer hohen Letalit¨ at verbundenen Infektionskrankheiten. Sie wird, wie alle
Aspergillosen, durch den humanpathogenen Schimmelpilz Aspergillus fumigatus ausgel¨ ost.
Bis heute ist die oft nicht effektive Therapie einer IA mit hohen Nebenwirkungen und
Kosten verbunden. Die Entwicklung von Pathogen-spezifischen Immuntherapien soll durch
die Forschung im Bereich der immunologischen Infektionsbiologie vorangetrieben werden.
Fur neuen Erkenntnisse wird die Interaktion von humanen Immunzellen mit ¨ A. fumigatus
analysiert.
In der vorliegenden Studie wurde mit dendritischen Zellen (DCs) gearbeitet, da diese
Pilzmorphologien von A. fumigatus phagozytieren k¨ onnen und uber Antigenpr ¨ ¨ asentation
das adaptive Immunsystem aktivieren. Es wurden aus humanen Monozyten differenzierte DCs (moDCs) verwendet, mit welchen viele Forschergruppen aufgrund ihrer verfugbar ¨
großen Anzahl arbeiten. Allerdings dauert die Generierung von moDCs funf Tage. Aus ¨
dem peripheren Blut entstammende CD1c-positive myeloide DCs (mDCs) oder CD303-
positive plasmazytoide DCs (pDCs) k¨ onnen dagegen direkt nach der Isolation verwendet
werden. Die beiden DC-Populationen werden aus verschiedenen Vorl¨ auferzellen des h¨ amatopoetischen Systems im Knochenmark gebildet. Ihr Ph¨ anotyp und ihre Immunfunktionen
unterscheiden sich untereinander und auch von denen der moDCs.
In Interaktionsstudien konnte analysiert werden, dass die drei verwendeten DC-Subtypen
(moDCs, mDCs, pDCs) unterschiedlich auf A. fumigatus reagieren. moDCs und mDCs reiften in direktem Kontakt zu Aspergillus, sie sekretierten ein relativ ¨ ahnliches Zytokinprofil
und exprimierten die bekannten Aspergillus-Rezeptoren Dectin-1, TLR2 und TLR4. Im
Kontrast dazu verblieben pDCs trotz Aspergillus-Kontakt unreif und sekretierten nahezu
keine Zytokine. Da moDCs und mDCs eine Immunreaktion auf den Pilz zeigten, wurden
sie mit verschiedenen Aspergillus-Antigenen beladen und n¨ aher untersucht.
Bevor verschiedene Aspergillus-Antigene zur Beladung der DCs eingesetzt werden konnten, wurden diese analysiert und aufgereinigt. Hierfur wurde ein routinierter Arbeitsprozess ¨
etabliert. Zwei der vier verfugbaren Proteinantigene waren mit Endotoxin kontaminiert. ¨
Da schon geringe Mengen an Endotoxinen auf DCs einen stimulatorischen Effekt ausubten, ¨
wurden die Proteine mittels Affinit¨ atschromatografie von den verunreinigenden Endotoxinen befreit.
In den Stimulationsexperimenten wirkten die beiden Proteinantigene CcpA und SHMT
immunogen auf moDCs und mDCs. CcpA induzierte eine st¨ arkere Maturierung und Zytokinfreisetzung als SHMT. Auff¨ allig war, dass mDCs im Vergleich zu moDCs die Expression
von MHC Klasse II st¨ arker erh¨ ohten und mehr IL18 freisetzten. Die mit CcpA oder SHMT
beladenen moDCs und mDCs aktivierten autologe T-Zellen zur IFNg Sekretion und zur
Proliferation. Zudem wurden durch die beiden Proteine Aspergillus-spezifische, CD154-
positive T-Zellen induziert. Diese Aspergillus-spezifische Immunogenit¨ at von CcpA und
SHMT macht die beiden Proteine zu interessanten Kandidaten fur einen Vakzinierungs- ¨
Cocktail einer DC-Immuntherapie. Aspergillus Lysat induzierte als weiteres Antigen eine
T-Zell Immunantwort mit CD154-positiven T-Zellen. Zudem war die Proliferation und
IFNg Sekretion von T-Zellen induziert, obwohl moDCs und mDCs nicht reiften und nur
wenige Zytokine sekretierten. Die beiden Aspergillus-Proteine CpcB und fg-gap induzierten die Reifung und Zytokinsekretion von moDCs und mDCs nicht. Demzufolge sind CpcB
und fg-gap fur eine DC-Immuntherapie nicht empfehlenswert. ¨
Ein Vakzinierungs-Cocktail enth¨ alt in der Regel Adjuvantien, welche die Immunreaktion
verst¨ arken. Adjuvante Effekte auf moDCs konnten die getesteten Aspergillus-RezeptorLiganden Zymosan, Pam3CSK4, LPS ultrapur und R848 ausl¨ osen. Hyalurons¨ aure konnte keine Verbesserung der Reifung oder Vitalit¨ at von moDCs und mDCs bewirken. Die
Antigen-bedingte Reifung der DCs fur n ¨ ¨ otige Restimulationen w¨ ahrend der Therapie konnte mittels einer tiefgekuhlten Lagerung in CryoStor Einfriermedium stabil beibehalten ¨
werden.
Die beiden immunogenen Aspergillus-Proteine, die adjuvanten Rezeptor-Agonisten und
die stabile Lagerung in CryoStor k¨ onnen als elementare Grundsteine fur einen Vakzinierungs- ¨
Cocktail einer anti-fungalen DC-Immuntherapie mit moDCs oder mDCs angesehen werden.
Fulminant myocarditis is rare but a potentially life-threatening disease. Acute or mild myocarditis following acute ischemia is generally associated with a profound activation of the host’s immune system. On one hand this is mandatory to protect the host’s heart by fighting the invading agents (i.e., bacteria, viruses or other microbial agents) and/or to induce healing and repair processes in the damaged myocardium. On other hand, uncontrolled activation of the immune system may result in the generation of auto-reactive (not always beneficial) immune cells.
Myocarditis or inflammatory cardiomyopathy is characterized by focal or diffuse infiltrates, myocyte necrosis and/or apoptosis and subsequent fibrotic replacement of the heart muscle. In humans, about 30% of the myocarditis-patients develop dilated cardiomyopathy. As the clinical picture of myocarditis is multifaceted, it is difficult to diagnose the disease. Therefore, the main goal of the present work was to test and further develop novel non-invasive methods for the detection of myocardial inflammation by employing both contrast enhanced MRI techniques as well as novel nuclear tracers that are suitable for in vivo PET/ SPECT imaging.
As a part of this thesis, a pre-clinical animal model was successfully established by immunizing female Lewis rats with whole-porcine cardiac myosin (CM). Induction of Experimental Autoimmune Myocarditis (EAM) is considered successful when anti-myosin antibody titers are increased more than 100-fold over control animals and pericardial effusion develops. In addition, cardiac tissues from EAM-rats versus controls were analyzed for the expression of various pro-inflammatory and fibrosis markers. To further exploit non-invasive MRI techniques for the detection of myocarditis, our EAM-rats were injected either with (1) ultra-small Paramagnetic iron oxide particles (USPIO’s; Feraheme®), allowing for in vivo imaging , (2) micron sized paramagnetic iron oxide particles (MPIO) for ex vivo inflammatory cell-tracking by cMRI, or (3) with different radioactive nuclear tracers (67gallium citrate, 68gallium-labeled somatostatin analogue, and 68gallium-labeled cyclic RGD-peptide) which in the present work have been employed for autoradiographic imaging, but in principle are also suitable for in vivo nuclear imaging (PET/SPECT). In order to compare imaging results with histology, consecutive heart sections were stained with hematoxylin & eosin (HE, for cell infiltrates) and Masson Goldner trichrome (MGT, for fibrosis); in addition, immuno-stainings were performed with anti-CD68 (macrophages), anti-SSRT2A (somatostatin receptor type 2A), anti-CD61 (β3-integrins) and anti-CD31 (platelet endothelial cell adhesion molecule 1).
Sera from immunized rats strongly reacted with cardiac myosin. In immunized rats, echocardiography and subsequent MRI revealed huge amounts of pericardial effusion (days 18-21). Analysis of the kinetics of myocardial infiltrates revealed maximal macrophage invasion between days 14 and 28. Disappearance of macrophages resulted in replacement-fibrosis in formerly cell-infiltrated myocardial areas. This finding was confirmed by the time-dependent differential expression of corresponding cytokines in the myocardium. Immunized animals reacted either with an early or a late pattern of post-inflammation fibrosis. Areas with massive cellular infiltrates were easily detectible in autoradiograms showing a high focal uptake of 67gallium-citrate and 68gallium labeled somatostatin analogues (68Ga DOTA-TATE). Myocardium with a loss of cardiomyocytes presented a high uptake of 68gallium labeled cyclic RGD-peptide (68Ga NOTA-RGD). MRI cell tracking experiments with Feraheme® as the contrast-agent were inconclusive; however, strikingly better results were obtained when MPIOs were used as a contrast-agent: histological findings correlated well with in vivo and ex vivo MPIO-enhanced MRI images.
Imaging of myocardial inflammatory processes including the kinetics of macrophage invasion after microbial or ischemic damage is still a major challenge in, both animal models and in human patients. By applying a broad panel of biochemical, histological, molecular and imaging methods, we show here that different patterns of reactivity may occur upon induction of myocarditis using one and the same rat strain. In particular, immunized Lewis rats may react either with an early or a late pattern of macrophage invasion and subsequent post-inflammation fibrosis. Imaging results achieved in the acute inflammatory phase of the myocarditis with MPIOs, 67gallium citrate and 68gallium linked to somatostatin will stimulate further development of contrast agents and radioactive-nuclear tracers for the non-invasive detection of acute myocarditis and in the near future perhaps even in human patients.
The impact of acquired severe motor impairments is pervasive and may lead to a complete loss of communication and voluntary motor control, rendering the patient behaviourally unresponsive. In routine clinical care it may thus be unclear, whether some of these patients are even conscious. Given that finding a cure is unlikely, care focuses on providing the best possible quality of life (QoL), and knowing its predictors might contribute to that aim. Patients who still can communicate often report a high QoL, and several predictors have been identified. However, many instruments used to assess QoL require at least residual verbal and motor abilities. Thus, a method to assess QoL independent of these requirements is desirable. In addition, many instruments assume QoL to be temporarily stable, and little information is available on predictors of instantaneous QoL, i.e. QoL as it fluctuates from moment to moment throughout the day.
The correct regulation of cell growth and proliferation is essential during normal animal development. Myc proteins function as transcription factors, being involved in the con-trol of many growth- and proliferation-associated genes and deregulation of Myc is one of the main driving factors of human malignancies.
The first part of this thesis focuses on the identification of directly regulated Myc target genes in Drosophila melanogaster, by combining ChIPseq and RNAseq approaches. The analysis results in a core set of Myc target genes of less than 300 genes which are mainly involved in ribosome biogenesis. Among these genes we identify a novel class of Myc targets, the non-coding small nucleolar RNAs (snoRNAs). In vivo studies show that loss of snoRNAs not only impairs growth during normal development, but that overexpression of several snoRNAs can also enhance tumor development in a neu-ronal tumor model. Together the data show that Myc acts as a master regulator of ribo-some biogenesis and that Myc’s transforming effects in tumor development are at least partially mediated by the snoRNAs.
In the second part of the thesis, the interaction of Myc and the Zf-protein Chinmo is described. Co-immunoprecipitations of the two proteins performed under endogenous and exogenous conditions show that they interact physically and that neither the two Zf-domains nor the BTB/POZ-domain of Chinmo are important for this interaction. Fur-thermore ChIP experiments and Myc dependent luciferase assays show that Chinmo and Myc share common target genes, and that Chinmo is presumably also involved in their regulation. While the exact way of how Myc and Chinmo genetically interact with each other still has to be investigated, we show that their interaction is important in a tumor model. Overexpression of the tumor-suppressors Ras and Chinmo leads to tu-mor formation in Drosophila larvae, which is drastically impaired upon loss of Myc.
The recently discovered human DREAM complex (for DP, RB-like, E2F and MuvB complex) is a chromatin-associated pocket protein complex involved in cell cycle- dependent gene expression. DREAM consists of five core subunits and forms a complex either with the pocket protein p130 and the transcription factor E2F4 to repress gene expression or with the transcription factors B-MYB and FOXM1 to promote gene expression.
Gas2l3 was recently identified by our group as a novel DREAM target gene. Subsequent characterization in human cell lines revealed that GAS2L3 is a microtubule and F-actin cross-linking protein, expressed in G2/M, plays a role in cytokinesis, and is important for chromosomal stability.
The aim of the first part of the study was to analyze how expression of GAS2L3 is regulated by DREAM and to provide a better understanding of the function of GAS2L3 in mitosis and cytokinesis.
ChIP assays revealed that the repressive and the activating form of DREAM bind to the GAS2L3 promoter. RNA interference (RNAi) mediated GAS2L3 depletion demonstrated the requirement of GAS2L3 for proper cleavage furrow ingression in cytokinesis. Immunofluorescence-based localization studies showed a localization of GAS2L3 at the mitotic spindle in mitosis and at the midbody in cytokinesis. Additional experiments demonstrated that the GAS2L3 GAR domain, a putative microtubule- binding domain, is responsible for GAS2L3 localization to the constriction zones in cytokinesis suggesting a function for GAS2L3 in the abscission process.
DREAM is known to promote G2/M gene expression. DREAM target genes include several mitotic kinesins and mitotic microtubule-associated proteins (mitotic MAPs). However, it is not clear to what extent DREAM regulates mitotic kinesins and MAPs, so far. Furthermore, a comprehensive study of mitotic kinesin expression in cancer cell lines is still missing.
Therefore, the second major aim of the thesis was to characterize the regulation of mitotic kinesins and MAPs by DREAM, to investigate the expression of mitotic kinesins in cancer cell line panels and to evaluate them as possible anti-cancer targets.
ChIP assays together with RNAi mediated DREAM subunit depletion experiments demonstrated that DREAM is a master regulator of mitotic kinesins. Furthermore, expression analyses in a panel of breast and lung cancer cell lines revealed that mitotic kinesins are up-regulated in the majority of cancer cell lines in contrast to non-transformed controls. Finally, an inducible lentiviral-based shRNA system was developed to effectively deplete mitotic kinesins. Depletion of selected mitotic kinesins resulted in cytokinesis failures and strong anti-proliferative effects in several human cancer cell lines.
Thus, this system will provide a robust tool for future investigation of mitotic kinesin function in cancer cells.
In der nuklearmedizinischen Therapie werden Radiopharmaka meist systemisch verabreicht. Primär werden dafür, wegen der kurzen Reichweite, beta-Strahler eingesetzt. Als Folge davon verteilt sich das Radiopharmakon im Körper, reichert sich in Organen und Zielstrukturen an und bestrahlt somit den Körper intern, im Gegensatz zur externen Bestrahlung bei der Strahlentherapie.
Das Verteilungsmuster der verabreichten Aktivität im Körper wird durch die chemischen und physikalischen Eigenschaften des Radiopharmakons bestimmt. Außerdem sind die Aktivität und die Art der Anreicherung ausschlaggebend für die durch ionisierende Strahlung deponierte Energie im Körper, der Energiedosis.
Gemeinsam haben externe und interne Bestrahlungsverfahren, dass der Patient ionisierender Strahlung ausgesetzt ist, die nicht nur die kranken Zellen zerstört, sondern auch gesunde Zellen schädigen kann. Dies geschieht durch direkte oder indirekte Wechselwirkung der Strahlung mit der DNA, die zur Schädigung der DNA-Struktur führt. Am häufigsten sind dabei Einzelstrangbrüche und Basenschäden. Die Doppelstrangbrüche sind im Vergleich zu Einzelstrangbrüchen und Basenschäden sehr selten aber sehr viel schädlicher für die Zelle, da die Reparatur komplizierter ist. Somit sind diese primär für den Zelltod oder für die Folgen nach fehlerhafter Reparatur verantwortlich.
Eine sehr schnelle Antwort auf strahleninduzierte oder durch andere Stoffe, wie z.B. zytotoxische Substanzen, induzierte Doppelstrangbrüche ist die Phosphorylierung der Histon H2 Variante H2AX, die gamma-H2AX genannt wird. Zusätzlich reichert sich das Protein 53BP1 nach dem Erkennen eines Doppelstrangbruches durch Sensorproteine sofort am Chromatin, das den Doppelstrang umgibt, an. Damit ist 53BP1 ein weiterer Biomarker, der strahleninduzierte Doppelstrangbrüche sehr effektiv nachweisen kann und der auf sehr verlässliche Weise mit gamma-H2AX kolokalisiert. Mittels Immunfluoreszenzfärbung lassen sich gamma-H2AX und 53BP1 als umschriebene „Foci“, im Zellkern mikroskopisch darstellen und zählen. Unter der Annahme, dass ein Focus einem Doppelstrangbruch entspricht, kann die Anzahl der Foci im Zellkern als quantitativer Biomarker für DNA Doppelstrangbrüche und damit für die Strahlenexposition und Strahlenwirkung verwendet werden.
Zudem zeigen Studien der Induktion von gamma-H2AX nach externer Bestrahlung von unterschiedlichen Gewebearten Linearität zwischen der Energiedosis und der Zahl der Foci im Zellkern. Weitere Studien beschäftigen sich mit den Auswirkungen externer Bestrahlung auf Patienten, aber nur wenige mit offenen radioaktiven Substanzen. Ziele dieser Arbeit waren daher:
1. Die Generierung einer bisher noch nicht beschriebenen in-vitro Kalibrierkurve nach interner Bestrahlung von Vollblut mit den in der Therapie eingesetzten beta-Strahlern.
2. Die gleichzeitige Bestimmung der physikalischen Dosis sowie der strahleninduzierten Anzahl der Foci in Lymphozyten, gewonnen aus Blutproben von Patienten nach Radiopeptidtherapie mit Lu-177 und Radioiodtherapie mit I-131.
3. Eine umfassende Beschreibung der Induktion und der Abnahme der Foci in den Lymphozyten aus den Blutproben der Patienten unter Einbeziehung der in-vitro Kalibrierung, um den dosis- und zeitabhängigen Verlauf der Anzahl der strahleninduzierten Foci zu bestimmen.
Für die in-vitro Kalibrierung mit I-131 und Lu-177 wurden bei Probanden Blutproben gewonnen und mit unterschiedlichen Aktivitätskonzentrationen ergänzt. Das Ziel war, eine Energiedosis bis 100mGy zu erhalten. Das Ergebnis war, dass sich die Zahl der strahleninduzierten Foci in Abhängigkeit von der Energiedosis gut durch eine lineare Funktion beschreiben lässt, so wie es auch für die externe Bestrahlung bereits gezeigt wurde.
Die Patientenstudien befassten sich mit dem Zusammenhang zwischen der im Blut deponierten Energiedosis und der Anzahl und dem zeitlichen Verlauf der induzierten Doppelstrangbrüche im peripheren Blut von Patienten unter Peptidrezeptor-Radionuklidtherapie mit Lu-177 DOTATATE/-TOC und Patienten unter Radioiodtherapie mit I-131 bei Ablationstherapien nach Operation eines differenzierten Schilddrüsenkarzinoms.
Die durchschnittliche Anzahl induzierter DSB-Foci zeigte in den frühen Zeitpunkten einen linearen dosisabhängigen Anstieg. In den ersten Stunden nach Therapie stimmten die in-vitro Kalibrierung und die Zahl der strahleninduzierten Foci sowohl für Lu-177 als auch für I-131 für die Patientendaten gut überein.
Die späteren Zeitpunkte werden durch eine Abnahme der Dosisrate und der Foci-Anzahl, bedingt durch Reparatur der DNA-Schäden, charakterisiert. Überstiegen die Blutdosiswerte in der ersten Stunde jedoch 20mGy (nur nach I-131-Gabe beobachtet), dann war die Induktion eines schnellen Reparaturprozesses festzustellen.
Diese experimentellen Ergebnissen und Modellierungen beschreiben erstmalig die Dosisabhängigkeit und den zeitlichen Verlauf der in-vitro und in-vivo DNA-Schadensantwort nach Inkorporation von beta-emittierenden Radionukliden.
Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach.
This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching.
Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect.
The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature.
Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.
Early life stress, including exposure to prenatal stress (PS), has been shown to affect the developing brain and induce severe effects on emotional health in later life, concomitant with an increased risk for psychopathology. However, some individuals are more vulnerable to early-life stress, while others adapt successfully, i.e. they are resilient and do not succumb to adversity. The molecular substrates promoting resilience in some individuals and vulnerability in other individuals are as yet poorly investigated. A polymorphism in the serotonin transporter gene (5HTT/SLC6A4) has been suggested to play a modulatory role in mediating the effects of early-life adversity on psychopathology, thereby rendering carriers of the lower-expressing short (s)-allele more vulnerable to developmental adversity, while long (l)-allele carriers are relatively resilient. The molecular mechanisms underlying this gene x environment interaction (GxE) are not well understood, however, epigenetic mechanisms such as DNA methylation and histone modifications have been discussed to contribute as they are at the interface of environment and the genome. Moreover, developmental epigenetic programming has also been postulated to underlie differential vulnerability/resilience independent of genetic variation.
The present work comprises two projects investigating the effects of prenatal maternal restraint stress in 5-HTT deficient mice. In the first study, we examined to which extent previously observed changes in behavior and hippocampal gene expression of female 5-Htt+/- prenatally stressed (PS) offspring were associated with changes in DNA methylation patterns. Additionally, we investigated the expression of genes involved in myelination in hippocampus and amygdala of those animals using RT-qPCR. The genome-wide hippocampal DNA methylation screening was performed using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip® Mouse Promoter 1.0R arrays. In order to correlate individual gene-specific DNA methylation, mRNA expression and behavior, we used hippocampal DNA from the same mice as assessed before. 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a part of which were also differentially expressed. More specifically, we identified a differentially methylated region in the Myelin basic protein (Mbp) gene, which was associated with Mbp expression in a 5-Htt-, PS- and 5-Htt x PS-dependent manner. Subsequent fine-mapping linked the methylation status of two specific CpG sites in this region to Mbp expression and anxiety-related behavior. We furthermore found that not only the expression of Mbp but of large gene set associated with myelination was affected by a 5-Htt x PS interaction in a brain-region specific manner. In conclusion, hippocampal DNA methylation patterns and expression profiles of female PS 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are associated with changes in gene promoter methylation, and processes associated with myelination contribute to the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction.
In the second study, we aimed at investing the molecular substrates underlying resilience to PS. For this purpose, we exposed 5-Htt+/+ dams to the same restraint stress paradigm and investigated the effects of PS on depression- and anxiety-like behavior and corticosterone (CORT) secretion at baseline and after acute restraint stress in female 5-Htt+/+ and 5-Htt+/- offspring. We found that PS affected the offspring’s social behavior in a negative manner. When specifically examining those PS animals, we grouped the PS offspring of each genotype into a social, resilient and an unsocial, vulnerable group. While anxiety-like behavior in the EPM was reduced in unsocial, but not social, PS 5-Htt+/+ animals when compared to controls, this pattern could not be found in animals of the other genotype, indicating that social anxiety and state anxiety in the EPM were independent of each other. We then assessed genome-wide hippocampal gene expression profiles using mRNA sequencing in order to identify pathways and gene ontology (GO) terms enriched due to 5-Htt genotype (G), PS exposure (E) and their interaction (GxE) as well as enriched in social, but not unsocial, PS offspring, and vice versa. Numerous genes were affected by 5-Htt genotype, PS and most of all a GxE-interaction. Enrichment analysis using enrichr identified that the genotype affected mitochondrial respiration, while GxE-interaction-affected processes associated primarily with myelination and chromatin remodeling. We furthermore found that 5-Htt+/- mice showed profound expression changes of numerous genes in a genomic region located 10 mio kb upstream of the 5 Htt locus on the same chromosome. When looking at social vs. unsocial mice, we found that a much higher number of genes was regulated in 5 Htt+/- animals than in 5-Htt+/+ animals, reflecting the impact of GxE-interaction. Double the number of genes was regulated in social PS vs. control mice when compared to unsocial PS vs. control in both genotypes, suggesting that the successful adaption to PS might have required more active processes from the social group than the reaction to PS from the unsocial group. This notion is supported by the up-regulation of mitochondrial respiration in social, but not in unsocial, PS 5-Htt+/- mice when compared to controls, as those animals might have been able to raise energy resources the unsocial group was not. Next to this, processes associated with myelination seemed to be down-regulated in social 5-Htt+/- mice, but not in unsocial animals, when compared to controls. Taken together, PS exposure affected sociability and anxiety-like behavior dependent on the 5-Htt genotype in female offspring. Processes associated with myelination and epigenetic mechanisms involved in chromatin remodeling seemed be affected in a GxE-dependent manner in the hippocampus of these offspring. Our transcriptome data furthermore suggest that mitochondrial respiration and, with this, energy metabolism might be altered in 5-Htt+/- offspring when compared to 5-Htt+/+ offspring. Moreover, myelination and mitochondrial respiration might contribute to resilience towards PS exposure in 5-Htt+/- offspring, possibly by affecting brain connectivity and energy capabilities.
In dieser Arbeit wurden Einzelmolekültechniken zur Untersuchung von G-Protein-gekoppelten Rezeptoren (GPCR) und G-Proteinen in der Zellmembran lebender Zellen etabliert und angewendet. GPCR stellen die größte Familie membrangebundener Rezeptoren dar und leiten Signale über heterotrimere G-Proteine in das Zellinnere weiter. Auch wenn jüngst sowohl inaktive, als auch aktive Konformationen von GPCR und G-Proteinen mittels Röntgenstrukturanalyse aufgelöst werden konnten, sind die Dynamiken ihrer Aktivierung und Deaktivierung bisher nur bruchstückhaft bekannt. In der Vergangenheit wurden die Schritte der Signalkaskade, beginnend mit der Bindung des Rezeptorliganden bis hin zur Bildung von sekundären Botenstoffen, erfolgreich mit Fluoreszenz-Resonanz-Energie-Transfer-Techniken aufgeklärt. Diesen experimentell bestimmten Aktivierungszeiten stehen Daten aus Modellierungsstudien gegenüber, die sehr viel schnellere Konformationsänderungen vorhersagen, welche bereits in Studien mittels Kernspinresonanzspektroskopie nachgewiesen werden konnten. Folglich ist anzunehmen, dass die Zeitdomäne, innerhalb der die Aktivierung der GPCR stattfindet, sehr breit gefächert ist.
Ein Ziel der vorliegenden Arbeit war es, diese mehrere Größenordnungen umfassenden Zeitskalen der GPCR-Aktivierung, welche in der Literatur beschrieben werden, mittels bildgebender Einzelmolekülverfolgung (SPT) und Fluoreszenz-Korrelations-Spektroskopie (FCS) zu untersuchen. Beide Verfahren liefern durch Einzelmolekülspuren oder Korrelationskurven eine Art Fingerabdruck des dynamischen Verhaltens des untersuchten Systems, was jeweils mit Vor- und Nachteilen verbunden ist. Die Stärke der Techniken zeigte sich bei dem vorliegenden Projekt vor allem in ihrer Kombination: Die klassische FCS bietet die Möglichkeit, Dynamiken über einen weiten Zeitraum von Mikrosekunden bis Sekunden auszuwerten, allerdings nur innerhalb eines kleinen, optisch definierten Detektionsvolumens. Die bildgebende Einzelmolekülverfolgung liefert hingegen ein großes Sichtfeld und ermöglicht somit die parallele Analyse vieler Einzelmolekülereignisse über die Zelle verteilt, jedoch auf Kosten der Zeitauflösung.
Durch die Anwendung von SPT und FCS konnte in dieser Arbeit ein Zeitbereich der Rezeptor- (und G-Protein-) Dynamiken von Mikrosekunden bis Sekunden gefunden und diskutiert werden. Um die selektive Anregung der Plasmamembran zu gewährleisten, wurde die Interne Totalreflexionsfluoreszenzanregung verwendet. Diese eignet sich ideal als Grundlage für die spätere Analyse mittels SPT und FCS, welche komplementär nutzbar sind und mit dem gleichen zellulären Assay und unter Verwendung der gleichen Fluoreszenzmarker betrieben werden können.
Die Studie am Beispiel der α2A- und β2-adrenergen Rezeptoren sowie des Gαi1-Proteins demonstrierte das enorme Potential dieser Einzelmolekültechniken für die Untersuchung von GPCR und skizziert die Komplexität deren Dynamik, wie sie auch durch neueste Modellierungsstudien vorhergesagt wird.
Effect of cytokine inhibition on peripheral memory B cells in patients with Rheumatoid arthtritis
(2015)
Objective: Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory autoimmune disease. Enhanced B cell activity has been proposed in the pathogenesis of RA along with different pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), critically involved in chronic inflammation. Biological agents targeting these cytokines IL-6 and TNF-α have considerably advanced treatment of autoimmunity. Enhanced B cell activity, particularly memory B cells gained particularly interest in evaluating response during therapies from biologics. Human peripheral memory B cells can be distinguished by the phenotypic expression of CD27 and IgD defining three major B cell subpopulations: CD27+IgD+ pre-switch, CD27+IgD- post-switch and CD27-IgD- double negative (DN) memory B cells. Therefore, we analyzed different memory populations during cytokine inhibition by using tocilizumab (anti-IL-6R, TCZ) and adalimumab (anti-TNF-α, ADA), with focus on DN B cells Suspended. DN B cells lacking the conventional memory marker CD27, but due to their mutational Ig repertoire (IgR) considered in the memory compartment. However, only scare data are available for this DN subpopulation in RA.
Methods: Phenotype analysis of activation markers (CD95 and ki-67) of B cell and their subsets were compared in RA patients (median age ~56 years) and in HD. DN memory B cells were phenotypically analyzed from RA patients during IL-6R or TNF-α inhibition at baseline week 12, week 24 and 1 year. Single B cell PCR approach was used to study Ig- receptors VH genes and isotype specific genes. Nonparametric Wilcoxon matched pair test and Mann-Whitney U test was used for statistical analysis by using GraphPadPrism 5. Univariate logistic regression was used to calculate odd ratios and correlation using Pearson r using SPSS statistics 22.
Results: Surface and intracellular staining of B cells showed a significantly higher percentage of CD95 and ki-67 expressions in RA, which was highest in post-switch memory B cells followed by pre-switch and DN memory B cells. During cytokines (IL-6R & TNF-α) inhibition, both CD95 and ki-67 expression were significantly reduced at week 12 and 24 along with reduction in their clinical parameters like DAS28, CRP, ESR. Furthermore, the phenotypic analysis in 107 RA patients and 49 healthy donors (HD) showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgA, IgG and IgM expressing cells with a clear dominance of IgG+ cells. Pre-therapy analysis of rearranged IgR sequences from patients (n=9) revealed that DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-alpha) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (p < 0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (p < 0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (p < 0.05) at week 12 and week 24 during TCZ. Patients with a good European league against rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (p = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (p = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05-2.06).
Conclusion: Both anti-TNF-α and anti-IL-6R could reduce higher B cell activity and improve disease activity tremendously in RA patients. The heterogeneous DN B cell compartment is expanded in RA and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.
Bei der Behandlung solider Tumoren spielen systemisch verabreichte Chemotherapeutika eine wich- tige Rolle. Allerdings akkumulieren diese Therapeutika besser in normalem Gewebe als in Tumoren. Als Ursache für diesen unzureichenden Transport von Medikamenten in den Tumor wurde bisher vor allem die dysfunktionale Tumorvaskulatur diskutiert. Diese befindet sich in einem chaotischen und unreifen Zustand ohne ausreichende Bedeckung der Gefäße mit stabilisierenden Perizyten. Aus dem Zustand der Vaskulatur resultierend erreichen Medikamente den Tumor nur in geringem Ausmaß und werden dort heterogen verteilt. Als Grund für den Zustand der Vaskulatur wur- de ein großer Überschuss an pro-angiogenetischen Faktoren im Tumor ausgemacht. Durch eine anti-angiogenetische Behandlung konnte in präklinischen Modellen für einen gewissen Zeitraum die Tumorvaskulatur „normalisiert“ werden. Dies zeichnete sich vor allem durch Veränderung von zwei wichtigen Parametern für die Medikamenteneinbringung aus: zum Einen kommt es zu einer Reduktion der Gefäßdichte. Zum Anderen zu einer Reifung der Blutgefäße. In einem Teil von Pati- enten scheint dabei der Effekt der Gefäßverbesserung zu überwiegen und es kann eine verbesserte Perfusion detektiert werden. Mutmaßlich führt dies auch zu einer verbesserten Einbringung von Therapeutika in den Tumor und so zu einer erhöhten Effizienz der Therapie. In einem weiteren Teil der Patienten scheint jedoch der Effekt der Gefäßreduktion zu überwiegen und die detektierte Perfusion im Tumor wird durch die Behandlung verringert.
Das in dieser Arbeit verwendete MT6-Fibrosarkom-Modell reagierte auf eine anti-angiogenetische Therapie nicht mit einer sonst in murinen Modellen beobachteten Wachstumsreduktion. Die- se ermöglichte eine so bisher nicht mögliche Untersuchung der sekundären Effekte einer anti- angiogenetischen Therapie wie die Medikamenteneinbringung in den Tumor. Die Vaskulatur in MT6-Tumoren zeigte dabei nach einer anti-angiogenetischen Vorbehandlung, die erwarteten Merk-male einer „normalisierten“ Vaskulatur wie eine Reduktion der Gefäßdichte bei gleichzeitiger Rei- fung der verbleibenden Gefäße. Dies führte jedoch nicht zu einer verbesserten Effizienz einer subsequenten Chemotherapie. Durch Vergleich mit einem weiteren Tumor-Modell, dem 4T1-Modell für ein metastasierendes Mammakarzinom, konnten signifikante Unterschiede im Gefäßbild beider Modelle ausgeschlossen werden. Durch mikroskopische Methoden konnte dabei beobachtet werden, dass die Diffusion von Medikamenten aus den Blutgefäßen des MT6-Modells im Vergleich zum 4T1-Modell verringert war. Weitere Untersuchungen deuten auf eine Differenz in der Qualität der extrazellulären Matrix der verwendeten Tumor-Modelle. Durch mRNA-Expressionsanalysen konnte die Enzymfamilie der Lysyloxidasen als mögliche Ursache für diesen Diffusionsunterschied identi- fiziert werden. Lysyloxidasen katalysieren vor allem die Quervernetzung von Proteinen der Extra- zellulärmatrix. Im Weiteren konnte gezeigt werden, dass die Quervernetzung von Matrixproteinen durch Lysyloxidasen ursächlich für die Diffusions-Inhibierung kleiner Moleküle wie das Chemo- therapeutikum Doxorubicin sein kann. Durch spezifische Inhibition der Lysyloxidasen mittels des Inhibitors βAPN konnte diese Diffusions-Inhibition sowohl in vitro als auch im MT6-Tumor-Modell nahezu vollständig verhindert werden. Die hohe Aktivität von Lysyloxidasen im MT6-Modell stell- te allerdings kein Alleinstellungsmerkmal dieses Modells dar. In weiteren Untersuchungen konnte gezeigt werden, dass Lysyloxidasen in einer Vielzahl von murinen und humanen Tumorzelllinien überexprimiert wird. Die Inhibition von Lysyloxidasen durch βAPN konnte dabei in allen unter- suchten Modellen die Einbringung von Medikamenten in den Tumor erhöhen und könnte so eine sinnvolle adjuvante Maßnahme zur Verbesserung bestehender Chemotherapien darstellen.
Die bipolare Störung ist eine psychische Erkrankung, die sich durch wiederkehrende depressive und (hypo-) manische Phasen auszeichnet. Neben Stimmungsschwankungen leiden viele Patienten unter kognitiven Beeinträchtigungen, die nicht nur während akuter Episoden, sondern auch in der Remission, d.h. in euthymer Stimmungslage persistieren. Die vorliegende Arbeit beschäftigte sich mit den klinischen Korrelaten von kognitiven Defiziten und der Effektivität eines kognitiven Trainings bei bipolaren Patienten (BP). In der ersten Teilstudie wurde untersucht, wie sich die kognitive Leistung der Patienten von der akuten Phase bis zur Remission verändert. Dazu wurden 55 akut depressive und (hypo-) manische BP und 55 gesunde Kontrollpersonen wiederholt mit einer neuropsychologischen Testbatterie untersucht. 29 Patienten konnten nach mindestens 3-monatiger Remission erneut getestet werden. Die Ergebnisse zeigen, dass die akut kranken BP domänenübergreifend kognitive Störungen im Vergleich zu gesunden Kontrollen aufweisen, wobei die depressiven Patienten eher in der Verarbeitungsgeschwindigkeit, der Aufmerksamkeit und dem Gedächtnis beeinträchtigt waren. Die akut manischen Patienten hatten hingegen auffällige Defizite in den exekutiven Funktionen. Die Performanz der BP besserte sich zwar in der Remission, es waren aber weiterhin im Vergleich zu den Kontrollen Defizite in der psychomotorischen Geschwindigkeit, dem Arbeitsgedächtnis und dem verbalen Gedächtnis festzustellen. Es zeigte sich außerdem, dass die Verarbeitungsgeschwindigkeit, die Aufmerksamkeit und das verbale Gedächtnis in Zusammenhang mit subdepressiven Symptomen und Schlafstörungen standen, wohingegen die exekutiven Testmaße nicht mit diesen „State“-Faktoren assoziiert waren. Diese Ergebnisse lassen vermuten, dass die exekutiven Funktionen als Trait-Merkmale der bipolaren Störung in Frage kommen, wohingegen Aufmerksamkeit und Gedächtnis durch das Vorliegen von Residualsymptomen beeinträchtigt sind.
Ziel des zweiten Teils dieser Arbeit war es, eine kognitive Defizit- vs. Nondefizit Subgruppe innerhalb der BP zu identifizieren, um herauszufinden welche soziodemographischen oder krankheitsrelevanten Charakteristika mit kognitiven Störungen in Zusammenhang stehen. Dazu wurde die neuropsychologische Testleistung von 79 euthymen BP und 70 gesunden Kontrollen verglichen. Es zeigte sich erwartungsgemäß, dass die BP in der psychomotorischen Geschwindigkeit, der Aufmerksamkeit, dem Arbeitsgedächtnis, dem verbalen Gedächtnis, der Wortflüssigkeit und dem problemlösenden Denken trotz stabiler Remission signifikant schlechtere Leistungen erbrachten als die gesunden Kontrollen. Im Anschluss wurde die bipolare Stichprobe anhand ihrer Testleistung in eine Defizit- und eine Nondefizit Gruppe aufgeteilt. Die Ergebnisse zeigen, dass 54% der BP in allen Tests eine völlig normgerechte Leistung erbrachten. Die Studie bestätigte demnach, dass nicht alle Patienten kognitive Defizite aufweisen, sondern Subgruppen bestehen, die sich in verschiedenen Variablen voneinander unterscheiden: Die Defizit-Subgruppe berichtete signifikant mehr subdepressive Symptome und es lagen häufiger persistierenden Schlafstörungen und die Diagnose einer komorbiden Erkrankung vor (Angststörung, ADHS und Migräne). Zudem zeigte sich ein Zusammenhang zwischen Polypharmazie und kognitiven Defiziten. Diese Ergebnisse demonstrieren, dass ein Teil der kognitiven Störungen bei BP durch eine nicht vollständige Remission und sekundäre Symptome bedingt sind. Es ergab sich keine Assoziation zwischen kognitiver Leistung und krankheitsrelevanten Variablen, wie z.B. Anzahl der Phasen, Bipolar-Subtyp oder Ersterkrankungsalter. Diese Daten widersprechen zwar nicht der Hypothese, dass kognitive Störungen durch neurodegenerative Prozesse bedingt sind, sie weisen jedoch darauf hin, dass bei der bipolaren Störung häufig Residualsymptome vorliegen, welche im Rahmen von Studie als auch bei der therapeutischen Arbeit stärker als bisher berücksichtigt werden müssen.
In beiden Teilstudien zeigte sich zudem, dass kognitive Störungen mit einem reduzierten psychosozialen Funktionsniveaus in Verbindung stehen. Dieses Ergebnis steht in Einklang mit bisherigen Untersuchungen, die berichten, dass Patienten mit kognitiven Defiziten soziale und berufliche Einschränkungen aufweisen, die wiederum mit einem schlechteren Krankheitsverlauf assoziiert ist. Aufgrund dessen wurde von einigen Autoren vorgeschlagen, mit Hilfe spezieller Interventionen wie der kognitiven Remediation (KR) die geistigen Funktionen zu rehabilitieren. In der vorliegenden Interventionsstudie wurde deshalb der Frage nachgegangen, ob die neurokognitive Leistungsfähigkeit und das psychosoziale Funktionsniveau der bipolaren Stichprobe durch KR verbessert werden kann. Zudem sollte untersucht werden, inwiefern kognitives Training zu Veränderungen der präfrontalen Hirnaktivität führt. Dafür wurde vor und nach dem Training eine Messung mit der Methode der funktionellen Nahinfrarotspektroskopie (fNIRS) durchgeführt. Das 3-monatige KR-Programm bestand aus einem computerisierten kognitiven Training und der Vermittlung von kognitiven Skills im Rahmen von 12-wöchentlichen Gruppensitzungen. Im Anschluss an das Training wurden die Teilnehmer (26 bipolare und als Vergleichsgruppe 13 unipolare Patienten) im Rahmen einer Post-Messung wiederholt untersucht. Zudem wurde zum Vergleich eine Kontrollgruppe von 10 BP im Abstand von 3 Monaten untersucht, die keine Intervention, sondern die Standardbehandlung erhielt. Aufgrund zahlreicher Drop-Outs konnten am Ende des Erhebungszeitraums die Daten von 16 bipolaren und 10 unipolar depressiven Patienten ausgewertet werden. Die Trainingsteilnehmer erbrachten im Gegensatz zu der Kontrollgruppe signifikante Leistungssteigerungen in den Tests zur Erfassung der psychomotorischen Geschwindigkeit, dem Arbeitsgedächtnisses, dem verbalen Gedächtnis und dem problemlösenden Denken. Zudem zeigte sich nach dem Training eine Verbesserung des psychosozialen Funktionsniveaus und eine Reduktion der subdepressiven Symptomatik. Eine Veränderung der präfrontalen Hirnaktivierung konnte jedoch nicht verifiziert werden. Die Ergebnisse lassen demnach schlussfolgern, dass Patienten mit affektiven Störungen von einem kognitiven Training profitieren, wobei die damit einhergehenden funktionalen Veränderungen der Hirnaktivität in Studien mit größeren Stichproben untersucht werden müssen.
Im Neuroblastom ist die Amplifikation des MYCN-Gens, das für den Transkriptionsfaktor N-Myc kodiert, der klinisch bedeutendste Faktor für eine schlechte Prognose. Als Mitglied der onkogenen Myc-Familie induziert N-Myc die Expression von Genen, die in vielen biologischen Prozessen wie Metabolismus, Zellzyklusprogression, Zellwachstum und Apoptose eine wichtige Rolle spielen. Die Deregulation der MYCN-Expression führt zu einem charakteristischen Genexpressionsprofil und einem aggressiven Phenotyp in den Tumorzellen.
In normalen neuronalen Vorläuferzellen wird N-Myc gewöhnlich sehr schnell proteasomal abgebaut. Während der Mitose wird N-Myc an Serin 62 phosphoryliert. Diese Phosphorylierung dient als Erkennungssignal für die Kinase GSK3β, die die Phosphorylierung an Threonin 58 katalysiert. Das Phosphodegron wird von Fbxw7, einer Komponente des E3-Ubiquitinligase-Komplex SCFFbxw7, erkannt. Die anschließende Ubiquitinierung induziert den proteasomalen Abbau des Proteins. Die Reduktion der N-Myc–Proteinlevel ermöglicht den neuronalen Vorläuferzellen den Austritt aus dem Zellzyklus und führt zu einer terminalen Differenzierung.
In einem shRNA Screen konnte AURKA als essentielles Gen für die Proliferation MYCN-amplifizierter Neuroblastomzellen identifiziert werden. Eine Aurora-A–Depletion hatte jedoch keinen Einfluss auf das Wachstum nicht-amplifizierter Zellen.
Während dieser Doktorarbeit konnte gezeigt werden, dass Aurora-A speziell den Fbxw7-vermittelten Abbau verhindert und dadurch N-Myc stabilisiert. Für die Stabilisierung ist zwar die Interaktion der beiden Proteine von entscheidender Bedeutung, überraschenderweise spielt die Kinaseaktivität von Aurora-A jedoch keine Rolle.
Zwei spezifische Aurora-A–Inhibitoren, MLN8054 und MLN8237, sind allerdings in der Lage, nicht nur die Kinaseaktivität zu hemmen, sondern auch die N-Myc-Proteinlevel zu reduzieren. Beide Moleküle induzieren eine Konformationsänderung in der Kinasedomäne von Aurora-A. Diese ungewöhnliche strukturelle Veränderung hat zur Folge, dass der N-Myc/Aurora-A–Komplex dissoziiert und N-Myc mit Hilfe von Fbxw7 proteasomal abgebaut werden kann. In MYCN-amplifizierten Zellen führt diese Reduktion an N-Myc zu einem Zellzyklusarrest in der G1-Phase. Die in vitro Daten konnten in einem transgenen Maus-Modell für das MYCN-amplifizierte Neuroblastom bestätigt werden. Die Behandlung mit MLN8054 und MLN8237 führte in den Tumoren ebenfalls zu einer N-Myc-Reduktion. Darüber hinaus konnte ein prozentualer Anstieg an differenzierten Zellen, die vollständige Tumorregression in der Mehrzahl der Neuroblastome und eine gesteigerte Lebenserwartung beobachtet werden.
Insgesamt zeigen die in vitro und in vivo Daten, dass die spezifischen Aurora-A–Inhibitoren ein hohes therapeutisches Potential gegen das MYCN-amplifizierte Neuroblastom besitzen.
Structural and Biochemical Characterization of the GABA(A) Receptor Interacting Protein Muskelin
(2015)
In a study from 2011, the protein muskelin was described as a central coordinator of the retrograde transport of GABA(A) receptors in neurons. As muskelin governs the transport along actin filaments as well as microtubules, it might be the first representative of a novel class of regulators, which coordinate cargo transport across the borders of these two independent systems of transport paths and their associated motorproteins. To establish a basis for understanding the mode of operation of muskelin, the aim of this thesis was an in-depth biochemical and structural characterization of muskelin and its interaction with the GABA(A) receptor.
One focus of the work was the analysis of the oligomerization of muskelin. As could be demonstrated, the oligomerization is based on two independent interactions mediated by different domains of the protein: a known interaction of the N-terminal discoidin domain with the C-terminal portion, termed head-to-tail interaction, and a dimerization of the LisH motif in muskelin that was so far neglected in the literature. For the detailed studies of both binding events, the solution of a crystal structure of a fragment of muskelin, comprising the Discoidin domain and the LisH motif, was an important basis. The fragment crystallized as a dimer, with dimerization being mediated solely by the LisH motif. Biochemical analysis corroborated that the LisH motif in muskelin serves as a dimerization element, and, moreover, showed that the C-terminal domain of the protein substantially stabilizes this dimerization. In addition, the crystal structure revealed the molecular composition of the surface of the head in the head-to-tail interaction, namely the discoidin domain. This information enabled to map the amino acids contributing to binding, which showed that the binding site of the head-to-tail interaction coincides with the generic ligand binding site of the discoidin domain.
As part of the analyses, residues that are critical for LisH-dimerization and the head-to-tail binding, respectively, were identified, whose mutation specifically interfered with each of the interactions separately. These mutations allowed to investigate the interplay of these interactions during oligomerization. It could be shown that recombinant muskelin assembles into a tetramer to which both interactions, the LisH-dimerization and the head-to-tail binding, contribute independently. When one of the two interactions was disturbed, only a dimer mediated via the respective other interaction could be formed; when both interactions were disturbed, the protein was present as monomer. Furthermore, Frank Heisler in the group of Matthias Kneussel was able to show the drastic impact of an impaired LisH-dimerization on muskelin in cells using these mutations. Disturbing the LisH-dimerization led to a complete redistribution of the originally cytoplasmic muskelin to the nucleus which was accompanied by a severe impairment of its function during GABA(A) receptor transport. Following up on these results in an analysis of muskelin variants, for which alterations of the subcellular localization had been published earlier, the crucial influence of LisH-dimerization to the subcellular localization and thereby the role of muskelin in the cell was confirmed.
The biochemical studies of the interaction of muskelin and the alpha1 subunit of the GABA(A) receptor demonstrated a direct binding with an affinity in the low micromolar range, which is mediated primarily by the kelch repeat domain in muskelin. For the binding site on the GABA(A) receptor, it was confirmed that the thirteen most C-terminal residues of the intracellular domain are critical for the binding of muskelin. In accordance with the strong conservation of these residues among the alpha subunits of the GABA(A) receptor, it could be shown that an interaction with muskelin in vitro is also possible for the alpha2 and alpha5 subunits. Based on the comparison of the binding sites between the homologous subunits, tentative conclusions can be drawn about the details of the binding, which may serve as a starting point for follow-up studies.
This thesis thereby makes valuable contributions to the understanding of muskelin, in particular the significance of its oligomerization. It furthermore provides an experimental framework for future studies that address related topics, such as the characterization of other muskelin interaction partners, or the questions raised in this work.
Brain-computer interfaces (BCIs) could provide a muscle-independent communication channel to persons with severe paralysis by translating brain activity into device commands. As a means of communication, in particular BCIs based on event-related potentials (ERPs) as control signal have been researched. Most of these BCIs rely on visual stimulation and have been investigated with healthy participants in controlled laboratory environments. In proof-of-principle studies targeted end users gained control over BCI systems; however, these systems are not yet established as an assistive technology for persons who would most benefit from them. The main aim of this thesis is to advance the usability of ERP-BCIs for target users. To this end, five studies with BCIs have been conducted that enabled users to communicate by focusing their attention on external stimuli.
Two studies were conducted in order to demonstrate the advantages and to further improve the practical application of visual BCIs. In the first study, mental workload was experimentally manipulated during prolonged BCI operation. The study showed the robustness of the visual ERP-BCI since users maintained a satisfactory level of control despite constant distraction in the form of background noise. Moreover, neurophysiological markers that could potentially serve as indicators of high mental workload or fatigue were revealed. This is a first step towards future applications in which the BCI could adapt to the mental state of the user (e.g. pauses if high mental workload is detected to prevent false selections). In the second study, a head-mounted display (HMD), which assures that stimuli are presented in the field of view of the user, was evaluated. High accuracies and information transfer rates, similar to a conventional display, were achieved by healthy participants during a spelling task. Furthermore, a person in the locked-in state (LIS) gained control over the BCI using the HMD. The HMD might be particularly suited for initial communication attempts with persons in the LIS in situations, where mounting a conventional monitor is difficult or not feasible.
Visual ERP-BCIs could prove valuable for persons with residual control over eye muscles and sufficient vision. However, since a substantial number of target users have limited control over eye movements and/or visual impairments, BCIs based on non-visual modalities are required. Therefore, a main aspect of this thesis was to improve an auditory paradigm that should enable motor impaired users to spell by focusing attention on different tones. The two conducted studies revealed that healthy participants were able to achieve high spelling performance with the BCI already in the first session and stress the importance of the choice of the stimulus material. The employed natural tones resulted in an increase in performance compared to a previous study that used artificial tones as stimuli. Furthermore, three out of five users with a varying degree of motor impairments could gain control over the system within the five conducted sessions. Their performance increased significantly from the first to the fifth session - an effect not previously observed for visual ERP-BCIs. Hence, training is particularly important when testing auditory multiclass BCIs with potential users.
A prerequisite for user satisfaction is that the BCI technology matches user requirements. In this context, it is important to compare BCIs with already established assistive technology. Thus, the fifth study of this dissertation evaluated gaze dependent methods (EOG, eye tracking) as possible control signals for assistive technology and a binary auditory BCI with a person in the locked-in state. The study participant gained control over all tested systems and rated the ease of use of the BCI as the highest among the tested alternatives, but also rated it as the most tiring due to the high amount of attention that was needed for a simple selection. Further efforts are necessary to simplify operation of the BCI.
The involvement of end users in all steps of the design and development process of BCIs will increase the likelihood that they can eventually be used as assistive technology in daily life. The work presented in this thesis is a substantial contribution towards the goal of re-enabling communication to users who cannot rely on motor activity to convey their thoughts.
The honeybee Apis mellifera is a social insect well known for its complex behavior and the ability to learn tasks associated with central place foraging, such as visual navigation or to learn and remember odor-reward associations. Although its brain is smaller than 1mm² with only 8.2 x 105 neurons compared to ~ 20 x 109 in humans, bees still show amazing social, cognitive and learning skills. They express an age – related division of labor with nurse bees staying inside the hive and performing tasks like caring for the brood or cleaning, and foragers who collect food and water outside the hive. This challenges foragers with new responsibilities like sophisticated navigation skills to find and remember food sources, drastic changes in the sensory environment and to communicate new information to other bees. Associated with this plasticity of the behavior, the brain and especially the mushroom bodies (MBs) - sensory integration and association centers involved in learning and memory formation – undergo massive structural and functional neuronal alterations. Related to this background my thesis on one hand focuses on neuronal plasticity and underlying molecular mechanisms in the MBs that accompany the nurse – forager transition.
In the first part I investigated an endogenous and an internal factor that may contribute to the nurse - forager phenotype plasticity and the correlating changes in neuronal network in the MBs: sensory exposure (light) and juvenile hormone (JH). Young bees were precociously exposed to light and subsequently synaptic complexes (microglomeruli, MG) in the MBs or respectively hemolymph juvenile hormone (JH) levels were quantified. The results show that light input indeed triggered a significant decrease in MG density, and mass spectrometry JH detection revealed an increase in JH titer. Interestingly light stimulation in young bees (presumably nurse bees) triggered changes in MG density and JH levels comparable to natural foragers. This indicates that both sensory stimuli as well as the endocrine system may play a part in preparing bees for the behavioral transition to foraging.
Considering a connection between the JH levels and synaptic remodeling I used gene knockdown to disturb JH pathways and artificially increase the JH level. Even though the knockdown was successful, the results show that MG densities remained unchanged, showing no direct effect of JH on synaptic restructuring.
To find a potential mediator of structural synaptic plasticity I focused on the calcium-calmodulin-dependent protein kinase II (CaMKII) in the second part of my thesis. CaMKII is a protein known to be involved in neuronal and behavioral plasticity and also plays an important part in structural plasticity reorganizing synapses. Therefore it is an interesting candidate for molecular mechanisms underlying MG reorganization in the MBs in the honeybee. Corresponding to the high abundance of CaMKII in the learning center in vertebrates (hippocampus), CaMKII was shown to be enriched in the MBs of the honeybee. Here I first investigated the function of CaMKII in learning and memory formation as from vertebrate work CaMKII is known to be associated with the strengthening of synaptic connections inducing long term potentiation and memory formation. The experimental approach included manipulating CaMKII function using 2 different inhibitors and a specific siRNA to create a CaMKII knockdown phenotype. Afterwards bees were subjected to classical olfactory conditioning which is known to induce stable long-term memory. All bees showed normal learning curves and an intact memory acquisition, short-term and mid-term memory (1 hour retention). However, in all cases long-term memory formation was significantly disrupted (24 and 72 hour retention). These results suggests the necessity of functional CaMKII in the MBs for the induction of both early and late phases of long-term memory in honeybees. The neuronal and molecular bases underlying long-term memory and the resulting plasticity in behavior is key to understanding higher brain function and phenotype plasticity. In this context CaMKII may be an important mediator inducing structural synaptic and neuronal changes in the MB synaptic network.
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.
Untersuchung der Rolle von Rhodopsin 7 und Cryptochrom im Sehprozess von Drosophila melanogaster
(2015)
Ausgangspunkt für 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 Äuglein 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 hinzugefügt. Bis heute fehlt allerdings jeglicher experimentelle Beleg für 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 Fähigkeit 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 für eine Beeinträchtigung der lichtinduzierten Antwort der Rezeptorzellen im Komplexauge. So zeigen die Intensitäts-Response Kurven der ERG Rezeptorpotentialamplitude von rh7 Knockout-Fliegen unter Weißlicht niedriger und mittlerer Intensität nach einer anfänglichen Dunkeladaptation von 15min eine insgesamt, im Vergleich zur Kontrolle erhöhte Rezeptorpotentialamplitude. Der Verlauf dieser Kurven deutet außerdem darauf hin, dass die Zunahme der Rezeptorpotentialamplitude mit steigender Lichtintensität größer wird. Zudem
zeigt das Aktionsspektrum für die Rezeptorpotentialamplitude der rh7 Knockout-Fliegen, dass diese Empfindlichkeitszunahme im gesamten Bereich von 370-648nm auftritt. Diese Beeinträchtigung 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 Intensität die Lichtantwort in den Rezeptorzellen des Komplexauges in Abhängigkeit von Intensität und Adaptationszustand beeinflusst und dass dieser Einfluss scheinbar nicht durch Licht eines eng begrenzten Wellenlängenbereichs induziert wird. Des weiteren legt die Untersuchung des ERG Nachpotentials nahe, dass Rh7 möglicherweise für eine normale Beendigung der Lichtantwort benötigt wird. Die allgemeine Funktion von Rh7 als Photorezeptor in Drosophila sowie die Eigenschaften der endogenen Funktion von Rh7 werden diskutiert.
Unabhängig davon wird in der vorliegenden Arbeit auch gezeigt, dass Fliegen ohne CRY zwar nach 15-minütiger, nicht jedoch nach 1-minütiger Dunkeladaptation bei Belichtung mit Weißlicht niedriger Intensität eine insgesamt geringere ERG Rezeptorpotentialamplitude aufweisen. Dies könnte auf eine Beeinträchtigung der Dunkeladaptationsprozesse bei Abwesenheit von CRY hindeuten.