WNT Signaling in Drosophila Neuromuscular Junctions

The Wnt -Wingless (Wg) in Drosophila- signaling is an evolutionary conserved, fundamental signal transduction pathway in animals, having a crucial role in early developmental processes. In the adult animal the Wnt cascade is mainly shut off; aberrant activation leads to cancer. One physiological exception in the adult animal is the activation of Wnt signaling in the nervous system. In the present work, we investigated Wg signaling in the Drosophila neuromuscular junctions (NMJs). The fly NMJs closely resemble the glutamatergic synapses in the mammalian central nervous system and serves as a model system to investigate the mechanism of synapse formation and stability. We demonstrate that the trimeric G-protein Go has a fundamental role in the presynaptic cell in the NMJ. It is implicated in the presynaptic Wg pathway, acting downstream of the ligand Wg and its receptor Frizzled2 (Fz2). Furthermore, we prove that the presynaptic Wg-Fz2-Gαo pathway is essential for correct NMJ formation. The neuronal protein Ankyrin2 (Ank2) localizes to the NMJ and has so far been considered to be a static player in NMJ formation, linking the plasma membrane to the cytoskeleton. We identify Ank2 as a direct target of Gαo. The physical and genetic interaction of Gαo with Ank2 represents a novel branch of the presynaptic Wg pathway, regulating the microtubule cytoskeleton in NMJ formation, jointly with the previously established Futsch-dependent branch, which controls microtubule stability downstream of the kinase Sgg (the homolog of GSK3ß). We moreover demonstrate that the Gαo-Ankyrin interaction to regulate the cytoskeleton is conserved in mammalian neuronal cells. Our findings therefore provide a novel, universally valid regulation of the cytoskeleton in the nervous system. Aberrant inactivation of the neuronal Wnt pathway is believed to be involved in the pathogenesis of the Aß peptide in Alzheimer's disease (AD). We modeled AD in Drosophila by expressing Aß42 in the nervous system and in the eye. Neuronal expression drastically shortens the life span of the flies. We prove that this effect depends on the expression specifically in glutamatergic neurons. However, Aß42 does not induce any morphological changes in the NMJ; therefore this synapse is not suitable to study the mechanism of Aß42 induced neurotoxicity. We furthermore demonstrate that genetic activation of the Wnt pathway does not rescue the Aß42 induced phenotypes - in opposition to the dominating view in the field. These results advice caution when interpreting data on the potential interaction of Wnt signaling and AD in other models. -- La voie de signalisation Wnt (Wingless (Wg) chez la drosophile) est conservée dans l'évolution et fondamentale pour le développement des animaux. Cette signalisation est normalement inactive chez l'animal adulte; une activation anormale peut provoquer le cancer. Or, ceci n'est pas le cas dans le système nerveux des adultes. La présente thèse avait pour but d'analyser le rôle de la voie de signalisation Wingless dans la plaque motrice de Drosophila melanogaster. En effet, cette plaque ressemble fortement aux synapses glutaminergiques du système nerveux central des mammifères et procure ainsi un bon modèle pour l'étude des mécanismes impliqués dans la formation et la stabilisation des synapses. Nos résultats montrent que la protéine trimérique Go joue un rôle fondamental dans la fonction de la cellule présynaptique de la plaque motrice. Go est en effet impliqué dans la voie de signalisation Wg, opérant en aval du ligand Wg et de son récepteur Frizzled2. Nous avons pu démontrer que cette voie de signalisation Wg-Fz2-Gαo est essentielle pour le bon développement et le fonctionnement de la plaque motrice. Fait intéressant, nous avons montré que la protéine neuronale Ankyrin2 (Ank2), qui est connue pour jouer un rôle statique en liant la membrane plasmique au cytosquelette dans la plaque motrice, est une cible directe de Gαo. L'interaction physique et génétique entre Gαo et Ank2 constitue ainsi une bifurcation de la voie de signalisation présynaptique Wg. Cette voie régule le cytosquelette des microtubules en coopération avec la branche liée à la protéine Futsch. Cette protéine est l'homologue de la protéine liant les microtubules MAP1B des mammifères et contrôle la stabilité des microtubules opérant en aval de la kinase Sgg (l'homologue de GSK3ß). De plus, la régulation du cytosquelette par l'interaction entre Gαo et Ankyrin est conservée chez les mammifères. Dans leur ensemble, nos résultats ont permis d'identifier un nouveau mode de régulation du cytosquelette dans le système nerveux, probablement valable de manière universelle. La voie de signalisation Wnt est soupçonnée d'être impliquée dans la toxicité provoquée par le peptide Aß dans le cadre de la maladie d'Alzheimer. Nous avons tenté de modéliser la maladie chez la drosophile en exprimant Aß42 spécifiquement dans le cerveau. Cette expérience a montré que l'expression neuronale d'Aß42 réduit la durée de vie des mouches de manière significative par un mécanisme impliquant les cellules glutamatergiques. Par contre, aucune modification morphologique n'est provoquée par Aß42 dans les plaques motrices glutamatergiques. Ces résultats montrent que ce modèle de Drosophile n'est pas adéquat pour l'étude de la maladie d'Alzheimer. De plus, l'activation génétique de la voie de signalisation Wg n'a pas réussi à restaurer les phénotypes de survie ou ceux des yeux causés par Aß42. Ces résultats indiquent que l'implication de la voie de signalisation Wg dans la maladie d'Alzheimer doit être considérée avec prudence

Entwicklung eines Prozesses zur schlickerbasierten additiven Fertigung von Hochleistungskeramik

Die Herstellung dreidimensionaler keramischer Bauteile mit etablierten schlickerbasierten additiven Fertigungsmethoden für Keramik, wie der Stereolithographie, ist bislang sehr zeitaufwändig. Dies ist vor allem den verwendeten Suspensionen mit einem hohen Anteil an organischen Hilfsstoffen geschuldet. Der dadurch benötigte zeitaufwendige Entbinderungsschritt hat zur Folge, dass nur dünnwandige Bauteile ökonomisch hergestellt werden können. Das in dieser Arbeit untersuchte und optimierte neue schlickerbasierte additive Fertigungsverfahren Laser Induzierter Schlickerguss (LIS) ermöglicht hingegen einen Aufbau von komplexen Bauteilen unter Verwendung von konventionellen Schlicker mit einem geringen Organikanteil, die den aufwendigen Entbinderungsschritt überflüssig machen. Hierdurch kann dieses additive Fertigungsverfahren einfacher in die keramische Prozesskette eingebettet werden. Bei additiven Fertigungsverfahren werden computergenerierte Konstruktionsdaten des gewünschten Bauteils schichtweise durch ein abwechselndes Wiederholen von Schichtauftrag und Belichtungsschritt aufgebaut. In dieser Arbeit wird gezeigt, dass die gewünschte Geometrie im Belichtungsschritt durch ein lokales, selektives Trocknen einer Schlickerschicht mit einem Laser verfestigt werden kann und damit keramische Grünkörper hergestellt werden können. Anhand von Experimenten mit keramischen Schlickern, in diesem Fall Siliziumnitrid, wurden unterschiedliche Arten der Laser-Belichtung und des lokalen Trocknens untersucht. Es konnte gezeigt werden, dass einfache keramische Grünkörper durch das Trocknen mittels eines fokussierten Laserstrahls aufgebaut werden können. Es lassen sich auf der Schlickerschicht präzise beliebige Geometrien abbilden. Die in den Versuchen realisierten Strukturen verändern sich in ihrer Geometrie in z-Richtung nicht, das heißt es wurden nur 2,5D-Strukturen aufgebaut. Das Herauslösen der Strukturen aus dem sie umgebenden Schlicker war jedoch aufwendig und induzierte Fehler wie Risse. In einer Weiterentwicklung wurde ein defokussierter Laserstrahl zur lokalen schichtweisen Trocknung eingesetzt, der die Oberfläche der gewünschten Geometrie erhitzte. Durch Optimierung des Prozesses ist es möglich, aus typischen Gießschlickern durch einen lokalen laserunterstützten Trocknungsprozess Grünkörper herzustellen. Damit war es erstmals erfolgreich möglich verschiedenen Geometrien wie Dreiecke und auch Bauteile mit größerer Komplexität, wie zum Beispiel Strukturen mit Überhängen, aufzubauen. Im Vergleich zu anderen Verfahren ist die verwendete Schichtstärke von 400 µm recht hoch, was eine vergleichsweise hohe Aufbaurate ermöglicht. Die mechanischen Eigenschaften der ersten mit diesem Verfahren hergestellten gesinterten Si3N4- Bauteile erreichen mit einer Biegefestigkeit von 275 MPa nicht die Werte von Si3N4-Bauteilen fertig entwickelter Verfahren wie beispielsweise Stereolithographie, sind aber besser als kommerziell erhältliche additive gefertigte Bauteile anderer Keramiken. Das Materialportfolio des Laser Induzierten Schlickergusses wurde anschließend auf die Baustoffe erweitert und Versuche mit alternativen Bindemitteln, den alkali-aktivierten Materialien durchgeführt. Mit Lithiumaluminat wurde ein alkali-aktiviertes Material gefunden, mit dem komplexe Geometrien, aufgebaut wurden. Die mechanische Charakterisierung der Bauteile ergab eine Druckfestigkeit von 49 MPa und eine Biegefestigkeit von 12 MPa. Die erhaltenen mechanischen Eigenschaften sind vergleichbar mit konventionell hergestellten alkali-aktivierten Materialien. Durch diese Arbeit konnte gezeigt werden, dass der Laser Induzierte Schlickerguss ein vielversprechendes neues Verfahren für die additive Fertigung istThe production of three-dimensional ceramic components using established slurry-based additive manufacturing methods for ceramics, such as stereolithography, has so far been very time-consuming. This is mainly due to the suspensions used, which contain a high proportion of organic additives. Therefore, the time-consuming debinding step required, results in the fact that only thin-walled components can be produced economically. This work investigates and optimizes the new slurry-based additive manufacturing process Laser Induced Slip Casting (LIS), which makes it possible to build complex components using conventional slurries with a low organic content, thus eliminating the need for the time-consuming debinding step. As a result, this additive manufacturing process can be more easily embedded in the ceramic process chain. In additive manufacturing processes, computer-generated design data of the desired component is built up layer by layer by alternately repeating the layer deposition and illumination step. In this work, it is shown that the desired geometry can be solidified in the illumination step by a local selective drying of a slip layer with a laser and thereby ceramic green bodies can be fabricated. Based on experiments with ceramic slips, in this case silicon nitride, different types of laser exposure and local drying were investigated. It was shown that simple ceramic green bodies can be built by drying using a focused laser beam. On the slip layer, precisely arbitrary geometries can be illuminated. The structures realized in the experiments do not change in their geometry in the z-direction, i.e. only 2.5D structures were built. However, extracting the structures from the surrounding slip was time consuming and induced defects such as cracks. In a further development, a defocused laser beam was used for local layer-by-layer drying, which heated the surface of the desired geometry. By optimizing the process, it is possible to produce green bodies from typical casting slurries by a local laser-assisted drying process. This made it possible for the first time to successfully build up different geometries such as triangles and also components with greater complexity, such as structures with overhangs. Compared to other processes, the layer thickness of 400 µm used is quite high, which enables a comparatively high build-up rate. The mechanical properties of the first sintered Si3N4 components produced using this process, with a flexural strength of 275 MPa, do not reach the values of Si3N4 components of fully developed processes such as stereolithography, but are better than commercially available additively manufactured components of other ceramics. The Laser Induced Slip Casting material portfolio was then expanded to include construction materials and experiments were conducted with alternative binders, alkali-activated materials. With lithium aluminate, an alkali-activated material was found with which complex geometries could be built. The mechanical characterization of the components showed a compressive strength of 49 MPa and a flexural strength of 12 MPa. The mechanical properties obtained are comparable to conventionally produced alkali-activated materials. Through this work, it was demonstrated that Laser Induced Slip Casting is a promising new process for additive manufacturing

How have Patients\u27 Experiences of Cancer Care Been Linked to Survival? A Systematic Review

Patient experience of care remains an important indicator of health care quality. Although studies show care experiences are associated with health outcomes for some conditions, the situation for cancer is unclear. New datasets on cancer patients in the US, Canada, and UK linking information on experiences and survival may enable an exploration of any association. This review aimed to identify studies linking any aspect of cancer patients’ experiences to their survival, to inform future analyses. We performed a systematic review using Medline database from January 1998 until March 2018. The settings included outpatient oncology clinics, primary care, hospitals, and cancer centres. The participants included adult patients from different demographic groups. 16 Studies (ten observational, two clinical trials, two qualitative, and two consecutive case series) describing a wide range of settings, populations and methods met our inclusion criteria. Patients’ experiences were mostly linked to survival in quantitative studies. Satisfaction with care and psychosocial support were the aspects of experience associated with survival. Although positive associations between experience and survival were more common, negative and lack of association findings were also reported. Overall, there was no agreement on the strength, direction of the association, and the type of measurements to use. In conclusion, a wide range of studies suggest a relationship may exist between patients’ experiences of cancer care and their survival. However, this relationship is complex and methodological challenging to study. Future research should carefully consider different aspects of patient experience and care and the way in which they may affect cancer survival

Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides.

Drosophila GoLoco motif-containing protein Pins is unusual in its highly efficient interaction with both GDP- and the GTP-loaded forms of the α-subunit of the heterotrimeric Go protein. We analysed the interactions of Gαo in its two nucleotide forms with GoLoco1-the first of the three GoLoco domains of Pins-and the possible structures of the resulting complexes, through combination of conventional fluorescence and FRET measurements as well as through molecular modelling. Our data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter. In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo. The sterical hindrance requiring such a rotation probably contributes to the guanine nucleotide exchange inhibitor activity of GoLoco1 and Pins as a whole. Our data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions

Survival of patients with small cell lung cancer undergoing lung resection in England, 1998–2009

Introduction: Chemotherapy or chemoradiotherapy is the recommended treatment for small cell lung cancer (SCLC), except in stage I disease where clinical guidelines state there may be a role for surgery based on favourable outcomes in case series. Evidence supporting adjuvant chemotherapy in resected SCLC is limited but this is widely offered. Methods: Data on 359 873 patients who were diagnosed with a first primary lung cancer in England between 1998 and 2009 were grouped according to histology (SCLC or non-SCLC (NSCLC)) and whether they underwent a surgical resection. We explored their survival using Kaplan-Meier analysis and Cox regression, adjusting for age, sex, comorbidity and socioeconomic status. Results: The survival of 465 patients with resected SCLC was lower than patients with resected NSCLC (5-year survival 31% and 45%, respectively), but much higher than patients of either group who were not resected (3%). The difference between resected SCLC and NSCLC diminished with time after surgery. Survival was superior for the subgroup of 198 'elective' SCLC cases where the diagnosis was most likely known before resection than for the subgroup of 267 'incidental' cases where the SCLC diagnosis was likely to have been made after resection. Conclusions: These data serve as a natural experiment testing the survival after surgical management of SCLC according to NSCLC principles. Patients with SCLC treated surgically for early stage disease may have survival outcomes that approach those of NSCLC, supporting the emerging clinical practice of offering surgical resection to selected patients with SCLC

Role of Comorbidity on Survival after Radiotherapy and Chemotherapy for Nonsurgically Treated Lung Cancer

BACKGROUND: Comorbidity, such as diseases of the cardiovascular, pulmonary, and other systems, may influence prognosis in lung cancer and complicate its treatment. The performance status of patients, which is a known prognostic marker, may also be influenced by comorbidity. Due to the close link between tobacco smoking and lung cancer, and because lung cancer is often diagnosed in advanced ages (median age at diagnosis in Denmark is 70 years), comorbidity is present in a large proportion of lung cancer patients. METHODS: Patients with any stage lung cancer who did not have surgical treatment were identified in the Danish Lung Cancer Registry. Danish Lung Cancer Registry collects data from clinical departments, the Danish Cancer Registry, Danish National Patient Registry, and the Central Population Register. A total of 20,552 patients diagnosed with lung cancer in 2005 to 2011 were identified. Comorbidity data were extracted from the Danish National Patient Registry, which is a register of all in- and outpatient visits to hospitals in Denmark. By record linkage, lung cancer patients who had previously been diagnosed with comorbid conditions were assigned a Charlson comorbidity index. Initial cancer treatment was categorized as chemotherapy, chemoradiation, radiotherapy, or no therapy. Data on Charlson comorbidity index, performance status, age, sex, stage, pulmonary function (forced expiratory volume in 1 second), histology, and type of initial treatment (if any) were included in univariable and multivariable Cox proportional hazard analyses. RESULTS: Treatment rates for chemotherapy and chemoradiation declined with increasing comorbidity and in particular increasing age. Women received treatment more often than men. In a univariable analysis of all patients combined, stage, performance status, age, sex, lung function, and comorbidity were all associated with survival. Apart from excess mortality among patients with unspecified histological subtypes (hazard ratio), there was no clear difference between the specified subtypes. When adjusting for the other factors, particularly age, sex, performance status, and stage proved to be robust while risk estimates for comorbidity were attenuated somewhat. When grouped by the three types of cancer treatment or no treatment, there was no influence of comorbidity on radiation therapy and modest influence on survival after chemotherapy and chemoradiation. In contrast, age remained a strong negative prognosticator after multivariate adjustment as did stage and performance status. CONCLUSION: Comorbidity has a limited effect on survival and only for patients treated with chemotherapy. It is rather the performance of the patient at diagnosis than the medical history that prognosticates survival in this patient group

Robustness of target dose coverage to motion uncertainties for scanned carbon ion beam tracking therapy of moving tumors

Beam tracking with scanned carbon ion radiotherapy achieves highly conformal target dose by steering carbon pencil beams to follow moving tumors using real-time magnetic deflection and range modulation. The purpose of this study was to evaluate the robustness of target dose coverage from beam tracking in light of positional uncertainties of moving targets and beams. To accomplish this, we simulated beam tracking for moving targets in both water phantoms and a sample of lung cancer patients using a research treatment planning system. We modeled various deviations from perfect tracking that could arise due to uncertainty in organ motion and limited precision of a scanned ion beam tracking system. We also investigated the effects of interfractional changes in organ motion on target dose coverage by simulating a complete course of treatment using serial (weekly) 4DCTs from six lung cancer patients. For perfect tracking of moving targets, we found that target dose coverage was high (V¯95 was 94.8% for phantoms and 94.3% for lung cancer patients, respectively) but sensitive to changes in the phase of respiration at the start of treatment and to the respiratory period. Phase delays in tracking the moving targets led to large degradation of target dose coverage (up to 22% drop for a 15° delay). Sensitivity to technical uncertainties in beam tracking delivery was minimal for a lung cancer case. However, interfractional changes in anatomy and organ motion led to large decreases in target dose coverage (target coverage dropped approximately 8% due to anatomy and motion changes after 1 week). Our findings provide a better understand of the importance of each of these uncertainties for beam tracking with scanned carbon ion therapy and can be used to inform the design of future scanned ion beam tracking systems

Social differences in lung cancer management and survival in South East England: a cohort study

OBJECTIVE: To examine possible social variations in lung cancer survival and assess if any such gradients can be attributed to social differences in comorbidity, stage at diagnosis or treatment. DESIGN: Population-based cohort identified in the Thames Cancer Registry. SETTING: South East England. PARTICIPANTS: 15 582 lung cancer patients diagnosed between 2006 and 2008. MAIN OUTCOME MEASURES: Stage at diagnosis, surgery, radiotherapy, chemotherapy and survival. RESULTS: The likelihood of being diagnosed as having early-stage disease did not vary by socioeconomic quintiles (p=0.58). In early-stage non-small-cell lung cancer, the likelihood of undergoing surgery was lowest in the most deprived group. There were no socioeconomic differences in the likelihood of receiving radiotherapy in stage III disease, while in advanced disease and in small-cell lung cancer, receipt of chemotherapy differed over socioeconomic quintiles (p<0.01). In early-stage disease and following adjustment for confounders, the HR between the most deprived and the most affluent group was 1.24 (95% CI 0.98 to 1.56). Corresponding estimates in stage III and advanced disease or small-cell lung cancer were 1.16 (95% CI 1.01 to 1.34) and 1.12 (95% CI 1.05 to 1.20), respectively. In early-stage disease, the crude HR between the most deprived and the most affluent group was approximately 1.4 and constant through follow-up, while in patients with advanced disease or small-cell lung cancer, no difference was detectable after 3 months. CONCLUSION: We observed socioeconomic variations in management and survival in patients diagnosed as having lung cancer in South East England between 2006 and 2008, differences which could not fully be explained by social differences in stage at diagnosis, co-morbidity and treatment. The survival observed in the most affluent group should set the target for what is achievable for all lung cancer patients, managed in the same healthcare system