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Targeting Aberrant STAT3 Signaling as a Therapeutic Strategy for Multiple MyelomaCroucher, Danielle 11 July 2013 (has links)
The oncogenic transcription factor STAT3 is aberrantly activated in over 70% of human tumours, including Multiple myeloma (MM). The present studies use both genetic and chemical tools to validate STAT3 as a therapeutic target, and demonstrate the anti-MM activity of a novel small molecule STAT3 inhibitor, BP-4-018. We show that shRNA-mediated STAT3 knockdown induces apoptosis in human myeloma cell lines (HMCLs). We translate these findings to a therapeutically relevant setting by demonstrating the broad anti-MM activity of BP-4-018 against HCMLs and primary patient samples, and demonstrate that BP-4-018 remains active against HMCLs co-cultured with bone marrow stroma. Inhibiting STAT3 via shRNA knockdown and BP-4-018 suppresses STAT3 transcriptional activity and down-regulates anti-apoptotic and proliferative STAT3 target genes. Finally, we show that BP-4-018 has activity in vivo, both alone and combined with subtherapeutic doses of bortezomib, without significant toxicities. Taken together, these data support the utility of STAT3 inhibitors for MM treatment.
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Improving Targeted Radionuclide Therapy Using Nuclear NanotechnologyEvans, Jordan Andrew 03 October 2013 (has links)
The objectives of this thesis are to produce radioactive antibody-conjugated gold nanoparticles to improve the efficacy of targeted radionuclide therapy for the treatment of cancer, and to demonstrate that this product can be produced at Texas A&M University.
We have proposed a method for determining the distribution of radioactive nuclei per nanoparticle, which is critical for determining radiotherapeutic efficacy. Using the distribution of radioactive nuclei per nanoparticle, we have produced methods for calculating the radiative dose to tissue using nano-improved targeted radionuclide therapy, but more importantly we propose procedures to experimentally determine the efficacy of targeted radionuclide therapy improved by application of radioactive nanomaterials in combination with immunotherapy, nanomaterial cytotoxicity, and other cancer therapies such as chemotherapy. These methods can also be used to determine the efficacy of combinatory treatments as a function of time.
Characterization of the antibody-nanoparticle attachment is critical; we have demonstrated successful antibody-nanoparticle conjugation using atomic force microscopy, dynamic light scattering, and agarose gel electrophoresis, providing more conclusive evidence of successful conjugation compared to flow cytometry.
We provide a mathematical derivation from basic electron-transport principles which demonstrates the theoretical dosimetric advantages of applying radioactive nanomaterials to targeted radionuclide therapy. The general formulae can be applied to any tumor size, any radionuclide, and any pharmacokinetic nanoparticle distribution throughout the body, ultimately allowing a quick method of approximating the necessary activation time and treatment dosage parameters for a specific patient without burdensome Monte Carlo computational simulations.
We further demonstrated that nano-TRT dosage to tumors should be considered as a function of radial position rather than average, as the dose across the tumor may be noticeably non-uniform causing some portions of the tumor to receive (potentially) significantly less dose than average.
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Bioinspired drug delivery of interleukin-4 / Bioinspirierte Wirkstofffreisetzung von Interleukin-4Spieler, Valerie January 2021 (has links) (PDF)
Chronic inflammatory diseases such as rheumatoid arthritis, type 2 diabetes and cardiovascular diseases, are associated with the homeostatic imbalance of one of several physiological systems combined with the lack of spontaneous remission, which causes the disease to persevere throughout patients’ lives. The inflammatory response relies mainly on tissue-resident, pro-inflammatory M1 type macrophages and, consequently, a chance for therapeutic intervention lies in driving macrophage polarization towards the anti-inflammatory M2 phenotype. Therefore, anti-inflammatory cytokines that promote M2 polarization, including interleukin-4 (IL4), have promising therapeutic potential. Unfortunately, their systemic use is hampered by a short serum half-life and dose-limiting toxicity. On the way towards cytokine therapies with superior safety and efficacy, this thesis is focused on designing bioresponsive delivery systems for the anti-inflammatory cytokine IL4.
Chapter 1 describes how anti-inflammatory cytokines are tightly regulated in chronic, systemic inflammation as in rheumatoid arthritis but also in acute, local inflammation as in myocardial infarction. Both diseases show a characteristic progression during which anti-inflammatory cytokine delivery is of variable benefit. A conventional, passive drug delivery system is unlikely to release the cytokines such that the delivery matches the dynamic course of the (patho-)physiological progress. This chapter presents a blueprint for active drug delivery systems equipped with a 24/7 inflammation detector that continuously senses for matrix metalloproteinases (MMP) as surrogate markers of the disease progress and responds by releasing cytokines into the affected tissues at the right time and place. Because they are silent during phases of low disease activity, bioresponsive depots could be used to treat patients in asymptomatic states, as a preventive measure. The drug delivery system only gets activated during flares of inflammation, which are then immediately suppressed by the released cytokine drug and could prevent the steady damage of subclinical chronic inflammation, and therefore reduce hospitalization rates.
In a first proof of concept study on controlled cytokine delivery (chapter 2), we developed IL4-decorated particles aiming at sustained and localized cytokine activity. Genetic code expansion was deployed to generate muteins with the IL4’s lysine 42 replaced by two different unnatural amino acids bearing a side chain suitable for click chemistry modification. The new IL4 muteins were thoroughly characterized to ensure proper folding and full bioactivity. Both muteins showed cell-stimulating ability and binding affinity to IL4 receptor alpha similar to those of wild type IL4. Copper-catalyzed (CuAAC) and strain-promoted (SPAAC) azide–alkyne cycloadditions were used to site-selectively anchor IL4 to agarose particles. These particles had sustained IL4 activity, as demonstrated by the induction of TF-1 cell proliferation and anti-inflammatory M2 polarization of M-CSF-generated human macrophages. This approach of site-directed IL4 anchoring on particles demonstrates that cytokine-functionalized particles can provide sustained and spatially controlled immune-modulating stimuli.
The idea of a 24/7 sensing, MMP driven cytokine delivery system, as described in the introductory chapter, was applied in chapter 3. There, we simulated the natural process of cytokine storage in the extracellular matrix (ECM) by using an injectable solution of IL4 for depot formation by enzyme-catalyzed covalent attachment to ECM components such as fibronectin. The immobilized construct is meant to be cleaved from the ECM by matrix-metalloproteinases (MMPs) which are upregulated during flares of inflammation. These two functionalities are facilitated by a peptide containing two sequences: a protease-sensitive peptide linker (PSL) for MMP cleavage and a sequence for covalent attachment by activated human transglutaminase FXIIIa (TGase) included in the injection mix for co-administration. This peptide was site-selectively conjugated to the unnatural amino acid at IL4 position 42 allowing to preserve wild type bioactivity of IL4. In vitro experiments confirmed the anticipated MMP response towards the PSL and TGase-mediated construct attachment to fibronectin of the ECM. Furthermore, the IL4-peptide conjugates were able to reduce inflammation and protect non-load bearing cartilage along with the anterior cruciate ligament from degradation in an osteoarthritis model in rabbits. This represents the first step towards a minimally invasive treatment option using bioresponsive cytokine depots with potential clinical value for inflammatory conditions.
One of the challenges with this approach was the production of the cytokine conjugate, with incorporation of the unnatural amino acid into IL4 being the main bottleneck. Therefore, in chapter 4, we designed a simplified version of this depot system by genetically fusing the bifunctional peptide via a flexible peptide spacer to murine IL4. While human IL4 loses its activity upon C-terminal elongation, murine IL4 is not affected by this modification. The produced murine IL4 fusion protein could be effectively bound to in vitro grown extracellular matrix in presence of TGase. Moreover, the protease-sensitive linker was selectively recognized and cleaved by MMPs, liberating intact and active IL4, although at a slower rate than expected. Murine IL4 offers the advantage to evaluate the bioresponsive cytokine depot in many available mouse models, which was so far not possible with human IL4 due to species selectivity.
For murine IL4, the approach was further extended to systemic delivery in chapter 5. To increase the half-life and specifically target disease sites, we engineered a murine IL4 variant conjugated with a folate-bearing PEG chain for targeting of activated macrophages. The bioactive IL4 conjugate had a high serum stability and the PEGylation increased the half-life to 4 h in vivo. Surprisingly, the folate moiety did not improve targeting in an antigen-induced arthritis (AIA) mouse model. IL4-PEG performed better in targeting the inflamed joint, while IL4-PEG-folate showed stronger accumulation in the liver. Fortunately, the modular nature of the IL4 conjugate facilitates convenient adaption of PEG chain length and the targeting moiety to further improve the half-life and localization of the cytokine.
In summary, this thesis describes a platform technology for the controlled release of cytokines in response to inflammation. By restricting the release of the therapeutic to the site of inflammation, the benefit-risk ratio of this potent class of biologics can be positively influenced. Future research will help to deepen our understanding of how to perfectly combine cytokine, protease-sensitive linker and immobilization tag or targeting moiety to tackle different diseases. / Chronische Entzündungskrankheiten wie rheumatoide Arthritis, Typ-2-Diabetes oder Herz-Kreislauf-Erkrankungen werden durch das Ungleichgewicht eines von mehreren physiologischen Systemen in Verbindung mit fehlender spontaner Remission verursacht, wodurch die Krankheiten lebenslang bestehen bleiben. Die zugrunde liegenden Entzündungsreaktionen beruhen hauptsächlich auf im Gewebe vorhandenen Makrophagen und deren Polarisation in Richtung des entzündlichen M1-Phänotyps, was gleichzeitig die Möglichkeit einer therapeutischen Intervention bietet. Entzündungshemmende Zytokine, einschließlich Interleukin-4 (IL4), haben ein großes therapeutisches Potenzial, da sie Makrophagen in Richtung des entzündungshemmenden M2-Phänotyps zu polarisieren vermögen. Leider ist ihre systemische Anwendung durch eine kurze Serumhalbwertszeit und dosislimitierende Toxizität eingeschränkt. Auf dem Weg zu Zytokintherapeutika mit verbesserter Sicherheit und Wirksamkeit konzentriert sich diese Arbeit auf die Entwicklung von bioresponsiven Freisetzungssystemen für das entzündungshemmende Zytokin IL4.
Kapitel 1 beschreibt, wie entzündungshemmende Zytokine bei chronischen systemischen Entzündungen wie rheumatoider Arthritis im Vergleich zu akuten lokalen Entzündungen wie dem Myokardinfarkt reguliert werden. Beide Erkrankungen zeigen einen charakteristischen Verlauf, währenddessen die Freisetzung von entzündungshemmenden Zytokinen von unterschiedlich großem Nutzen ist. Gewöhnliche, passive Arzneimittelfreisetzungssysteme sind nicht in der Lage, Zytokine in idealer Menge zur optimalen Unterdrückung des dynamischen, (patho-)physiologischen Verlaufs der Krankheit freizusetzen. In diesem Kapitel werden deshalb aktive Arzneimittelfreisetzungssysteme vorgestellt, die mit einer Sensorik für die Entzündung ausgestattet sind, mit der sie kontinuierlich die Konzentration von Matrix-Metalloproteinasen (MMP) als Indikatoren für den Krankheitsverlauf erfassen können. Somit kann das aktive Arzneimittelfreisetzungssystem krankes Gewebe zum richtigen Zeitpunkt und am richtigen Ort mit Zytokinen behandeln. Solche bioresponsiven Depots können zur vorbeugenden Behandlung von asymptomatischen Patienten eingesetzt werden, da sie während Phasen geringer Krankheitsaktivität inaktiv sind. Das Freisetzungssystem wird erst durch Entzündungsschübe aktiviert, die dann sofort durch die freigesetzten Zytokine unterdrückt werden. Dadurch könnte die dauerhafte Schädigung durch subklinische, chronische Entzündung verhindert und als Konsequenz die Hospitalisierungsrate gesenkt werden.
In einer ersten Machbarkeitsstudie wurden in Kapitel 2 IL4-dekorierte Partikel mit dem Ziel entwickelt, eine langanhaltende und lokalisierte Zytokinaktivität zu gewährleisten. Dazu wurden IL4-Muteine erzeugt, bei denen das Lysin 42 mittels Erweiterung des genetischen Codes durch zwei verschiedene unnatürliche Aminosäuren ersetzt wurde, die jeweils eine für Klick-Chemie geeignete Seitenkette tragen. Die IL4-Muteine wurden ausführlich charakterisiert, um eine korrekte Faltung und volle Bioaktivität sicherzustellen. Beide Muteine zeigten zellstimulierende Fähigkeit und Bindungsaffinität an IL4-Rezeptor-alpha, die mit der von Wildtyp-IL4 vergleichbar ist. Anschließend wurde kupferkatalysierte (CuAAC) und kupferfreie (SPAAC) Azid-Alkin-Cycloaddition verwendet, um IL4 ortsspezifisch auf Agarosepartikeln zu verankern. Die Partikel waren in der Lage, die IL4-Aktivität über längere Zeit aufrecht zu erhalten, was durch TF-1-Zellproliferation und M2-Polarisation von M-CSF-generierten, humanen Makrophagen gezeigt werden konnte. Dieser Ansatz der ortsspezifischen Verankerung von IL4 auf Agarosepartikeln zeigt, dass zytokinfunktionalisierte Partikel anhaltende und räumlich kontrollierte, immunmodulierende Stimuli liefern können.
Die Idee eines MMP-gesteuerten Zytokinfreisetzungssystems mit 24/7-Sensorik, das im Einleitungskapitel vorgestellt wurde, wurde in Kapitel 3 umgesetzt. Der natürliche Prozess der Zytokinspeicherung in der extrazellulären Matrix (EZM) wurde mithilfe einer injizierbaren IL4-Lösung zur enzymatischen Depotbildung durch kovalente Bindung an EZM-Komponenten, z. B. Fibronektin, simuliert. Nach der Bindung soll das Konstrukt durch Matrix-Metalloproteinasen (MMPs), die während Entzündungsschüben hochreguliert werden, aus der EZM freigesetzt werden können. Eine Peptidsequenz, die ein Protease-sensitives Verbindungsstück und eine Sequenz, mit der das Zytokin bei gleichzeitiger Injektion von aktivierter menschlicher Transglutaminase FXIIIa (TGase) kovalent auf der EZM immobilisiert wird enthält, wurde ortsspezifisch über eine unnatürliche Aminosäure an Position 42 von IL4 gekoppelt. Dadurch wurde die Bioaktivität von IL4 vollständig erhalten, während das Protease-sensitive Verbindungsstück auf MMPs reagierte und das Konstrukt durch TGase an das Fibronektin der EZM gebunden werden konnte. Die IL4-Peptid-Konjugate waren in einem Osteoarthritis-Modell bei Kaninchen in der Lage, die Entzündung des Kniegelenks zu verringern und den nicht-tragenden Knorpel sowie das vordere Kreuzband vor Degradation zu schützen. Dies ist der erste Schritt in Richtung einer minimalinvasiven Behandlung durch Verwendung von bioresponsiven Zytokindepots mit potenziellem klinischem Nutzen bei Entzündungserkrankungen.
Eine der Herausforderungen bei diesem Vorgehen war die Herstellung der Zytokinkonjugate, wobei der Einbau der unnatürlichen Aminosäure in IL4 den größten Engpass darstellte. Deshalb wurde in Kapitel 4 eine vereinfachte Version dieses Depotsystems entworfen, indem das bifunktionelle Peptid über eine flexible Verbindungssequenz mit murinem IL4 genetisch fusioniert wurde. Während humanes IL4 bei C-terminaler Verlängerung an Aktivität verliert, ist murines IL4 durch die Modifikation nicht beeinflusst. Die murinen IL4-Fusionsproteine konnten in Gegenwart von TGase wirksam an in vitro generierte extrazelluläre Matrix gebunden werden. Darüber hinaus wurde das Protease-sensitive Verbindungsstück selektiv von MMPs erkannt und gespalten, wobei intaktes und aktives IL4 freigesetzt wurde, wenn auch mit einer langsameren Rate als erwartet. Murines IL4 bietet die Möglichkeit das bioresponsive Zytokindepot in den vielen verfügbaren Mausmodellen zu testen, was mit humanem IL4 aufgrund der Speziesselektivität nicht möglich ist.
Für murines IL4 wurde die Entwicklung in Kapitel 5 auf die systemische Applikation ausgeweitet. Um die Serumhalbwertszeit zu erhöhen und eine Wirkstofflokalisation im entzündeten Gewebe zu erreichen, wurde eine murine IL4-Variante entwickelt, die mit einer Folat-tragenden PEG-Kette konjugiert wurde, um aktivierte M1 Makrophagen zu adressieren. Das bioaktive IL4-Konjugat wies eine hohe Serumstabilität auf und die PEGylierung erhöhte die Halbwertszeit in vivo auf 4 h. Allerdings konnte durch die Konjugation der Folatgruppe an IL4 die Wirkstofflokalisation in einem Mausmodell mit Antigen-induzierter Arthritis (AIA) nicht verbessert werden. IL4-PEG akkumulierte sich stärker im entzündeten Gelenk, während IL4-PEG-Folat eine stärkere Anreicherung in der Leber zeigte. Erfreulicherweise erleichtert der modulare Aufbau des IL4-Konjugats die bequeme Anpassung der PEG-Kettenlänge und der zielorientierten Einheit, um die Halbwertszeit und Lokalisierung des Zytokins weiter zu verbessern.
Zusammenfassend beschreibt diese Arbeit eine Plattformtechnologie zur kontrollierten Freisetzung von Zytokinen als Reaktion auf Entzündungen. Durch die Beschränkung der Freisetzung des Therapeutikums auf den Ort der Entzündung kann das Nutzen-Risiko-Verhältnis dieser potenten Klasse von Biologika positiv beeinflusst werden. Zukünftige Forschungen werden dazu beitragen zu verstehen, wie Zytokin, Protease-sensitives Verbindungsstück und Immobilisierungsanhängsel oder etwaige zielorientierte Einheiten zur Bekämpfung verschiedener Krankheiten perfekt kombiniert werden können.
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Development and use of an adoptive transfer method for detecting radiation-induced bystander effects in vivoBlyth, Benjamin John, benjamin.blyth@flinders.edu.au January 2009 (has links)
Ionising radiation can cause damage to DNA that can result in gene mutations contributing to carcinogenesis. Radiation-protection policy currently estimates cancer risks from exposures to radiation in terms of excess risk per unit dose. At very low radiation dose-rates, where not all cells are absorbing radiation energy, this formula carries the inherent assumption that risk is limited to those cells receiving direct energy depositions. Numerous studies have now called this assumption into question. Such low dose-rates are in the relevant range that the public receives from natural background and man-made sources, and, if this fundamental assumption proves unfounded, current estimations of radiation-induced cancer risk at low doses will be incorrect. Accurate predictions of stochastic cancer risks from low-dose radiation exposures are crucial to evaluating the safety of radiation-based technologies for industry, power generation and the increasing use of radiation for medical diagnostic and screening purposes.
This thesis explores phenomena known as radiation-induced bystander effects. The term bystander effects, as used here, describes biological responses to ionising radiation (hitherto observed in vitro) in cells not directly traversed by an ionising track, due to intercellular signals received from neighbouring cells that did receive energy depositions. This study aimed to determine whether radiation effects are communicated between irradiated and unirradiated cells in vivo, and if so, whether this effect alters current estimations of cancer risk following low-dose radiation exposures. In order to answer these questions, an in vivo experimental system for studying bystander effects in mice was developed. The method was based on the adoptive transfer of irradiated splenocytes into unirradiated hosts with simultaneous identification of irradiated donor cells, and biological endpoints in unirradiated bystander cells in situ using fluorescence microscopy and image analysis.
Splenocytes from donor mice were radiolabelled with 3H-thymidine or received an acute X-ray dose. The irradiated donor cells, labelled with a fluorescent probe, were then adoptively transferred into unirradiated recipient mice via the tail vein, whilst control mice received sham-irradiated donor cells. A proportion of the cells lodged in the recipient mouse spleens where they remained for a period before the tissues were cryopreserved. The locations of donor cells were identified in frozen spleen sections by the fluorescent probe, and the levels of apoptosis and proliferation were simultaneously evaluated in situ in the surrounding unirradiated bystander cells using fluorescence-based assays. Transgenic pKZ1 recipient mice were also used to quantify chromosomal inversions in bystander cells. Since three-dimensional spatial relationships were preserved, responses could be measured in the local area surrounding irradiated cells as well as further afield. Following the development of the irradiated-cell adoptive transfer protocol and validation of the sensitivity and reproducibility of the biological assays in situ, a series of experiments was performed. In the initial experiments, 500,000 radiolabelled cells (0.33 mBq.cell-1) were injected into recipient mice and the spleen tissues were isolated 22 h later. No changes in apoptosis or proliferation were detected in local bystander spleen cells or throughout the spleen, compared to mice receiving sham-radiolabelled donor cells. In subsequent experiments, the effects of a number of experimental conditions were explored including the injection of tenfold more donor cells, analysis of spleen tissues after three days lodging in vivo, radiolabelling of donor cells with 100-fold higher 3H dose-rate and irradiation of donor cells ex vivo with 0.1 or 1 Gy X-rays. In each case, no changes in apoptosis or proliferation were observed.
The in vivo method described here was designed to simulate the conditions of a bystander scenario from low dose-rate exposures relevant to public radiation protection. Contrary to the many reports of bystander effects in vitro, experiments using this sensitive method for examining the in vivo responses of unirradiated cells to neighbouring low-dose irradiated cells, have so far shown no changes in bystander cells in the spleen. This adoptive transfer method is the first in vivo method for examining the effects of known irradiated cells exposed to low radiation doses at low dose-rates, on neighbouring cells in situ that are truly unirradiated. Both the irradiated and bystander cells are normal, non-transformed primary spleen cells functioning in their natural environment. This in vivo experimental system allows the examination of tens of thousands of bystander cells and has shown a remarkable sensitivity, with statistical power to rule out changes in apoptosis <10% from the control.
The relevance of in vitro bystander findings is unclear. Many reported bystander effects are more analogous to the systemic communication of abscopal effects from highly irradiated tissues. Disagreement between experimental systems and difficulty in reproducing key results between laboratories further complicate the translation of bystander data in vitro to human risk-estimation. The radiation protection community has expressed its need for in vivo validation of the bystander phenomenon before it can be included into the appraisal of carcinogenic risk. This adoptive transfer method is now available to study a range of bystander endpoints and potential signalling mechanisms in vivo, and provides a way to translate the wealth of data previously collected in vitro into findings directly relevant to human risk-estimation.
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Entwicklung und Charakterisierung sprühgetrockneter Mikropartikel mit Gelatine und Transportuntersuchungen von Gelatinehydrolysaten /Kälkert, Katrin. January 2004 (has links)
Universiẗat, Diss--Heidelberg, 2004. / Zusfassung in engl. Sprache.
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Synthese säurelabil detoxifizierter seco-CI-Acetale für eine selektive Tumortherapie unter Anwendung radikalischer Synthesemethoden /Starck, Dorothea. January 1993 (has links) (PDF)
Univ., Diss.--Göttingen, 1993.
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Hydroxylapatit Keramiken als "drug delivery" Systeme /Joschek, Steffi Karen. January 1999 (has links) (PDF)
Univ., Diss.--Regensburg, 1999.
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Infected biomaterials new strategies for local anti-infective treatmentMatl, Florian January 2008 (has links)
Zugl.: München, Univ., Diss., 2008
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Synthese und Untersuchung von Dextranestern als filmbildende Hilfsstoffe zur gezielten Arzneistofffreisetzung im Dickdarm /Kesselhut, Jan-Frederic. January 1994 (has links) (PDF)
Universiẗat, Diss.--Freiburg/Br., 1994.
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Porphyrinplatin(II)-Komplexe in der Tumortherapie : systematische Synthese und Testung neuer multifunktionaler Wirkstoffe /Bart, Karl-Christian. January 2001 (has links) (PDF)
Univ., Diss.--Regensburg, 2001.
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