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.

<p>The ABL family of non-receptor tyrosine kinases, ABL1 (also known as c-ABL) and ABL2 (also known as Arg), links diverse extracellular stimuli to signaling pathways that control cell growth, survival, adhesion, migration and invasion. ABL tyrosine kinases play an oncogenic role in human leukemias. However, the role of ABL kinases in solid tumors including breast cancer progression and metastasis is just emerging. </p><p>To evaluate whether ABL family kinases are involved in breast cancer development and metastasis, we first analyzed genomic data from large-scale screen of breast cancer patients. We found that ABL kinases are up-regulated in invasive breast cancer patients and high expression of ABL kinases correlates with poor prognosis and early metastasis. Using xenograft mouse models combined with genetic and pharmacological approaches, we demonstrated that ABL kinases are required for regulating breast cancer progression and metastasis to the bone. Using next generation sequencing and bioinformatics analysis, we uncovered a critical role for ABL kinases in promoting multiple oncogenic pathways including TAZ and STAT5 signaling networks and the epithelial to mesenchymal transition (EMT). These findings revealed a role for ABL kinases in regulating breast cancer tumorigenesis and bone metastasis and provide a rationale for targeting breast tumors with ABL-specific inhibitors.</p> / Dissertation

Submitted by admin tede (tede@pucgoias.edu.br) on 2017-02-06T16:52:19Z No. of bitstreams: 1 Caio Bruno Quinta de Souza Leal.pdf: 116273125 bytes, checksum: 5e247a988672d255dad21fa0aedcc5da (MD5) / Made available in DSpace on 2017-02-06T16:52:19Z (GMT). No. of bitstreams: 1 Caio Bruno Quinta de Souza Leal.pdf: 116273125 bytes, checksum: 5e247a988672d255dad21fa0aedcc5da (MD5) Previous issue date: 2014-01-31 / Monitoring of minimal residual disease (MRD) in patients with Chronic Myeloid Leukemia (CML) who receive treatment with Imatib mesylate (IM) is extremely important, because it enables the evaluation of the response to the treatment and the early diagnosis of possible recurrences. The objective of this study was to standardize molecular methods used in order to monitor MRD in patients with CML, on therapy with IM. Peripheral blood samples were collected from 11 patients diagnosed with CML in October 2012 to September 2013, in the Department of Hematology of Hospital Araújo Jorge of the Association to Combat Cancer in Goiás. Three months after starting treatment, patients underwent a new peripheral blood collection for evaluation of MRD. Detecting bcr-abl transcripts and endogenous controls (abl and β2m) employed reverse transcription methods associated with polymerase chain reaction (RT-PCR), while quantification of bcr-abl transcripts was achieved by using reverse transcription associated with real-time PCR (RQ-PCR) and Taqman probes. Specific oligonucleotides and probes recognizing e13a2 and e14a2 junctions of bcr-abl transcripts and to the abl endogenous control were used in this study. By the time of diagnosis, three patients (27.3%) expressed the b2a2 transcript, five patients (45.5%) expressed the b3a2 transcript, two patients (18.2%) expressed both transcripts and one patient (9%) did not express any of the transcripts. The endogenous controls analysis resulted in better amplification for the abl transcript, which was used in the RQ-PCR reactions. The assessment of DRM was possible in only eight patients, due to the loss of follow-up. Three months after starting treatment with IM, all patients presented complete hematologic response. However, only one patient (12.5%) presented the undetectable transcript, reaching the full molecular response, while the other seven patients (87.95%) presented MRD. One (12.5%) of the seven patients who presented MRD, reached complete molecular response, while six patients (75%) presented a reduction of two logs, achieving minor molecular response, and one patient (12.5%) presented only partial molecular response. By using molecular biology methods, our results have enabled the standardization and the establishment of a laboratory routine, according to the international guidelines, for monitoring MRD in patients with CML. / O monitoramento de doença residual mínima (DRM) em pacientes com leucemia meilóide crônica (LMC) que recebem tratamento com mesilato de imatibe (MI) é extremamente relevante, pois possibilita o acompanhamento da resposta e o diagnóstico precoce de eventuais recidivas da doença. O objetivo deste estudo foi padronizar métodos moleculares utilizados na avaliação de DRM em pacientes com LMC, em tratamento com MI. Amostras de sangue periférico foram coletadas de 11 pacientes diagnosticados com LMC, no período de outubro de 2012 a setembro de 2013, no Setor de Hematologia do Hospital Araújo Jorge da Associação de Combate ao Câncer em Goiás. Três meses após o início do tratamento, os pacientes foram submetidos a uma nova coleta de sangue periférico para avaliação de DRM. A detecção dos transcritos bcr-abl e controles endógenos (abl e β2m) empregaram os métodos de transcrição reversa associados à reação em cadeia da polimerase (RT-PCR), enquanto a quantificação dos transcritos bcr-abl foi feita por meio de transcrição reversa associada à PCR em tempo real (RQ-PCR), utilizando a metodologia de sondas de hidrólise (TaqMan). Oligonucleotídeos e sondas Taqman específicos para as junções e13a2 e e14a2 dos transcritos bcr-abl e para o controle endógeno (abl) foram usados neste estudo. Ao diagnóstico, três pacientes (27,3%) expressaram o transcrito b2a2, cinco pacientes (45,5%) o transcrito b3a2, dois pacientes (18,2%) expressaram ambos os transcritos e um paciente (9%) não expressou nenhum dos transcritos. A amplificação dos controles endógenos resultou em melhor amplificação para o transcrito abl, que foi usado nas reações de RQ-PCR. A avaliação de DRM foi possível em oito pacientes, devido à perda de seguimento dos demais. Três meses após o início do tratamento com MI, todos os pacientes apresentaram resposta hematológica completa. No entanto, apenas um paciente (12,5%) apresentou o transcrito bcr-abl indetectável, alcançando a reposta molecular completa, enquanto os outros sete pacientes (87,95%) apresentaram DRM. Dentre os sete pacientes que apresentaram DRM, seis (75%) apresentaram redução de um a dois logs, alcançando resposta molecular menor, enquanto um (12,5%) apresentou resposta molecular parcial. Nossos resultados possibilitaram a padronização e o estabelecimento de uma rotina segundo as diretrizes internacionais, para monitoramento da DRM em pacientes com LMC, utilizando métodos de biologia molecular.

Murine plasmacytoma is induced by plastic implants, injection of paraffin oil or pristane, or through viral infection, and Myc is invariably overexpressed in the tumour cells. Although translocation and juxtaposition of the Myc locus to an immunoglobulin locus is the prominent nonrandom cytogenetic aberration observed, the significance of other karyotypic instabilities in murine plasmacytoma is not clear, including the previously observed occurrence of trisomy 11. As well as identifying new cytogenetic mutations in murine plasmacytomagenesis, this study provides evidence for their combined and sequential accumulation that may offer new parallels to human B-cell malignancies. Plasmacytomas were induced in Balb/c Rb6.15 mice by intraperitoneal (i.p.) pristane injection prior to infection with the ABL-MYC retrovirus, and confirmed by histological examination. Spectral karyotype analysis of tumour samples identified frequent aneuploidy, tetraploidy, and amplification of chromosomes 11, 12 and 16. In contrast, control mice treated by i.p. pristane injection did not develop plasmacytoma, and lipopolysaccharide-stimulated splenocytes from control mice had mainly normal diploid karyotypes. However, karyotypic instability in a minority of splenocytes indicated that control mice showing no signs of plasmacytoma development nevertheless are prone to numerical and structural cytogenetic mutations that may possibly result in plasmacytoma initiation and progression under favourable conditions, such as infection with ABL-MYC virus with the resulting high expression of v-abl and Myc in target cells. These results indicate the possible existence of proto-oncogenes present on murine chromosomes 11, 12, and 16 that are important for plasmacytoma initiation and/or progression. There are also indications that T(1;6) and monosomy of the X chromosome may also play roles in plasmacytomagenesis, and that trisomy 12 may only occur in cells with pre-existing nonrandom mutations, thereby acting as a late mutation event. As other experimental models of murine plasmacytoma have not shown a similar karyotypic etiology, there appears to be several possible redundant cytogenetic mutation events that lead to plasmacytoma. Also, as tumours in this study present various combinations of the aforementioned amplified chromosomes, their combined amplification may serve redundant purposes as well. / May 2008

Murine plasmacytoma is induced by plastic implants, injection of paraffin oil or pristane, or through viral infection, and Myc is invariably overexpressed in the tumour cells. Although translocation and juxtaposition of the Myc locus to an immunoglobulin locus is the prominent nonrandom cytogenetic aberration observed, the significance of other karyotypic instabilities in murine plasmacytoma is not clear, including the previously observed occurrence of trisomy 11. As well as identifying new cytogenetic mutations in murine plasmacytomagenesis, this study provides evidence for their combined and sequential accumulation that may offer new parallels to human B-cell malignancies. Plasmacytomas were induced in Balb/c Rb6.15 mice by intraperitoneal (i.p.) pristane injection prior to infection with the ABL-MYC retrovirus, and confirmed by histological examination. Spectral karyotype analysis of tumour samples identified frequent aneuploidy, tetraploidy, and amplification of chromosomes 11, 12 and 16. In contrast, control mice treated by i.p. pristane injection did not develop plasmacytoma, and lipopolysaccharide-stimulated splenocytes from control mice had mainly normal diploid karyotypes. However, karyotypic instability in a minority of splenocytes indicated that control mice showing no signs of plasmacytoma development nevertheless are prone to numerical and structural cytogenetic mutations that may possibly result in plasmacytoma initiation and progression under favourable conditions, such as infection with ABL-MYC virus with the resulting high expression of v-abl and Myc in target cells. These results indicate the possible existence of proto-oncogenes present on murine chromosomes 11, 12, and 16 that are important for plasmacytoma initiation and/or progression. There are also indications that T(1;6) and monosomy of the X chromosome may also play roles in plasmacytomagenesis, and that trisomy 12 may only occur in cells with pre-existing nonrandom mutations, thereby acting as a late mutation event. As other experimental models of murine plasmacytoma have not shown a similar karyotypic etiology, there appears to be several possible redundant cytogenetic mutation events that lead to plasmacytoma. Also, as tumours in this study present various combinations of the aforementioned amplified chromosomes, their combined amplification may serve redundant purposes as well.

Murine plasmacytoma is induced by plastic implants, injection of paraffin oil or pristane, or through viral infection, and Myc is invariably overexpressed in the tumour cells. Although translocation and juxtaposition of the Myc locus to an immunoglobulin locus is the prominent nonrandom cytogenetic aberration observed, the significance of other karyotypic instabilities in murine plasmacytoma is not clear, including the previously observed occurrence of trisomy 11. As well as identifying new cytogenetic mutations in murine plasmacytomagenesis, this study provides evidence for their combined and sequential accumulation that may offer new parallels to human B-cell malignancies. Plasmacytomas were induced in Balb/c Rb6.15 mice by intraperitoneal (i.p.) pristane injection prior to infection with the ABL-MYC retrovirus, and confirmed by histological examination. Spectral karyotype analysis of tumour samples identified frequent aneuploidy, tetraploidy, and amplification of chromosomes 11, 12 and 16. In contrast, control mice treated by i.p. pristane injection did not develop plasmacytoma, and lipopolysaccharide-stimulated splenocytes from control mice had mainly normal diploid karyotypes. However, karyotypic instability in a minority of splenocytes indicated that control mice showing no signs of plasmacytoma development nevertheless are prone to numerical and structural cytogenetic mutations that may possibly result in plasmacytoma initiation and progression under favourable conditions, such as infection with ABL-MYC virus with the resulting high expression of v-abl and Myc in target cells. These results indicate the possible existence of proto-oncogenes present on murine chromosomes 11, 12, and 16 that are important for plasmacytoma initiation and/or progression. There are also indications that T(1;6) and monosomy of the X chromosome may also play roles in plasmacytomagenesis, and that trisomy 12 may only occur in cells with pre-existing nonrandom mutations, thereby acting as a late mutation event. As other experimental models of murine plasmacytoma have not shown a similar karyotypic etiology, there appears to be several possible redundant cytogenetic mutation events that lead to plasmacytoma. Also, as tumours in this study present various combinations of the aforementioned amplified chromosomes, their combined amplification may serve redundant purposes as well.

Made available in DSpace on 2016-08-10T10:38:55Z (GMT). No. of bitstreams: 1 CAIO BRUNO QUINTA DE SOUZA LEAL - PARTE 1.pdf: 34304765 bytes, checksum: 476dda15021939ae8be633403ae9dabe (MD5) Previous issue date: 2014-01-31 / Monitoring of minimal residual disease (MRD) in patients with Chronic Myeloid Leukemia (CML) who receive treatment with Imatib mesylate (IM) is extremely important, because it enables the evaluation of the response to the treatment and the early diagnosis of possible recurrences. The objective of this study was to standardize molecular methods used in order to monitor MRD in patients with CML, on therapy with IM. Peripheral blood samples were collected from 11 patients diagnosed with CML in October 2012 to September 2013, in the Department of Hematology of Hospital Araújo Jorge of the Association to Combat Cancer in Goiás. Three months after starting treatment, patients underwent a new peripheral blood collection for evaluation of MRD. Detecting bcr-abl transcripts and endogenous controls (abl and &#946;2m) employed reverse transcription methods associated with polymerase chain reaction (RT-PCR), while quantification of bcr-abl transcripts was achieved by using reverse transcription associated with real-time PCR (RQ-PCR) and Taqman probes. Specific oligonucleotides and probes recognizing e13a2 and e14a2 junctions of bcr-abl transcripts and to the abl endogenous control were used in this study. By the time of diagnosis, three patients (27.3%) expressed the b2a2 transcript, five patients (45.5%) expressed the b3a2 transcript, two patients (18.2%) expressed both transcripts and one patient (9%) did not express any of the transcripts. The endogenous controls analysis resulted in better amplification for the abl transcript, which was used in the RQ-PCR reactions. The assessment of DRM was possible in only eight patients, due to the loss of follow-up. Three months after starting treatment with IM, all patients presented complete hematologic response. However, only one patient (12.5%) presented the undetectable transcript, reaching the full molecular response, while the other seven patients (87.95%) presented MRD. One (12.5%) of the seven patients who presented MRD, reached complete molecular response, while six patients (75%) presented a reduction of two logs, achieving minor molecular response, and one patient (12.5%) presented only partial molecular response. By using molecular biology methods, our results have enabled the standardization and the establishment of a laboratory routine, according to the international guidelines, for monitoring MRD in patients with CML. / O monitoramento de doença residual mínima (DRM) em pacientes com leucemia meilóide crônica (LMC) que recebem tratamento com mesilato de imatibe (MI) é extremamente relevante, pois possibilita o acompanhamento da resposta e o diagnóstico precoce de eventuais recidivas da doença. O objetivo deste estudo foi padronizar métodos moleculares utilizados na avaliação de DRM em pacientes com LMC, em tratamento com MI. Amostras de sangue periférico foram coletadas de 11 pacientes diagnosticados com LMC, no período de outubro de 2012 a setembro de 2013, no Setor de Hematologia do Hospital Araújo Jorge da Associação de Combate ao Câncer em Goiás. Três meses após o início do tratamento, os pacientes foram submetidos a uma nova coleta de sangue periférico para avaliação de DRM. A detecção dos transcritos bcr-abl e controles endógenos (abl e &#946;2m) empregaram os métodos de transcrição reversa associados à reação em cadeia da polimerase (RT-PCR), enquanto a quantificação dos transcritos bcr-abl foi feita por meio de transcrição reversa associada à PCR em tempo real (RQ-PCR), utilizando a metodologia de sondas de hidrólise (TaqMan). Oligonucleotídeos e sondas Taqman específicos para as junções e13a2 e e14a2 dos transcritos bcr-abl e para o controle endógeno (abl) foram usados neste estudo. Ao diagnóstico, três pacientes (27,3%) expressaram o transcrito b2a2, cinco pacientes (45,5%) o transcrito b3a2, dois pacientes (18,2%) expressaram ambos os transcritos e um paciente (9%) não expressou nenhum dos transcritos. A amplificação dos controles endógenos resultou em melhor amplificação para o transcrito abl, que foi usado nas reações de RQ-PCR. A avaliação de DRM foi possível em oito pacientes, devido à perda de seguimento dos demais. Três meses após o início do tratamento com MI, todos os pacientes apresentaram resposta hematológica completa. No entanto, apenas um paciente (12,5%) apresentou o transcrito bcr-abl indetectável, alcançando a reposta molecular completa, enquanto os outros sete pacientes (87,95%) apresentaram DRM. Dentre os sete pacientes que apresentaram DRM, seis (75%) apresentaram redução de um a dois logs, alcançando resposta molecular menor, enquanto um (12,5%) apresentou resposta molecular parcial. Nossos resultados possibilitaram a padronização e o estabelecimento de uma rotina segundo as diretrizes internacionais, para monitoramento da DRM em pacientes com LMC, utilizando métodos de biologia molecular

<p>I den här uppsatsen har vi undersökt och behandlat låneförbudet som återfinns i ABL 12:7 och i kap 21 i nya ABL. Vi har undersökt om lagen uppfyller sina syften samt vilka syften som lagstiftaren har haft. Intressant är att en ny ABL träder i kraft den 1 januari 2006. Vi har utgått från lagstiftningen och sedan följt upp med rättspraxis och doktrin.</p><p>Under arbetets gång upptäckte vi luckor i lagstiftningen vilket medför att syftena bakom lagstiftningen inte kom till sin fulla rätt. Luckorna öppnar möjligheter att kringgå låneförbudet med tämligen enkla metoder. Vi har bland annat undersökt kringgående av lagstiftningen med hjälp utav efterföljande finansiering samt ett kringgående med hjälp av andra rättsobjekt.</p><p>Med efterföljande finansiering menas att ett bolag köps med bolagets egna pengar, med hjälp av undantaget för koncernlån. Ett kringgående med hjälp utav andra rättsobjekt kan se ut på lite olika sätt. I vårt arbete har vi använt av oss utav en fysisk person samt ett handelsbolag. Ett kringgående av lagstiftningen möjliggörs genom att andra rättsobjekt än aktiebolag ej lyder under aktiebolagslagen i stora drag.</p><p>Dessa handlingar rör sig inom ett grått område inom juridiken och gör låneförbudet till ett tämligen trubbigt redskap.</p><p>Eftersom låneförbudet tillhör specialstraffrätten möjliggörs kringgående av lagstiftningen då restriktiv lagtolkning måste användas. Faller en handling inte in ordagrant i vad som står i lagtexten är kringgåendet av låneförbudet både i nya och gamla ABL ett faktum.</p>

I den här uppsatsen har vi undersökt och behandlat låneförbudet som återfinns i ABL 12:7 och i kap 21 i nya ABL. Vi har undersökt om lagen uppfyller sina syften samt vilka syften som lagstiftaren har haft. Intressant är att en ny ABL träder i kraft den 1 januari 2006. Vi har utgått från lagstiftningen och sedan följt upp med rättspraxis och doktrin. Under arbetets gång upptäckte vi luckor i lagstiftningen vilket medför att syftena bakom lagstiftningen inte kom till sin fulla rätt. Luckorna öppnar möjligheter att kringgå låneförbudet med tämligen enkla metoder. Vi har bland annat undersökt kringgående av lagstiftningen med hjälp utav efterföljande finansiering samt ett kringgående med hjälp av andra rättsobjekt. Med efterföljande finansiering menas att ett bolag köps med bolagets egna pengar, med hjälp av undantaget för koncernlån. Ett kringgående med hjälp utav andra rättsobjekt kan se ut på lite olika sätt. I vårt arbete har vi använt av oss utav en fysisk person samt ett handelsbolag. Ett kringgående av lagstiftningen möjliggörs genom att andra rättsobjekt än aktiebolag ej lyder under aktiebolagslagen i stora drag. Dessa handlingar rör sig inom ett grått område inom juridiken och gör låneförbudet till ett tämligen trubbigt redskap. Eftersom låneförbudet tillhör specialstraffrätten möjliggörs kringgående av lagstiftningen då restriktiv lagtolkning måste användas. Faller en handling inte in ordagrant i vad som står i lagtexten är kringgåendet av låneförbudet både i nya och gamla ABL ett faktum.