Malgré l'amélioration des traitements, environ 20% des patients atteints de leucémie aigue lymphoblastiques (LAL) rechutent dans la moelle osseuse ou dans des sites extra-médullaires tels que les ovaires et les testicules, ce qui est particulièrement fréquent dans les rechutes tardives de LAL-B présentant un remaniement ETV6/RUNX1. Les travaux réalisés par Virginie Gandemer en 2007, ont montré que l'expression de CD9 permettait de distinguer les leucémies ETV6/RUNX1 des autres types de leucémie. Le gène CD9 code pour une protéine de la famille des tétraspanines dont l'expression a été corrélée avec le risque métastatique et la survie des patients. Par ailleurs il a été démontré que la protéine CD9 était impliquée dans le homing et la prise de greffe des cellules souches hématopoïétiques et leucémiques. Nous avons donc émis l'hypothèse qu'à travers ses propriétés fonctionnelles sur la migration et le homing, CD9 pourrait être un acteur clé des rechutes de LAL-B. Le but de ce travail de thèse était donc premièrement de déterminer le mode de régulation de CD9 dans les LAL-B ETV6/RUNX1 et deuxièmement de déterminer les effets de l'expression de CD9 sur la motilité et la prise de greffe des LAL-B. Les analyses préalablement réalisées au laboratoire avaient suggéré que CD9 pouvait être régulé par des miARNs. Nous avons identifié un cluster de 3 miARNs potentiellement impliqués dans la régulation de CD9 dans les LAL-B ETV6/RUNX1. Ces résultats doivent cependant être complétés par d'autres analyses fonctionnellles afin d'être confirmés. Nous avons étudié le rôle de la protéine CD9 dans la dissémination des cellules de LAL-B. Nous avons démontré que CD9 était un régulateur potentiel de l'adhésion et un nouveau facteur impliqué dans la migration et le homing dépendants de CXCR4 en favorisant l'activation de RAC1 et les réarrangements de l'actine en réponse au CXCL12. Enfin, nous avons décrit pour la première fois l'influence de CD9 sur la migration et le homing dans les testicules via RAC1. Nos résultats montrent donc que CD9 favorise la dissémination des cellules de LAL-B dans les testicules et suggèrent que cette protéine pourrait constituer un acteur majeur des rechutes tardives de LAL-B dont les mécanismes d'apparitions sont peu connus. / Despite improvements in survival rates, approximately 20% of children suffering from acute lymphoblastic leukemia (B-ALL) present relapses from bone marrow or from B-extramedullary sites, such as the testes or ovaries, particularly in cases of late relapse of ETV6/RUNX1-ALL. Virgine Gandemer showed in 2007, that the expression of CD9, a protein from the tetraspanin superfamily, can be used to distinguish ETV6/RUNX1 lymphoblastic leukemia from other types of ALL. CD9 expression has been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer. Moreover CD9 has been implicated in hematopoietic and leukemic stem cell homing. We hypothesized, that CD9 protein, through its functional properties on migration and homing, could be a key actor of B-ALL relapses. The purpose of our study was then to investigate, first the transcriptional regulation of CD9 in ETV6/RUNX1 B-ALL and secondly, the effect of CD9 expression on motility and engrafment of B lymphoblasts. The analysis of CD9 transcriptional regulation previously made in the team, suggested that it could be regulated by miRNAs. We identified a cluster of 3 miRNAs potentially implicated in the regulation of CD9 expression in ETV6/RUNX1 B-ALL. This result has to be confirmd by more functional analysis. We investigated the role of CD9 in the dissemination of B-ALL. We identified CD9 as a potential regulator of B-ALL cell adhesion and a new factor involved in CXCR4-mediated migration and homing, through the promotion of actin rearrangement in response to CXCL12. We also characterized the effect of CD9 protein expression on RAC1 activation, which had an impact on blast migration and engraftment. Finally, we described, for the first time, the influence of CD9, mediated by RAC1 signaling, on B-cell chemotactic migration and homing in the testis. Our work provides evidence for an impact of CD9 on the ability of pre-B leukemic cells to disseminate to testes, through its effects on migration and homing, and suggests that CD9 may be a key player in late relapses of B-ALL, which are currently poorly understood.

Este estudo teve como objetivo avaliar o estado nutricional relativo ao selênio (Se) de pacientes na fase de pós-tratamento da leucemia linfóide aguda (LLA) e sua relação com o estresse oxidativo. Foram selecionados 24 pacientes no pós-tratamento da LLA (9,2 ± 1,9 anos) atendidos no Instituto de Oncologia Pediátrica da Universidade Federal de São Paulo e 60 indivíduos saudáveis (9,5 ± 1,3 anos) da Escola de Aplicação da Universidade de São Paulo. Foram coletados 10 mL de sangue venoso para análise de Se plasmático e eritrocitário, glutationa peroxidase (GPx), superóxido dismutase (SOD), α- tocoferol, malondialdeído (MDA) e 8-oxo-desoxiguanosina (8-oxo-dGuo). A urina de 24 horas foi coletada para análise da excreção de Se, e três recordatórios de consumo alimentar de 24 horas para avaliação do Se ingerido. Os resultados obtidos quanto aos parâmetros bioquímicos de avaliação de Se não apresentaram diferença significativa entre os grupos de pacientes e controles, e foram respectivamente: Se plasmático, 44,4 ± 9,0 µg/L e 48,7 ± 12,0 µg/L (p = 0,122); Se eritrocitário, 49,9 ± 15,9 µg/L e 45,0 ± 15,9 µg/L (p = 0,202); Se urinário, 19,6 ± 14,8 µg Se/g de creatinina e 18,6 ± 9,6 µg Se/g de creatinina (p = 0,820). O consumo médio de Se foi de 27,4 ± 8,7 µg/dia e 28,0 ± 1,5 µg/dia (p = 0,756), respectivamente. Os grupos estudados foram considerados deficientes em Se, considerando os pontos de corte adotados. A atividade da GPx foi significativamente menor nos pacientes do que nos controles (33,3 ± 11,1 U/g Hb e 76,9 ± 25,9 U/g Hb) (p = 0,000), e a atividade da SOD não diferiu entre pacientes e controles (1796,9 ± 257,8 U/g Hb e 1915,9 ± 473,9 U/g Hb) (p = 0,145), assim como as concentrações de MDA (1,7 ± 0,3 µmol/L e 1,8 ± 0,4 µmol/L) (p = 0,053). A concentração de α-tocoferol foi estatisticamente maior nos pacientes que nos controles (17,7 ± 4,7 µmol/L e 10,6 ± 3,2 µmol/L) (p =0,000), bem como a concentração de 8-oxo-dGuo (43,6 ± 28,0 8-oxo/106 dG e 21,3 ± 22,9 8-oxo/106 dG) (p = 0,014). Os resultados apresentados apontam que os participantes deste estudo estão deficientes em Se e, em especial os pacientes no pós-tratamento da LLA estão sujeitos a um aumento do estado de estresse oxidativo, pois apesar das concentrações de MDA serem semelhantes entre os pacientes e os controles, a atividade da GPx dos pacientes foi reduzida e a concentração de 8-oxo-dGuo e α-tocoferol estavam aumentadas em relação aos controles. / This study aimed to evaluate the nutritional status of selenium (Se) in patients in post-treatment of acute lymphoblastic leukemia (ALL) and its relationship with oxidative stress. We selected 24 patients in post-treatment of ALL (9.2 ± 1.9 years) at the Pediatric Oncology Institute of Federal University of São Paulo and 60 healthy individuals (9.5 ± 1.3 years) of the School of Application at the University of São Paulo. We collected 10 mL of venous blood for analysis of Se in plasma and erythrocytes, glutathione peroxidase (GPx), superoxide dismutase (SOD), α-tocopherol, malondialdehyde (MDA) and 8-oxo-deoxyguanosine (8-oxo-dGuo). The 24-hour urine was collected for analysis of Se excretion and Se intake was evaluated by using three non-consecutive days of 24- hour recall. The results regarding biochemical evaluation of Se did not differ significantly between patients in post-treatment of ALL and controls, and the results were respectively: Se in plasma, 44.4 ± 9.0 µg/L and 48.7 ± 12.0 µg/L (p = 0.122); Se in erythrocytes, 49.9 ± 15.9 µg/L and 45.0 ± 15.9 µg/L (p = 0.202); Se in urine, 19.6 ± 14.8 µg Se/g creatinine and 18.6 ± 9.6 µg Se/g creatinine (p = 0.820). The average intake of selenium was 27.4 ± 8.7 mg/day and 28.0 ± 1.5 mg/day (p = 0.756), respectively. Both groups were considered deficient in selenium, according to the cut-off points adopted. The GPx activity was significantly lower in patients than in controls (33.3 ± 11.1 U/g Hb and 76.9 ± 25.9 U/g Hb) (p = 0.000), and no difference in SOD activity was observed between groups (1796.9 ± 257.8 U/g Hb and 1915.9 ± 473.9 U/g Hb) (p = 0.145). MDA concentrations were not different between patients and controls (1.7 ± 0.3 µmol/L and 1.8 ± 0.4 µmol/L) (p = 0.053) and α-tocopherol concentration was statistically higher in patients (17.7 ± 4.7 µmol/L and 10.6 ± 3.2 µmol/L) (p = 0.000), as well the concentration of 8-oxo-dGuo (43.6 ± 28.0 8-oxo/106 dG and 21.3 ± 22.9 8-oxo/106 dG) (p = 0.014). These results indicate that the participants in this study are deficient in Se mainly those who are in post-treatment of ALL are exposed to an increased state of oxidative stress, because although the concentrations of MDA were similar between patients and controls, the GPx activity of the patients was reduced and the concentration of 8-oxo-dGuo and α-tocopherol were increased compared to controls.

Orientador: José Andrés Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:04:03Z (GMT). No. of bitstreams: 1 Laranjeira_AngeloBrunelliAlbertoni_D.pdf: 13248620 bytes, checksum: 729ec8e23331934ddd1e5803361b6fe6 (MD5) Previous issue date: 2012 / Resumo: A Leucemia Linfóide Aguda (LLA) é o tipo de câncer mais comum que acomete crianças. Sabe-se que a interação do tumor com o contexto celular do hospedeiro (microambiente tumoral) é recíproca, ou seja, na medida em que o tumor estimula o seu microambiente, este potencializa a sobrevivência, proliferação e invasividade tumoral. A interação da LLA com as células estromais da medula óssea tem um impacto positivo na resistência das células leucêmicas à quimioterapia. No presente estudo foi investigado a modulação de uma série genes de sensibilidade e resistência à asparaginase em células de LLA-B precursoras após co-cultura com as células estromais. Mostramos o aumento da expressão e secreção da IGFBP7 pelas células leucêmicas após co-cultivo com células do estroma da medula óssea. Em ensaios com o silenciamento do IGFBP7 em células leucêmicas e células estromais, mostramos que a IGFBP7 atua regulando positivamente o crescimento celular e aumenta a resistência a asparaginase. A IGFBP7 'leucêmica' junto com IGF/insulina atua sobre as células estromais, induzindo nestas células o aumento da produção de asparagina, e diminuindo a ação da asparaginase. Além deste mecanismo de resistência dependente das células estromais, mostramos que a IGFBP7 em conjunto com IGF/insulina promove a resistência das células leucêmicas à ação de outros compostos quimioterápicos (dexametasona e metotrexato) de forma independente da interação leucemia-estroma. Ainda pode ser observado que o plasma de crianças com LLA ao diagnóstico, apresenta maiores níveis de IGFBP7 do que em amostras controles. É importante ressaltar que níveis mais altos de mRNA IGFBP7 foram associados com menor sobrevida livre de leucemia (Modelo de regressão de Cox, P = 0,003), em células de LLAB Ph(-) presursoras / Abstract: Acute Lymphoblastic Leukemia (ALL) is the most common type of cancer that affects children. It is known that the interaction between tumor and the cellular context of the host (tumor microenvironment) is reciprocal, ie, to the extent that the tumor stimulates their microenvironment, this enhances the survival, proliferation and tumor invasiveness. The interaction of ALL with bone marrow stromal cells has a positive impact on leukemia resistance to chemotherapy. In the present study, we investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL upon co-culture with stromal cells. We showed an increase expression and secretion of IGFBP7 in leukemic cells after co-culture with BMSCs. Assays with IGFBP7 knockdown in leukemic cells and stromal cells, showed that IGFBP7 acts as a positive regulator of cell growth and increases resistance to asparaginase. 'Leukemic' IGFBP7 together with IGF/insulin acts on stromal cells, increasing asparagine production, thus reducing the asparaginase effect. Besides this mechanism of resistance dependent of stromal cells, we showed that IGFBP7 in conjunction with IGF/insulin promotes the resistance of leukemia cells to the action of other chemotherapeutic compounds (dexamethasone and methotrexate) independently of the interaction leukemia-stroma. We still observed that diagnostic BM plasma from children with ALL at diagnosis, have higher levels of IGFBP7 than control samples. Importantly, higher levels of IGFBP7 mRNA were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-ALL Ph (-) patients / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Orientador: José Andrés Yunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T08:28:04Z (GMT). No. of bitstreams: 1 Milani_Mateus_M.pdf: 3959646 bytes, checksum: a36674e099fefbc487286cdc33006c0a (MD5) Previous issue date: 2014 / Resumo: A leucemia linfoide aguda (LLA) é o câncer mais comum na infância. O transplante de células primárias de LLA humana em camundongos imunosuprimidos tem sido de suma importância para o entendimento da fisiopatologia da doença e para o teste de novos fármacos. Ao contrário de modelos animais de tumores sólidos, cujo volume é facilmente medido na superfície dos animais, a LLA infiltra órgãos inacessíveis ao exterior, daí a necessidade de definir métodos adequados para o monitoramento da progressão da doença. Resultados aqui apresentados indicam que proteínas secretadas pela LLA podem servir como marcadores quantitativos da carga leucêmica, facilmente aferidos por ELISA de amostras de plasma sanguíneo. Dentre três proteínas testadas (B2M, IGFBP2 e Hsp90), o ELISA de Hsp90 apresentou sensibilidade superior à análise da porcentagem de células leucêmicas no sangue dos animais, por citometria de fluxo de células marcadas com anti-huCD45. Os níveis de Hsp90 humano no plasma sanguíneo mostraram-se positivamente correlacionados com o porcentual de células leucêmicas na medula óssea e fígado e em menor grau com os níveis do baço e sangue periférico (SP) ao longo do tempo, tanto nas LLA de linhagem B quanto nas LLA-T. O ELISA de Hsp90 permite detectar a instauração da leucemia nos animais transplantados, até duas semanas antes da detecção pelo método tradicional de análise de sangue periférico por citometria de fluxo. Ao contrário do observado para IGFBP2, o tratamento dos animais leucêmicos com Dexametasona ou um inibidor da PI3K não interferiu nos níveis de Hsp90, que se mantiveram proporcionais à porcentagem de células leucêmicas huCD45+ no sangue periférico. No conjunto, os resultados demonstram que a análise do plasma dos animais por ELISA de Hsp90 é um método melhor do que os atualmente utilizados, para diagnóstico precoce e acompanhamento de LLA humana quando em níveis de doença residual mínima, ou seja, quando a porcentagem de células de LLA é inferior a 5% do total de células da medula óssea / Abstract: Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer . The transplantation of human primary ALL cells in immunodeficient mice has been of much importance for understanding the disease's pathophysiology and testing new drugs. Unlike animal models of solid tumors whose volume is easily measured on the animal surface, the ALL infiltrates organs that are inaccessible to external antigens, hence the need to define more suitable methods for monitoring the disease's progression. Results presented here indicate that proteins secreted by the ALL can serve as quantitative markers of leukemic burden and are easily measured by ELISA of blood plasma samples. Among three tested proteins (B2M, IGFBP2 and Hsp90), Hsp90 ELISA analysis showed higher sensitivity than the analysis of leukemic cells on animal blood by flow cytometry of anti- huCD45 labeled cells. The levels of Hsp90 in human blood plasma were shown to be positively correlated with the percentage of leukemic cells in the bone marrow and liver and to a lesser extent with the levels in the spleen and peripheral blood (PB) over time, both in B-lineage ALL as in ALL-T. The Hsp90 ELISA allows the leukemia's engraftment detection in transplanted animals up to two weeks prior to detection by the traditional method of peripheral blood analysis by flow cytometry. Unlike observed for IGFBP2, treatment of leukemic animals with Dexamethasone or PI3K inhibitors did not interfere in Hsp90 levels, which remained proportional to the percentage of huCD45+ leukemic cells in the peripheral blood. Taken together, the results demonstrate that the analysis of animal plasma by Hsp90 ELISA is a better method than those currently used for early diagnosis and monitoring of human ALL on minimal residual disease levels, when the percentage of ALL cells is less than 5 % of the total bone marrow cells / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Hintergrund: Der Phospholipase A2 Rezeptor 1 (PLA2R1) ist ein Typ 1 Transmembranrezeptor, welcher der Mannose-Rezeptor-Familie zugeordnet werden kann. Die Bedeutung von PLA2R1 für physiologische und pathologische Vorgänge ist noch weitestgehend unbekannt. Jedoch wird die Regulation wichtiger zellulärer Prozesse, wie Proliferation, Apoptose/ Seneszenz, Adhäsion, Migration/ Invasion und Inflammation im Zusammenhang mit dem Rezeptor diskutiert. Darüber hinaus ist eine Änderung der PLA2R1-Expression bei der Entstehung verschiedenster Krebserkrankung nachweisbar. Hierbei wird der Rezeptor einerseits mit einer pro-onkogenen und pro-migratorischen Wirkung in Verbindung gebracht. Andererseits ist ein tumorsuppressiver Effekt von PLA2R1 und eine Induktion der mitochondrialen Apoptose in Tumorzellen beschrieben. Zudem ist die Expression von PLA2R1 durch epigenetische Mechanismen kontrolliert und eine Promotor-Hypermethylierung ist assoziiert mit einer Repression der Rezeptor-Expression in der Prostatakarzinom (PCa)-Zelllinie LNCaP und der pädiatrischen, akuten lymphoblastischen Leukämie (ALL)-Zelllinie Jurkat. Vorangegangene Arbeiten zeigten eine Hypermethylierung innerhalb eines definierten Bereiches des PLA2R1-Promotors bei adulten Patienten mit akuter myeloischer Leukämie und Myelodysplastischem Syndrom (MDS) sowie eine Korrelation der PLA2R1-Promotormethylierung mit dem Krankheitsstadium und der Klassifizierung nach dem Internationalen Prognostischen Scoring System (IPSS). Fragestellung/ Hypothese: Das Ziel der vorliegenden Arbeit war einerseits die Untersuchung der Funktion von PLA2R1 in den PCa-Zelllinien LNCaP und PC-3. Während in LNCaP die Rezeptor-Expression durch Promotor-Hypermethylierung unterdrückt ist, kann in PC-3-Zellen eine Hochregulation von PLA2R1 im Vergleich zu normalen Prostataepithelzellen nachgewiesen werden. Durch in vitro Transfektionsexperimente sollte der Effekt einer Re-expression von PLA2R1 in LNCaP-Zellen sowie die Auswirkungen einer Reduktion der PLA2R1-Expression in PC-3-Zellen untersucht werden. Der Einfluss der veränderten PLA2R1-Expressionslevel auf wichtige Zellparameter wurde evaluiert. Die in vitro Daten der PCa-Zelllinien wurden mit den in vivo Ergebnissen des Tumorwachstums von transfizierten LNCaP- und PC-3-Zellen in Xenograft-Mausmodellen verglichen. Andererseits sollte basierend auf den Ergebnissen von adulten Patienten mit AML- und MDS-Diagnose und der dabei festgestellten Hypermethylierung des Rezeptor-Promotors der Methylierungsstatus des Rezeptors bei der pädiatrischen ALL untersucht werden. Überdies sollte die Eignung der PLA2R1-Methylierungsanalyse als sensitiver Biomarker für die Therapiekontrolle, Überwachung der minimalen Resterkrankung (MRD) und Risikostratifizierung der pädiatrischen ALL evaluiert werden. Die Funktion des Rezeptors im Kontext der pädiatrischen ALL wurde durch eine transfektionsbasierte Re-expression von PLA2R1 in der Jurkat-ALL-Zelllinie untersucht. Durch in vitro Experimente wurden die Auswirkungen der verschiedenen PLA2R1-Expressionslevel auf Proliferation und Apoptose/ Nekrose in transfizierten Jurkat-Zellen analysiert. Material und Methoden: Durch Transfektion mit einem PLA2R1-Expressionsvektor konnte eine stabile Überexpression des Rezeptors in LNCaP- (LNCaP-PLA2R1) und Jurkat-Zellen (Jurkat-PLA2R1) erreicht und die Ergebnisse mit Kontrollvektor-transfizierten LNCaP- (LNCaP-Ctrl) und Jurkat-Zellen (Jurkat-Ctrl) verglichen werden. Mittels CRISPR/Cas9-Knockdown konnte eine Verminderung der PLA2R1-Expression in PC-3-Zellen (PC-3-KD) im Vergleich zu Kontrollvektor-transfizierten PC-3-Zellen (PC-3-Ctrl) erreicht werden. Genexpressionsanalysen wurden mittels quantitativer PCR nach reverser Transkription (RT-qPCR) durchgeführt und die Proteinsynthese des Rezeptors durch Western Blot Analyse überprüft. In vitro sollten die Auswirkungen der differenziellen PLA2R1-Expression der transfizierten Zellen auf wichtige proliferative und metastatische Zellparameter untersucht werden. Die Zellviabilität/ Proliferation wurde mittels WST-1 Assay für adhärente Zellen und Zellwachstumskurven-Analyse mit Trypanblau-Färbung bei Suspensionszellen analysiert. Zellmotilität und Proliferation wurden bei transfizierten PCa-Zelllinien mithilfe des Wundheilungsassays beurteilt. Apoptose konnte durch Wasserstoffperoxid stimuliert und mittels Caspase-Glo® 3/7 Assay und RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay für transfizierte PCa-Zelllinien sowie durchflusszytometrische Analysen nach Annexin-V-FLUOS/ Hoechst 33258 Färbung für transfizierte Jurkat-Zellen untersucht werden. Die klonogene Überlebensrate und das Koloniewachstum der transfizierten PCa-Zelllinien sollten mithilfe des klonogenen Assays analysiert werden. In einer in vivo Pilotstudie wurde der Effekt von PLA2R1 auf das Tumorwachstum mittels Xenograft-Mausmodellen (männliche SCID/beige Mäuse) durch subkutane Injektion der transfizierten LNCaP- (n = 5) und PC-3-Zellen (n = 9) überprüft. Die PLA2R1-Promotormethylierung als sensitiver Biomarker für die pädiatrische ALL wurde durch Isolation und Bisulfit-Behandlung der genomischen DNA von Knochenmark (KM)-Aspiraten und Leukozyten des peripheren Blutes (PB) von ALL-diagnostizierten Kindern (n = 44) sowie einer anschließenden Analyse mittels digitaler PCR (dPCR) evaluiert. Die Ergebnisse konnten mit dem Methylierungsstatus einer gesunden Kontrollgruppe (n = 20) verglichen werden. Ergebnisse und Schlussfolgerungen: In LNCaP-PLA2R1 und Jurkat-PLA2R1 konnte im Gegensatz zu den dazugehörigen Kontrollzellen eine stabile Überexpression des Rezeptors auf Ebene der Genexpression und Proteinsynthese detektiert werden. Bei PC-3-KD-Zellen war eine Reduktion der PLA2R1-Genexpression und eine Repression der Proteinsynthese unterhalb der Nachweisgrenze des Western Blot Assays zu verzeichnen, während PC-3-Ctrl-Zellen eine Genexpression und Proteinsynthese des Rezeptors zeigten. Die Zellviabilität/ Proliferation und Motilität war signifikant erhöht in LNCaP-PLA2R1 und PC-3-Ctrl im Vergleich zu LNCaP-Ctrl- und PC-3-KD-Zellen. Demgegenüber war eine Verminderung von Apoptose und Koloniewachstum in LNCaP-PLA2R1 und PC-3-Ctrl-Zellen nachweisbar. Durch Genexpressionsanalysen konnte eine Induktion der Expression von Fibronektin 1 (FN1), TWIST Homolog 1 (TWIST1) und Cyclin-abhängige Kinase 6 (CDK6) in LNCaP-PLA2R1-Zellen identifiziert werden. In vivo schien die PLA2R1-abhängige negative Regulation des Koloniewachstums die pro-onkogenen Eigenschaften des Rezeptors zu überwiegen. Dies resultierte in einem verminderten Tumorwachstum von LNCaP-PLA2R1 und einer tumorsuppressiven Rolle des Rezeptors in dieser PCa-Zelllinie. Im Gegensatz dazu zeigten PC-3-Ctrl-Zellen ein schnelleres Tumorwachstum im Xenograft-Mausmodell, was für einen pro-onkogenen Effekt der endogenen PLA2R1-Expression in PC-3-Zellen sprechen würde. Der differenzielle Einfluss von PLA2R1 auf die Regulierung des Tumorzellwachstums könnte im Zusammenhang mit der veränderten Expression von FN1, TWIST1 und CDK6 stehen, jedoch sind weiterführende Experimente nötig, um die Beteiligung dieser Gene in der PLA2R1-Signaltransduktion zu untersuchen. Die Analyse der Zellwachstumskurve der transfizierten Jurkat-Zellen zeigte eine Abnahme der Proliferationsrate und eine Zunahme des Anteils an toten Zellen bei Jurkat-PLA2R1 im Vergleich zu Jurkat-Ctrl-Zellen. Durchflusszytometrische Analysen bestätigten eine Abnahme des Anteils gesunder sowie eine vermehrte Repräsentation von apoptotischen und nekrotischen Jurkat-PLA2R1-Zellen im Vergleich zur Kontrolle, was einen tumorsuppressiven Einfluss des Rezeptors bei der pädiatrischen ALL suggeriert. Die Funktion von PLA2R1 als Tumorsuppressor steht im Einklang mit der festgestellten Hypermethylierung des Rezeptor-Promotors in KM-Aspiraten und PB-Proben von pädiatrischen Patienten mit prä-B und common ALL zum Zeitpunkt der Diagnose der primären Krebserkrankung und des ALL-Rezidives im Vergleich zu der Kontrollgruppe. Der parallele Abfall der PLA2R1-Promotormethylierung und der relativen Blastenzahl im Verlauf der ALL-Induktionstherapie sowie eine signifikante, positive Korrelation beider Größen in KM- und PB-Proben ließen auf die leukämischen Blasten als Quelle der Hypermethylierung des PLA2R1-Promotors schließen. Überdies wiesen Hochrisikopatienten der pädiatrischen ALL eine signifikant höhere PLA2R1-Promotormethylierung am Tag 15 der ALL-Induktionstherapie auf im Vergleich zu Patienten mit einem geringeren Risiko. Zusammenfassend deuteten die in vitro und in vivo Daten auf eine wichtige Funktion des Rezeptors bei der Regulation von Proliferation und Apoptose bei der pädiatrischen ALL hin. Die Analyse der PLA2R1-Promotormethylierung könnte als sensitiver Biomarker zu einer verbesserten ALL-Therapiekontrolle, MRD-Überwachung und Risikostratifizierung während der ALL-Induktionstherapie beitragen.:Inhaltsverzeichnis 1 Zusammenfassung 4 2 Abstract 8 3 Einführung in die Thematik 11 4 Publikation 1: “Diverse Effects of Phospholipase A2 Receptor Expression on LNCaP and PC-3 Prostate Cancer Cell Growth in vitro and in vivo” 24 5 Publikation 2: “Methylation of the Phospholipase A2 Receptor 1 Promoter Region in Childhood B Cell Acute Lymphoblastic Leukaemia” 25 6 Diskussion und Ausblick 26 7 Literaturverzeichnis 32 8 Danksagung 41 9 Anlagen 42 / Background: The phospholipase A2 receptor 1 (PLA2R1) is a type I transmembrane receptor and a member of the mannose receptor family. Physiological and pathophysiological functions of PLA2R1 are still not completely understood. However, PLA2R1 expression is discussed to have an impact on proliferation, apoptosis/ senescence, adhesion, migration/ invasion as well as inflammatory cell responses and divergent PLA2R1 expression is detectable in different types of cancer compared to corresponding normal tissues. In this context, receptor expression is linked to both a pro-oncogenic/ pro-migratory and a tumour-suppressive/ pro-apoptotic impact in different cancer cells. Moreover, PLA2R1 expression is controlled by epigenetic mechanisms and hypermethylation of the PLA2R1 promoter is associated with silenced expression of the receptor in the prostate carcinoma (PCa) cell line LNCaP and the paediatric, acute lymphocytic leukaemia (ALL) cell line Jurkat. Previous work revealed a defined hypermethylated region of the PLA2R1 promoter in adult patients with acute leukaemia and myelodysplastic syndrome (MDS). PLA2R1 promoter methylation correlated with disease stage and International Prognostic Scoring System (IPSS) classification. Aim: The aim of the present study was to evaluate the function of PLA2R1 in PCa cell lines LNCaP and PC-3. The receptor expression is silenced in LNCaP but upregulated in PC-3 cells compared to normal prostate epithelial cells. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Based on previous findings of PLA2R1 promoter hypermethylation in adult ALL and MDS patients, the aim of the present study was to analyse the methylation status of the PLA2R1 promoter in paediatric ALL patients compared to healthy individuals. PLA2R1 methylation analysis was evaluated as sensitive biomarker for ALL treatment response, minimal residual disease (MRD) monitoring, and risk stratification. The impact of the receptor in childhood ALL was investigated by transfection-based re-expression of PLA2R1 in the paediatric ALL cell line Jurkat and the effect of different PLA2R1 expression levels on proliferation and apoptosis/ necrosis was analysed in in vitro experiments. Material and Methods: Stable PLA2R1 overexpression was achieved by transfection of LNCaP (LNCaP-PLA2R1) and Jurkat cells (Jurkat-PLA2R1) with a PLA2R1 plasmid vector. Results were compared to control vector transfected LNCaP (LNCaP-Ctrl) and Jurkat cells (Jurkat-Ctrl). Alternatively, PLA2R1 was knocked down using CRISPR/Cas9 in PC-3 cells (PC-3-KD) and compared to the corresponding control-transfected cells (PC-3-Ctrl). Gene expression analysis was conducted by quantitative reverse transcription PCR (RT-qPCR). PLA2R1 protein synthesis was analysed by western blot. The impact of the differential PLA2R1 expression on proliferative and metastatic parameters of transfected cancer cells was investigated in vitro. Cell viability/ proliferation was assessed by means of WST-1 Assay for adherent cells and via cell growth curve analysis after trypan blue staining for suspension cells. Cell motility and proliferation of transfected PCa cell lines were estimated by wound healing assay. Hydrogen peroxide-stimulated apoptosis was analysed by Caspase-Glo® 3/7 Assay and RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay for transfected PCa cell lines and flow cytometric analysis after Annexin-V-FLUOS/ Hoechst 33258 staining for transfected Jurkat cells. Colony formation of transfected PCa cell lines was evaluated by clonogenic assay. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP (n = 5) and PC-3 cells (n = 9). Evaluating PLA2R1 promoter methylation as sensitive biomarker for paediatric ALL, genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of 44 paediatric ALL patients. After bisulfite treatment of isolated DNA samples, PLA2R1 methylation was analysed using digital PCR and compared to 20 healthy controls. Results and Conclusions: PLA2R1 gene expression and protein synthesis were detectable in LNCaP-PLA2R1, PC-3-Ctrl, and Jurkat-PLA2R1 cells but not in LNCaP-Ctrl and Jurkat-Ctrl cells. In PC-3-KD cells, PLA2R1 gene expression was significantly reduced compared to PC-3-Ctrl and PLA2R1 protein synthesis of PC-3-KD cells was below the limit of detection of western blot analysis. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3-Ctrl compared to LNCaP-Ctrl and PC-3-KD cells, respectively. However, levels of apoptosis and clonogenicity were reduced in LNCaP-PLA2R1 and PC-3-Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1 compared to control cells. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor’s pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3-Ctrl xenografts exhibited faster tumour growth compared to PC-3-KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required. Cell growth curve analyses of transfected Jurkat cells revealed a decreased proliferation and increased cell death of Jurkat-PLA2R1 compared to Jurkat-Ctrl cells. Flow cytometry confirmed the reduced fraction of healthy cells and an increase of the apoptotic and necrotic fractions in Jurkat-PLA2R1 cells compared to control cells, suggesting a tumour-suppressive effect of the receptor in paediatric ALL. PLA2R1’s tumour-suppressive function is in accordance with hypermethylation of the receptor promoter in BM aspirates and PB samples of paediatric patients diagnosed with pre-B and common ALL as well as in patients with disease relapse in comparison to healthy controls. PLA2R1 methylation decreased along with leukaemic blast cell reduction during ALL induction treatment and significant positive correlations between PLA2R1 methylation and leukaemic blast cell numbers of BM and PB samples were observable. Therefore, our data suggests that leukaemic blasts are the origin of PLA2R1 hypermethylation in BM and PB samples. Moreover, high risk paediatric ALL patients exhibited increased levels of PLA2R1 promoter methylation compared to non-high risk groups on day 15 of ALL induction treatment. Collected data indicates that PLA2R1 promoter methylation quantitation can be used as biomarker for ALL induction treatment control, risk stratification, and early detection of ALL relapse.:Inhaltsverzeichnis 1 Zusammenfassung 4 2 Abstract 8 3 Einführung in die Thematik 11 4 Publikation 1: “Diverse Effects of Phospholipase A2 Receptor Expression on LNCaP and PC-3 Prostate Cancer Cell Growth in vitro and in vivo” 24 5 Publikation 2: “Methylation of the Phospholipase A2 Receptor 1 Promoter Region in Childhood B Cell Acute Lymphoblastic Leukaemia” 25 6 Diskussion und Ausblick 26 7 Literaturverzeichnis 32 8 Danksagung 41 9 Anlagen 42

Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l’enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues.L’objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d’incubation, elles sont fortement tuées après 25h.Cette cytotoxicité est contact-dépendante mais ni la voie de l’exocytose des granules cytotoxiques ni celle des récepteurs de mort n’y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l’amplifie partiellement. Interférer avec les dérivés de l’oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l’X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l’expression par les NK des composants clefs d’une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l’existence d’une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires. / Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and anti-tumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood.In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h.Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with X-linked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway.

Bakgrund: Akut lymfatisk leukemi (ALL) är den vanligaste cancerformen bland barn och ungdomar och kan innebära stor fysisk och psykisk påfrestning hos föräldrar och barn. När barnet drabbas av ALL kan föräldrar känna oro och rädsla för att förlora barnet. Syfte: Syftet med studien var att belysa föräldrars upplevelser när barnet har drabbats av akut lymfatisk leukemi (ALL). Metod: En litteraturstudie med induktiv ansats genomfördes där elva artiklar granskades och valdes till resultat. Resultat: I resultatet framkom huvudkategorin: Upplevelse av att vara förälder till barn som drabbats av ALL med underkategorier: att känna livet krascha, att känna otillräcklighet, att oroas över ekonomin, att känna behov av förändrade föräldrastrategier och att blicka framåt. Andra huvudkategorin var Upplevelse av sjuksköterskans betydelse med underkategorier: att känna emotionellt stöd och att uppleva behov av information och undervisning. Föräldrar upplevde kommunikation med sjuksköterska som viktigt och hjälpte föräldrar hantera påfrestningen. Vid bristande kommunikation upplevde föräldrar att sjuksköterskan undanhöll information och tilliten till sjuksköterskan försvann. Slutsats: Studien kan ge kunskap om föräldrars upplevelser när barnet drabbas av ALL och vilka behov som finns av hjälp och stöd. Mer forskning krävs för att undersöka på vilket sätt sjuksköterskan kan underlätta för föräldrar under den påfrestande tiden. / Background: Acute lymphocytic leukemia (ALL) is the most common cancer in children and youth and may cause great physical and psychological burden on parents and children. Parents might worry and fear losing the child when their child has ALL. Aim: The aim of this study was to explore parent's experiences when their child suffered from acute lymphocytic leukemia (ALL). Method: A literature review with an inductive structure was performed where eleven articles were analyzed and chosen for results. Result: The result presents main category: Experience of being a parent off a child affected by ALL with subcategories: to feel life crashing, to feel insufficient, to worry about economics, to feel the need of changing parent strategies, to focus ahead. The second main category was The experience of the importance of the nurse with subcategories: to feel emotional support and to experience the need for information and education. Parents experienced that communication with the nurse was important and helped parents manage burden. Lack of communication made parents experience that the nurse withheld information and the trust disappeared. Conclusions: The study can provide knowledge about parent's experiences when their child has ALL and the need of help and support. More research is required to investigate in which way the nurse can ease parent's burden during the stressful time.

Cette étude s’intéresse au lien entre les dynamiques cérébrales et les capacités cognitives, cette problématique a déjà été explorée auparavant en imagerie cérébrale, notamment à l’aide de tâches effectuées pendant l’imagerie. Cependant la caractérisation de l’activité spontanée a principalement été faite soit avec une faible précision spatiale (capteur EEG/MEG), soit en IRMf qui a une faible résolution temporelle. L’objectif de cette thèse est de caractériser l’activité spontanée au repos au niveau cortical associée à différents processus cognitifs et leur performance. Le second chapitre cherche à établir les corrélats neuronaux de la performance de la mémoire au repos à l’aide des puissances spectrales localisées au niveau des sources corticales. Le troisième chapitre cherche à répliquer les méthodes utilisées dans l’article 1 avec les mêmes participants, mais dans un autre domaine cognitif afin d’établir les corrélats neuronaux de la fluence verbale ainsi que de discriminer une composante verbale et exécutive. Ces deux composantes ont été mises en évidence en utilisant une factorisation avec un test purement exécutif (Trail making test- condition 4) et un autre purement verbal (richesse du vocabulaire). Dans le quatrième chapitre, nous répliquons encore la méthode de l’article 1 avec les mêmes sujets, mais sur un test d’apprentissage verbal. Lors de l’apprentissage verbal, deux stratégies d’apprentissage (sériel et sémantique) possibles sont utilisées de manière concurrente, nous avons cherché à établir si des différences comportementales se traduisaient par des patrons d’activation différents. Dans le cinquième chapitre, nous avons cherché à établir des différences fonctionnelles entre les survivants de la leucémie et des sujets contrôles, puis à établir un lien entre la neurotoxicité et le déficit cognitif rencontré chez cette population, finalement nous avons établi un modèle intégrant neurotoxicité, performance cognitive et marqueur neurophysiologique fonctionnel cérébral. Cette recherche aura approfondi les connaissances sur l’état de repos et principalement fourni les premiers travaux qui mettent en lien l’activité cérébrale spontanée au repos au niveau des sources corticales avec plusieurs tests neuropsychologiques comportementaux. Les résultats ont amené des patrons d’activation spatio-fréquentielle différents, démontrant des spécificités reliées à certains tests comportementaux ou des traitements de l’information (sériel ou sémantique). Finalement les travaux sur les survivants de la leucémie ont montré que l’état de repos pouvait caractériser le fonctionnement des déficits cognitifs à long terme et être un marqueur de remédiation pour de futurs traitements. / This study is interested in the link between brain dynamics and cognitive abilities. This problem has already been explored before in brain imaging, notably with the help of task performed during imaging. However, the characterization of spontaneous activity has mainly been done either with weak spatial resolution (EEG/MEG sensor) or in fMRI which has a low temporal resolution. The objective of this thesis is to characterize the spontaneous activity at rest at the cortical level associated with different cognitive processes and their performance. The second chapter seeks to establish the neural correlates of resting memory performance using spectral powers localized at cortical sources. The third chapter seeks to replicate the methods used in article 1 with the same participants but in another cognitive domain in order to establish the neural correlates of verbal fluency as well as to discriminate a verbal and an executive component. These two components were highlighted using a factorization with a purely executive test (Trail making test-condition 4) and another purely verbal one (vocabulary richness). In the fourth chapter, we replicate the method of article 1 with the same subjects, but on a verbal learning test. During verbal learning, two possible learning strategies (serial and semantic) are used concurrently, we sought to establish whether behavioural differences translate into different activation patterns. In the fifth chapter, we sought to establish functional differences between leukemia survivors and control subjects, then to search for a link between neurotoxicity and the cognitive deficit encountered in this population; finally we established a model integrating neurotoxicity, cognitive performance and functional neurophysiological brain markers. This research will have deepened the knowledge on the resting state and mainly provided the first works that link the spontaneous brain activity at rest at the level of cortical sources with several behavioural neuropsychological tests. The results led to different spatio-frequential activation patterns, showing specificities related to certain behavioural tests or information processing (serial or semantic). Finally, work on leukemia survivors has shown that resting states could characterize the functioning of long-term cognitive deficits and be a remediation marker for future treatments.

Acute lymphoblastic leukemia is a hematological malignancy that gains a proliferative advantage and originates in the bone marrow. One of the more common genetic alterations in ALL is KMT2A-rearrangement which constitutes 80% of the cases of ALL in infants. Patients carrying the KMT2A rearrangement have a poor prognosis and will eventually develop drug resistance. This project aimed to find new therapeutic targets which would help in the development of novel drugs. We designed a model which uses gene expression data, to infer expressions of oncogenes and the genes which could be associated with immune pathways. The data was extracted and transformed by removing the batch effects and identifying the biotypes of these genes for more focused research. Here we utilized exome RNA-seq,  hence it was necessary to reduce the high dimensionality of the data. The dimensionality reduction was performed using Monte Carlo Feature Selection. After the feature selection, a list of highly significant genes was obtained. These genes were used in a machine learning model, R.ROSETTA, which produces rule-based results centered on rough sets theory. The rules were visualized using VisuNet, an interactive tool that creates networks from the rules. Among others, we identified levels of expressions of genes such as JAK3, TOX3, and DMRTA1 and their relations to other genes  using the machine learning model. These significant genes were also used to do pathway analysis using pathfindR which allowed us to infer the oncogenic pathways. The pathway analysis helped us deduce pathways such as immunodeficiency and other signaling pathways that could be potential drugs