ß-Amyloid-vermittelte, Mitochondrien-abhängige Zelltodmechanismen in der Alzheimer Demenz

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterised by the presence of amyloid plaques, intracellular neurofibrillary tangles and pronounced cell death. The aim of this thesis was to investigate pathways involved in the Aß cascade of neurodegeneration. Since novel findings indicate that already this Aß species exerts neurotoxic effects long before hyperphosphorylated tau, neurofibrillary tangles and extracellular Aß plaques appear, the investigations were accomplished with specific regard to the effects of intracellular Aß. The Swedish double mutation in the APP gene results in six- to eightfold increased Aß production of both Aß1-40 and Aß1-42 compared to human wildtype APP cells (APPwt). Data obtained from PC12 cells indicate that it is possible to specifically increase the Aß load without enhancing APP expression levels. On the basis of these findings, it seemed possible to investigate dose-dependent effects of Aß in multiple experimental designs. These assay designs were created in order to mimick different in-vivo situations that are discussed to occur in AD patients: APPsw PC12 cells exhibit low physiological concentrations of Aß within picomolar range in contrast to APPsw HEK cells, expressing Aß levels within the nanomolar range. Of note, the APPsw HEK cells showed a specific and highly significant increase in the intracellular accumulation of insoluble Aß1-42. Moreover, an intracellular accumulation of Aß and APP was found in the mitochondria of the HEK APPsw cells suggesting a direct impact on mitochondrial function on these cells. This effect might finally lead to disturbances in the energy metabolism of the cell or to increased cell death. Furthermore, baseline g- and ß-secretase activity was assessed since these enzymes represent promising therapeutic targets to slow or halt the disease process. As expected, ß-secretase activity was significantly elevated in all APPsw cell lines. This might be due to the proximity of the Swedish double mutation next to the N-terminus of the Aß sequence. Interestingly, g-secretase activity was similarly increased in PC12 APPsw cells. In addition, the toxicity of different Aß species was investigated in SY5Y and PC12 cells with regard to their effect on cellular viability mirrored by mitochondrial activity using MTT assay. Here, it turned out that not monomers, but already dimers are neurotoxic correlates. Fibrillar Aß species showed the highest toxicity. In the next step, SY5Y cells forming endogenous, dimeric APP and Aß were investigated. In accordance with previous findings, these cells showed a decreased MTT reduction potential in comparison to APPwt and control SY5Y cells reflecting a decrease of cellular viability. The impaired energy metabolism of the cells was even more drastically mirrored by reduced baseline ATP levels. In the second part of this thesis, the expression and intracellular distribution of Bcl-2 family proteins and pro-apoptotic mitochondrial factors under baseline conditions and during oxidative stress were analyzed in the APPwt and APPsw bearing cells. The most prominent finding was the reduction of expression levels of the anti-apoptotic factor Bcl-xL in the cytosolic fractions of APPwt and APPsw PC12 cells. This might indicate that a lack of anti-apoptotic factors or their altered intracellular distribution, rather than an increase in caspase-dependent pro-apoptotic factors, could be responsible for the increased vulnerability of APPwt- and APPsw-transfected PC12 cells against oxidative stress. Since total Bcl-xL expression was unaffected in PC12 cells, in contrast to APPwt and APPsw-expressing SY5Y and HEK cells revealing significantly decreased Bcl-xL expression levels. Thus, alterations in Bcl-xL distribution seem to be an early event in the disease process. Increasing Bcl-xL expression might potentially be one promising strategy for AD modification. PC12 and HEK cells bearing APPsw or APPwt were treated with the potent g-secretase inhibitor DAPT. Of note, DAPT did not only efficiently block Aß production, but additionally led to an elevation of the MTT reduction potential, reflecting an increase in cellular viability. As another disease-modifying strategy, several efforts are undertaken to ameliorate AD-relevant symptoms by the treatment with nerve growth factor (NGF). Generally, it is known that substituted pyrimidines have modest growth-promoting effects. Here, KP544, a novel substituted pyrimidine, was characterised. This drug increased MTT reduction potential in terminally differentiated and undifferentiated PC12 cells. Furthermore, treatment with KP544 led to a reduction in Aß1-40 secretion. Thus, one may conclude that the target of KP544, GSK-3ß, represents a connecting link between the two main pathological hallmarks of AD and might thus be a very promising therapeutic target for AD.Alzheimer Demenz (AD) ist die häufigste neurodegenerative Erkrankung, welche durch einen kontinuierlichen Verlust der Gedächtnisleistung und der kognitiven Fähigkeiten charakterisiert ist. Das Ziel dieser Arbeit war die Untersuchung zellulärer Signaltransduktionswege, die an der Amyloidkaskade beteiligt sind. Im ersten Teil der Arbeit wurde die APP-Prozessierung und Aß-Produktion in verschiedenen in-vitro-Modellen untersucht. PC12-, HEK- und SY5Y-Zellen, die stabil mit der Schwedischen APP-Doppelmutation (APPsw) und mit wildtyp APP (APPwt) transfiziert wurden, dienten als Zellkulturmodelle. Die Schwedische Doppelmutation auf dem APP-Gen führt zu einer sechs- bis achtfach erhöhten Produktion von Aß1-40 und Aß1-42 im Vergleich zu APPwt-Zellen. Anhand der Daten aus PC12-Zellen konnte abgeleitet werden, dass durch diese Mutation die sekretierten Aß-Spiegel ohne Beeinflussung der APP-Expression gesteigert werden. Basierend auf diesem Ergebnis erscheint es zumindest möglich, dosisabhängige Aß-Effekte in verschiedenen experimentellen Ansätzen zu untersuchen. Diese Versuchsdesigns sollten möglichst authentisch verschiedene Stadien in Alzheimer-Gehirnen abbilden: Beispielsweise dient das APPsw PC12-Zellmodell als Modell für niedrige physiologische Aß-Konzentrationen im picomolaren Bereich, im Gegensatz zu APPsw HEK-Zellen, die Aß-Spiegel im nanomolaren Bereich zeigen. APPsw HEK-Zellen zeigten eine spezifische und hochsignifikante Anreicherung von unlöslichem Aß1-42 im intrazellulären Kompartiment. Außerdem wurde eine Akkumulation von Aß und APP in Mitochondrien von APPsw HEK-Zellen beobachtet. Dies läßt eine direkte Beeinflussung mitochondrialer Funktionen vermuten, welche letztendlich zu Störungen im Engergiehaushalt der Zelle und zu gesteigertem Zelltod führen könnte. Die Toxizität verschiedener Aß-Spezies wurde in SY5Y- und PC12-Zellen im Hinblick auf die zelluläre Viabilität, dargestellt durch die Messung der mitochondrialen Aktivität im MTT-Assay, untersucht. Hier zeigte sich, dass nicht Monomere, sondern Dimere erste toxische Korrelate darstellen, und dass fibrilläres Aß die höchste Toxizität aufweist. Im nächsten Schritt wurden SY5Y-Zellen untersucht, die dimeres APP und Aß bilden. In Übereinstimmung mit anderen Daten besaßen diese Zellen im Vergleich zu APPwt und Kontroll-Zellen ein erniedrigtes MTT-Reduktionspotential und somit eine reduzierte zelluläre Viabilität. Im zweiten Teil der Arbeit wurde die Expression und die intrazelluläre Verteilung von Proteinen der Bcl-2-Familie und pro-apoptotischer mitochondrialer Faktoren untersucht. Die Proteine wurden unter basalen Bedingungen und unter oxidativem Stress in APPwt und APPsw Zellen untersucht. Interessanterweise sind die Expressionsspiegel des anti-apoptotischen Faktors Bcl-xL in zytosolischen Fraktionen von APPwt und APPsw Zellen erniedrigt. Dies könnte darauf hinweisen, dass ein Mangel an anti-apoptotischen Faktoren oder ihre veränderte intrazelluläre Verteilung, eher noch als eine Erhöhung caspase-abhängiger, pro-apoptotischer Faktoren, für die gesteigerte Empfindlichkeit der APPwt- und APPsw-transfizierten Zellen gegenüber oxidativem Stress verantwortlich ist. Eine Erhöhung der Bcl-xL-Expression eine potentielle krankheitsmodulierende Strategie darstellen. PC12- und HEK-Zellen wurden mit dem potenten g-Sekretase-Inhibitor DAPT behandelt. Die Behandlung mit DAPT führte nicht nur zu einer effizienten Hemmung der Aß-Produktion, sondern auch zu einer Steigerung des MTT-Reduktionspotentials. Es wurden schon einige Versuche unternommen, AD-relevante Symptome durch die Behandlung mit Nervenwachstumsfaktor (NGF) zu behandeln. In dieser Arbeit wurde KP544, ein neuartiges substituiertes Pyrimidin, näher charakterisiert. Der genaue Wirkmechanismus von KP544 ist unbekannt. Die Behandlung mit KP544 führt zu einer Steigerung des MTT-Reduktionspotentials in undifferenzierten und differenzierten PC12-Zellen sowie zu einer verminderten Aß1-40-Sekretion. Zusätzlich wurde eine Inaktivierung der Glykogen-Synthase-Kinase-3ß (GSK-3ß) in Gegenwart von KP544 beobachtet. Dieser Befund ist sehr interessant, da GSK-3ß nicht nur die APP-Prozessierung, sondern auch die Hyperphosphorylierung von Tau, einem weiteren Merkmal der Alzheimer Demenz, moduliert. Daher könnte man schlussfolgern, dass das Target von KP544, GSK-3ß, ein Bindeglied zwischen den zwei Hauptmerkmalen der Alzheimer Demenz darstellt und somit ebenfalls ein sehr interessanter therapeutischer Angriffspunkt darstellt

Pasar Tunggal ASEAN 2015: Diplomasi Indonesia dan Penguatan Kapasitas Tenaga Kerja Terdidik

This paper argues that in order to prepare ASEAN Economic Community 2015, Indonesia should improve their competence in service sector through skilled labourempowerment. Within five priorities in service sector; health, e-commerce, tourism, flight service and logistics, the role of skilled labourplays an essential factor for the achievement of Indonesian national interest. In order to discuss this issue, the explanation about ‘top-down approach’ and ‘bottom-up approach’ of Indonesian policy toward ASEAN Single Market 2015, should be perceived. National Interest which has been formulated in the national level should be feasible to be implemented in the local level. Meanwhile, the society ought to plays their role as activator network to support Indonesian diplomacy. Indonesia can use their mechanism of diplomacy to empower the worker. Further, this paper will also try to elaborate the role of society as an important variable for Indonesian diplomacy

Analysis of the dynamic performance of self-excited induction generators employed in renewable energy generation

Incentives, such as the Feed-in-tariff are expected to lead to continuous increase in the deployment of Small Scale Embedded Generation (SSEG) in the distribution network. Self-Excited Induction Generators (SEIG) represent a significant segment of potential SSEG. The quality of SEIG output voltage magnitude and frequency is investigated in this paper to support the SEIG operation for different network operating conditions. The dynamic behaviour of the SEIG resulting from disconnection, reconnection from/to the grid and potential operation in islanding mode is studied in detail. The local load and reactive power supply are the key factors that determine the SEIG performance, as they have significant influence on the voltage and frequency change after disconnection from the grid. Hence, the aim of this work is to identify the optimum combination of the reactive power supply (essential for self excitation of the SEIG) and the active load (essential for balancing power generation and demand). This is required in order to support the SEIG operation after disconnection from the grid, during islanding and reconnection to the grid. The results show that the generator voltage and speed (frequency) can be controlled and maintained within the statuary limits. This will enable safe disconnection and reconnection of the SEIG from/to the grid and makes it easier to operate in islanding mode

Risk-minimal routes for emergency cars

The computation of an optimal route for given start and destination in a static transportation network is used in many applications of private route planning. In this work we focus on route planning for emergency cars, such as for example police, fire brigade and ambulance. In case of private route planning typical quantities to be minimized are travel time or route length. However, the idea of this paper is to minimize the risk of a travel time exceeding a certain limit. This is inspired by the fact that the emergency cars have to reach the destination within a legal time. We consider mainly two approaches. The first approach takes into account relevant information to determine the weight, i.e. the desirability of certain edges of a graph during the minimization procedure. One possible risk factor to be aware of would be a suddenly jammed single-lane road on which the emergency car has no chance to make use of the benefits of the siren for instance. The same holds for full-closure situations and railroad crossings. We present a catalogue of risk factors along with an appropriate algorithm for practical route planning in emergency situations. The second one takes into account a weekly updated set of probe-vehicle data for each minute of the week along with data of current travel times. Comparing those travel-time data allows calculation of the associated risk for traveling certain edges of a route in a road network. We expect our algorithm to be a major advancement especially for destinations that lie outside the typical region travelled weekdays. In this case the automatic route planning naturally goes along with an additional gain of time

Amyloid-beta Leads to Impaired Cellular Respiration, Energy Production and Mitochondrial Electron Chain Complex Activities in Human Neuroblastoma Cells

Evidence suggests that amyloid-beta (Aβ) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Aβ can lead to neuronal dysfunction. Here we investigated the specific effects of Aβ on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Aβ protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demis

Revival of the side-to-side approach for distal coronary anastomosis

Side-to-side anastomosis was employed by just ten proportional stitches while performing distal anastomosis during coronary artery surgery. This technique is simple and quick. Here this simple technique is described in detail and the postoperative status of grafted conduits is reported

Event traffic forecast for metropolitan areas based on microscopic simulation

It is shown that a traditional travel demand forecast combined with a simulationbased approach can serve as a short-term forecast for the traffic situation. The approach presented was developed and tested during the Soccer World Cup 2006 in the city of Cologne as a service for the action forces to react as fast as possible to developing aberrations. This paper discusses the merits and the short-comings of the approach

Wall Orientation and Shear Stress in the Lattice Boltzmann Model

The wall shear stress is a quantity of profound importance for clinical diagnosis of artery diseases. The lattice Boltzmann is an easily parallelizable numerical method of solving the flow problems, but it suffers from errors of the velocity field near the boundaries which leads to errors in the wall shear stress and normal vectors computed from the velocity. In this work we present a simple formula to calculate the wall shear stress in the lattice Boltzmann model and propose to compute wall normals, which are necessary to compute the wall shear stress, by taking the weighted mean over boundary facets lying in a vicinity of a wall element. We carry out several tests and observe an increase of accuracy of computed normal vectors over other methods in two and three dimensions. Using the scheme we compute the wall shear stress in an inclined and bent channel fluid flow and show a minor influence of the normal on the numerical error, implying that that the main error arises due to a corrupted velocity field near the staircase boundary. Finally, we calculate the wall shear stress in the human abdominal aorta in steady conditions using our method and compare the results with a standard finite volume solver and experimental data available in the literature. Applications of our ideas in a simplified protocol for data preprocessing in medical applications are discussed.Comment: 9 pages, 11 figure

Clinical effectiveness and cost-effectiveness of pegvisomant for the treatment of acromegaly: a systematic review and economic evaluation

Background: Acromegaly, an orphan disease usually caused by a benign pituitary tumour, is characterised by hyper-secretion of growth hormone (GH) and insulin-like growth factor I (IGF-1). It is associated with reduced life expectancy, cardiovascular problems, a variety of insidiously progressing detrimental symptoms and metabolic malfunction. Treatments include surgery, radiotherapy and pharmacotherapy. Pegvisomant (PEG) is a genetically engineered GH analogue licensed as a third or fourth line option when other treatments have failed to normalise IGF-1 levels. Methods: Evidence about effectiveness and cost-effectiveness of PEG was systematically reviewed. Data were extracted from published studies and used for a narrative synthesis of evidence. A decision analytical economic model was identified and modified to assess the cost-effectiveness of PEG. Results: One RCT and 17 non-randomised studies were reviewed for effectiveness. PEG substantially reduced and rapidly normalised IGF-1 levels in the majority of patients, approximately doubled GH levels, and improved some of the signs and symptoms of the disease. Tumour size was unaffected at least in the short term. PEG had a generally safe adverse event profile but a few patients were withdrawn from treatment because of raised liver enzymes. An economic model was identified and adapted to estimate the lower limit for the cost-effectiveness of PEG treatment versus standard care. Over a 20 year time horizon the incremental cost-effectiveness ratio was pound81,000/QALY and pound212,000/LYG. To reduce this to pound30K/QALY would require a reduction in drug cost by about one third. Conclusion: PEG is highly effective for improving patients' IGF-1 level. Signs and symptoms of disease improve but evidence is lacking about long term effects on improved signs and symptoms of disease, quality of life, patient compliance and safety. Economic evaluation indicated that if current standards (UK) for determining cost-effectiveness of therapies were to be applied to PEG it would be considered not to represent good value for money