The COP9 signalosome (CSN) contains eight-subunits and is a highly conserved protein complex implicated in ubiquitin-mediated proteolysis. Jun activation domain-binding protein 1 (Jab1) is the fifth component of CSN (CSN5) with a molecular weight of 38 kDa. Jab1 overexpression is observed in many human cancers, but there is no clear evidence how Jab1 contributes to malignant transformation in human cancers. Bcr-Abl is a cytoplasmic chimeric oncoprotein produced by Philadelphia chromosome translocation and found in more than 90% of patients with chronic myelogenous leukemia (CML). Recent studies have shown that the Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase and may facilitate cell-cycle progression. In this study, we found that inhibition of Bcr-Abl kinase by STI-571 downregulated 50% of full length human Jab1 promoter activity and gene expression. Promoter deletion assay indicated that the responsive element for Bcr-Abl is located between -405/-223 region of human Jab1 promoter. Treatment of LY294002 also reduced Jab-405/-223 promoter activity and Jab1 expression. Promoter mutagenesis assay and ChIP assay suggest that Bcr-Abl stimulated both £]-catenin /TCF complex and STAT1 bind to the consensus binding sites of Jab-405/-223 promoter. Taken together, Bcr-Abl oncogene may regulate Jab1 via PI3K/AKT signal pathway, £]-catenin /TCF and STAT1 transcription factors.

Die vorliegende Arbeit handelt von einer in vitro Untersuchung der Stoffwechselveränderun-gen durch die Expression von BCR/ABL bei BaF3-Zellen, einer murinen, IL-3-abhängigen B-Zelllinie. Die Zellen wurden in nährstoffreichem Standard- und in durch Titrationen ermittel-tem nährstoffarmem Minimalmedium auf unterschiedliche Stoffwechselaktivität in Abhän-gigkeit von BCR/ABL-Expression untersucht sowie auf die zusätzliche Beeinflussbarkeit durch IL-3. Danach wurden vergleichend zwischen den 2 Zelllinien (BaF3 und BaF3-BCR/ABL) im Minimalmedium und im Standardmedium Metabolite wie Glukose, Laktat und Aminosäuren bestimmt, wobei BaF3-BCR/ABL sowohl mit als auch ohne IL-3 kultiviert wur-de. Um den Einfluss von Nährstoffrestriktion auf die Therapie zu zeigen, wurden anschlie-ßend vergleichend in den beiden Medien die Tyrosinkinaseinhibitoren Imatinib und Nilotinib titriert. Die Ergebnisse zeigen, dass BaF3-BCR/ABL einen Wachstumsvorteil im Minimalmedium hat, welcher im Standardmedium nicht vorliegt. Während die bereits bekannte Verstärkung der Glukoseaufnahme durch BCR/ABL im Standardmedium bestätigt wurde, konnte im Minimal-medium Gegenteiliges gezeigt werden. Zudem wurde ein Unterschied im Aminosäurestoff-wechsel zwischen BaF3 + IL-3 und BaF3-BCR/ABL + IL-3 im Minimalmedium deutlich. Die therapeutische Relevanz des gezeigten Einflusses der Nährstoffrestriktion konnte anschlie-ßend in der Tyrosinkinaseinhibitortitration dargestellt werden, da die Medikamente in Abhän-gigkeit vom Medium unterschiedliche Wirkungen zeigen. Insgesamt bieten die Ergebnisse einen metabolischen Erklärungsansatz für das Überleben von Tumorstammzellen in nährstoffarmen Arealen des Knochenmarks unter Therapie und Raum für neue Therapieansätze.

L'empreinte génomique, une régulation épigénétique unique entraînant une expression génique spécifique aux parents d'origine, est essentielle au développement et à la croissance des mammifères. H19 est un ARN long non codant exprimé en milieu maternel qui est un régulateur central du réseau de gènes à empreinte contrôlant le développement. H19 est exprimé pendant le développement embryonnaire dans de nombreux tissus, y compris toutes les cellules hématopoïétiques. Le rôle de H19 au cours du développement embryonnaire n'a été documenté que pour le placenta où il contrôle la croissance. Le rôle de H19 dans la lymphopoïèse n'a pas été étudié. Notre laboratoire a précédemment trouvé H19 comme principal transcrit exprimé sélectivement par les proB du foie foetal (FF), et exprimé de façon différentielle par des immigrants thymiques précoces et tardives. Cependant, le rôle du gèneH19 dans celui du développement des cellules B, ou même dans le système immunitaire, reste méconnu. Ici, nous avons réalisé une caractérisation complète des perturbations du développement et de la fonction du système immunitaire des souris pour lesquelles un grand segment du locus H19 a été supprimé. Dans cette étude, nous avons constaté que le mutant H19 avait un impact spécifique sur le développement des cellules du FF induisant une augmentation sélective du nombre des cellules proB BP1+ présentant des perturbations importantes du réarrangement du locus IgH.On observe également chez les animaux H19-/- adultes une expansion anormale du compartiment B mature. Bien que H19 ne soit plus exprimé après la naissance, les lymphocytes B des souris adultes mutantes présentent un phénotype altéré. On observe en effet des perturbations importantes du profil d'expression du marqueur B220. Les souris H19-/-présentent un défaut d'expansion des lymphocytes B du centre germinatif, ainsi qu'une chute de la production des IgM spécifiques dans le sérum après immunisation. Indiquant une réponse défectueuse des cellules B. De manière cohérente, nous avons trouvé une réactivité réduite au BCR des cellules B naïves H19-/-, associée les expériences de reconstitution compétitive ont mis en évidence un altération cellule-intrinsèque de la réponse humorale chez les animaux mutants. Un défaut d'induction de l'expression des molécules du CMHII, CD40,et CD86, qui pourrait être à l'origine des perturbations de la réponse humorale observée chez les souris H19-/-. Les analyse de transcriptome réalisées sur les lymphocytes B du centre germinal des animaux mutants ont mis en évidence une expression différentielle des gènes impliqués dans la régulation de l'intensité du signal émanant du récepteur B à l'antigène. Au total ce travail nous a permis de démontrer l'activité régulatrice exercée par l'ARN non codant H19 sur le développement et la fonction du système immunitaire. / Genomic imprinting, a unique epigenetic regulation resulting in a parent-of-origin specificgene expression, is essential for normal mammalian development and growth. H19 is amaternally expressed long non-coding RNA that is a central regulator of the imprinting gene network controlling development and growth. H19 is expressed throughout embryonic development in multiple tissues including all hematopoietic cells. The role of H19 during embryonic development has only been documented for the placenta where it controls growthand the role of H19 in lymphopoiesis has not been investigated. The laboratory has previouslyfound H19 as the major differentially expressed transcript in two microarrays comparing fetalliver (FL) and bone marrow (BM) derived pro-B cells, as well as between early and latethymic settling progenitors. However, a role for imprinting gene H19 in B cell development,or even in immune system remains elusive. Here we sought to analyze mice where a large segment of the H19 locus has been deleted. In our work, we found that loss of H19 have specific impact on the FL B cell development byproducing increased numbers of BP1+ proB cell. Although BP1+ proB cells from H19-/- FLshowed impaired Ig heavy chain V-D-J rearrangement, that increase resulted in a net enlarged B cell compartment in the adult periphery of H19 mutant. In adult mice, although H19 is notexpressed in B lymphocytes after birth, B cells from H19-/- mice exhibited altered B cellsurface phenotype, represented by an upregulated B220 expression on all B cell subsets. After immunization with different T cell dependent antigens, H19-/- exhibits reduced GC B cells, and impaired specific IgM titer in the serum, indicating a defected B cell response in H19-/-mice. Competitive reconstitution analysis showed a B cell autonomous impairment in the Bcell response. Consistently we found a reduced BCR responsiveness of H19-/- naïve B cells that together with less efficient upregulation of MHCII and CD40 expression after immunization might be responsible for the impaired immune response in H19-/- mice. Genome-wide transcription analysis revealed differential expression of genes involved inregulating the intensity of B cell receptor signaling. This work brings new insights on the regulation role of long non-coding RNA H19 in the early B cell development and immune system

3BP2 was originally identified through its interaction with the ABL kinase. Fusion of ABL with the BCR gene forms the BCR-ABL onco-protein, which is causative in Chronic Myeloid Leukemia (CML) and acute lymphoid leukemia (ALL). Due to the ability of 3BP2 to regulate ABL activity in osteoblasts, we hypothesize that 3BP2 modulates BCR-ABL signalling. Overexpression of 3BP2 in the CML-T1 cell line produced a marked decrease in global tyrosine phosphorylation. 3BP2 overexpression also resulted in a significant increase in CML-T1 cell growth, accompanied by altered ERK1/2, AKT, SYK, LYN, HCK, and CBL phosphorylation and expression. A phospho-SRC family protein and a 116 kDa phospho-protein were identified as 3BP2 interaction partners in response to BCR-ABL activation. BCR-ABL bone marrow transplantation (BMT) models in 3bp2-/- mice exhibit accelerated disease compared to wild-type mice, with altered leukemic phenotype. In conclusion, 3BP2 is able to modulate signalling through BCR-ABL and affect BCR-ABL-induced disease outcome.

3BP2 was originally identified through its interaction with the ABL kinase. Fusion of ABL with the BCR gene forms the BCR-ABL onco-protein, which is causative in Chronic Myeloid Leukemia (CML) and acute lymphoid leukemia (ALL). Due to the ability of 3BP2 to regulate ABL activity in osteoblasts, we hypothesize that 3BP2 modulates BCR-ABL signalling. Overexpression of 3BP2 in the CML-T1 cell line produced a marked decrease in global tyrosine phosphorylation. 3BP2 overexpression also resulted in a significant increase in CML-T1 cell growth, accompanied by altered ERK1/2, AKT, SYK, LYN, HCK, and CBL phosphorylation and expression. A phospho-SRC family protein and a 116 kDa phospho-protein were identified as 3BP2 interaction partners in response to BCR-ABL activation. BCR-ABL bone marrow transplantation (BMT) models in 3bp2-/- mice exhibit accelerated disease compared to wild-type mice, with altered leukemic phenotype. In conclusion, 3BP2 is able to modulate signalling through BCR-ABL and affect BCR-ABL-induced disease outcome.

The natural history of CML has been transformed in recent years by the introduction of Glivec[superscript TM] (imatinib mesylate), an ABL kinase inhibitor, which provides the new treatment paradigm for chronic phase CML. While the majority of patients with CP-CML respond very well to imatinib, there are approximately 15% of patients who fail to respond, or respond suboptimally. While the major cause of secondary imatinib resistance can be attributable to kinase domain mutations, the underlying cause of primary resistance is yet to be elucidated. Utilizing the phosphorylation of the adaptor protein Crkl, an immediate downstream partner of BCRABL, as a surrogate measure of BCR-ABL kinase activity, a large interpatient variation in the degree of imatinib induced kinase inhibition achieved in-vitro, was observed in previously untreated CP-CML patients. The observed in-vitro sensitivity was a good predictor of molecular response in patients treated with 600mg imatinib as front line therapy. Furthermore, analysis of the in-vivo reduction in p-Crkl mediated measured in blood cells in response to imatinib over the first 28 days of therapy, revealed that patients with higher % reductions respond significantly better over a two year period, than those with lower % reductions. Using 14-C labelled imatinib, it was demonstrated that this intrinsic sensitivity correlated to the amount of drug which was retained within the target haemopoietic cell, and furthermore, that a critical determinant of the active influx of imatinib, was the functional activity of the human organic cation transporter -1 (OCT-1), as determined by a prazosin (potent inhibitor of OCT-1) inhibition assay. Patients with high OCT-1 Activity had superior molecular responses when compared to those with low OCT-1 Activity, but in those patients who could tolerate increased imatinib dose, these inferior responses could be largely overcome. In contrast, Nilotinib, a more potent second generation tyrosine kinase inhibitor, is not dependent on OCT-1 for influx, making it a possible treatment choice for patients with low OCT-1 Activity. Both imatinib and nilotinib interact with the efflux transporters ABCB1, and ABCG2. In combination studies imatinib results in a significantly increased intracellular concentration of nilotinib, most likely through interaction with these efflux transporters. Furthermore, commonly used therapies such as proton pump inhibitors also interact with ABCB1 and ABCG2, and demonstrable changes in intracellular drug concentrations were observed in-vitro with concomitant administration of these agents and imatinib or nilotinib at clinically relevant concentrations. In conclusion, these data demonstrate that the degree of kinase inhibition mediated in-vitro and in-vivo by imatinib, is a critical determinant of subsequent molecular response. This intrinsic sensitivity to imatinib induced kinase inhibition is related to the activity of the OCT-1 protein. This protein is not involved in the transport of nilotinib, suggesting it as a possible treatment alternative in those patients with low OCT-1 Activity. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1319077 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

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Magíster en Minería / Los métodos de extracciones secuenciales se utilizan comúnmente para evaluar el riesgo potencial de los residuos, suelos o sedimentos una vez expuestos a la intemperie. Cada una de las estas etapas del protocolo simulan distintos ambientes (ej: reductor, oxidante) y cada elemento es susceptible a estos ambientes dependiendo del mineral que formen. En el presente estudio se puso a prueba el método de extracción secuencial BCR (Community Bureau of Reference), intentando extender su aplicación a residuos mineros. Se eligió este método, ya que es uno de los más aceptado, por certificar algunos materiales (BCR-701), además, de su frecuente utilización en estudios medioambientales en el campo minero. Se tratará de optimizar el método, mediante la evaluación de la condición experimental peso de la muestra vs volumen de reactivo, utilizando las siguientes proporciones de masa: 1.000 mg (proporción original), 500 mg y 200 mg, observando la disolución de los minerales presentes en las distintas muestras. Cabe mencionar, que las muestras se seleccionaron con base al criterio que tuvieran la variedad de minerales suficientes para poder observar la disolución selectiva de los minerales en los pasos (S1, S2, S3) de la BCR, con base a esto, se eligieron las siguientes muestras: 1) Lodo pirítico de Monte Romero, 2) Residuo concentrador de Riotinto (RT1A), 3) Residuo en pilas de lixiviación de Tharsis (HLT1C), además, una muestra estándar (BCR 701), para determinar la calidad del experimento. De acuerdo a los resultados obtenidos en el análisis de control de calidad, se pudo argumentar que el experimento realizado es confiable, ya que, los valores experimentales obtenidos en la muestra BCR-701 están de acuerdo con los valores certificados de esta muestra, en los elementos Cr, Cu, Ni, Pb y Zn. Con respecto a los resultados de las tres primeras muestras, se concluyó que extender la aplicación de la BCR a residuos mineros, sería imposible, ya que se observó que los minerales contenidos en las distintas muestras no se disolvían selectivamente en cada una de las etapas para la cual fue diseñada. Sin embargo, se puede destacar que ciertos componentes minerales de los residuos (i.e., barita en muestra RT1A o yeso en muestra HLT1C) si mostraron una disolución selectiva y completa en los pasos de la BCR diseñados con tal fin. Por lo cual, parte de la información referente a la liberación de metales puede ser útil e interpretable desde un punto de vista ambiental. Finalmente, se proponen alternativas al uso de la BCR en pro de mejorar estudios ambientales en el campo minero. Estas posibles mejoras tienen su fundamento en la aplicación de nuevas tecnologías (QEMSCAN-MLA) en las diferentes etapas (i.e., muestreo representativo, caracterización mineral y evaluación mineralógica) que conforman un estudio predictivo de movilidad de contaminantes.

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