Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001. / "December 2001." Includes bibliographical references (leaves 178-225).

L'utilisation d'un agent déméthylant induit la réexpression de la protéine RIP3, une sérine-thréonine kinase, dans un modèle leucémique murin exprimant BCR-ABL humain. La réexpression de RIP3 conduit rapidement les cellules vers la nécroptose. Le mutant délété du domaine kinase est de façon surprenante plus "apoptogène" et induit le clivage de p65/RelA sur le résidu d'acide aspartique D361 par la caspase 6. Pour déterminer l'impact de ce clivage, nous avons construit un mutant non clivable p65/RelA D361E, ainsi que des plasmides exprimant chacun des fragments p65/RelA 1-361 ou p65/RelA 362-549, ou un plasmide exprimant simultanément p65/RelA 1-361 + p65/RelA 362-549. Ces différents plasmides codant pour les différentes formes de la protéine p65/RelA sont incorporés par transfection dans les cellules leucémiques ou de mélanome pour lesquels le gène RIP3 est respectivement méthylé ou exprimé. In vivo, nous mettons en évidence une différence de tumorigénicité entre les deux modèles. Elle est accrue par la présence de p65/RelA D361E par rapport à celle de p65/RelA WT et de p65/RelA 1-361 + p65/RelA 362-549 dans le modèle leucémique. Elle est au contraire faible dans le modèle du mélanome pour lequel la surexpression des fragments p65/RelA 1-361 +362-549 induit la tumorigenèse la plus forte. L'agressivité du mutant non clivable in vivo n'est pas corrélée à l'activité de NF-kB mesurée in vitro. Les fragments comme le mutant p65/RelA D361E induisent des profils d'expression différents dans le modèle murin de leucémie avec la modulation notable d'expression génique de la famille d'inhibiteurs de protéases à cystéine Stefins, ainsi que le transporteur de bicarbonate de sodium SLC4A5 qui joue un rôle majeur dans la régulation du pH intracellulaire. Le mutant p65/RelA D361E induit une expression importante du transporteur de bicarbonate de sodium SLC4A5 dans le modèle leucémique responsable de l'augmentation du pH intracellulaire qui participe au développement tumoral. Par contre, ce sont les deux fragments p65/RelA 1-361 + p65/RelA 362-549 qui induisent simultanément une expression plus forte de la molécule d'immunoéchappement PDL1, vraisemblablement par un mécanisme post-traductionnel. L'étude de la "souchitude" des modèles montre une différence d'activité du mutant p65/RelA D361E selon le modèle. On observe une augmentation de l'activité ALDH dans le modèle leucémique et une diminution de la formation de sphères dans le modèle de mélanome. En conclusion, ces résultats indiquent que les fragments issus du clivage de p65/RelA par l'activité de RIP3 indépendante de la kinase possèdent un rôle différent de celui de la forme sauvage sur la souchitude des cellules cancéreuses, et qu'elle dépend du modèle étudié. Ils confirment que le mutant non clivable possède la plus forte activité tumorigénique. Ils laissent également supposer que les fragments Nter et Cter puissent avoir une activité dans des cellules tumorales possédant une protéine RIP3 fonctionnelle et active, probablement par des mécanismes inflammatoires ou autres qui doivent être caractérisés. / The receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation, and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces p65/RelA caspase-mediated cleavage resulting in N-terminal 1-362 and C-terminal 362-549 fragments. We show here that a non-cleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decrease mouse survival and that coexpressed p65/RelA fragments increase tumoriginicty of B16/F1 melanoma cells that did not correlated with in vitro measured Nf-kB activity. Fragments and p65/RelA fragments display different expression profiles in DA1-3b leukemic cells, with the notable modulation of gene expression of the Stefin cysteine protease inhibitor family and of SLC4A5, a Na+-coupled HCO−3 transporter. DA1-3b cells expressing p65/RelA D361E mutant showed more basic intracellular pH. p65/RelA fragments induced ovexpression of PD-L1 immunoescape molecule in DA1-3b cells. Markers of stemness were also affected: p65/RelA D361E induced increased ALDH activity in DA1-3b cells and fragments expression resulted in increased melanoma sphere formation in B16/F1 cells. Thus, far from being neutral, p65/RelA cleavage initiated by kinase independent activity of RIPK3 induced a pleiotropic range of effects in vitro and in vivo in cancer cells, that may vary across tumor types.

A leucemia mielóide aguda (LMA) acomete pacientes de todas as faixas etárias com freqüência aumentada em idosos. Nosso estado não apresenta registros sistemáticos da patologia, justificando-se desta forma o levantamento através dos centros de diagnóstico e tratamento, os quais concentram a maioria dos casos. Foram incluídos todos os pacientes com diagnóstico de LMA “de novo” levantados a partir dos registros dos centros de diagnóstico e tratamento da patologia no estado no período entre 1996 e 2000. Foram computados no período 532 pacientes (taxa estimada de 1,04 casos / 100.000 habitantes / ano). A idade média ao diagnóstico foi 41,0 anos e 46,6 % dos casos eram do sexo masculino. Houve distribuição semelhante nas 7 mesorregiões geográficas do estado. Em relação ao número de casos por faixa etária, observou-se uma taxa estimada em 0,5 – 1 caso / 100.000 habitantes até a faixa etária dos 45 anos, atingindo 3,5 casos / 100.000 habitantes na faixa etária igual ou superior a 70 anos. A sobrevida média considerando todos os casos foi de 17%, aumentando para 25% considerando a população abaixo de 60 anos e alcançando 40% na população entre 10 e 20 anos. Tais dados evidenciam uma distribuição semelhante por região, estimativa de incidência na faixa infantil semelhante a países desenvolvidos e bastante menor na população idosa. A mortalidade geral foi semelhante àquela relatada na literatura. / Acute myeloid leukemia (AML) affects patients in all age groups, but it is more frequent among elderly people. The state of Rio Grande do Sul does not have systematic data on AML; thus these data have to be collected from hospitals or centers where the disease is diagnosed and treated. Patients with de novo acute myeloid leukemia were included based on the information retrieved from hospital registries between 1996 and 2000. At the time, 532 patients had been registered (estimated rate of 1.04 cases per 100,000 inhabitants / year). Mean age at diagnosis was 41.0 years, and 46.6% of the patients were male. Cases were similarly distributed across the seven meso-regions of Rio Grande do Sul. There was an estimated incidence of 0.5 to 1 case per 100,000 inhabitants up to the age of 45 years, and of 3.5 cases per 100,000 inhabitants for individuals aged 70 years and older. The mean five-year survival rate was 17% for all cases, 25% for patients under the age of 60 years, and 40% for those aged between 10 and 20 years. There was a similar distribution of AML cases per region, an incidence rate in the pediatric population similar to that of developed countries, and a much lower incidence among elderly patients. Overall mortality was comparable to that reported in the literature.

Made available in DSpace on 2016-08-29T15:34:30Z (GMT). No. of bitstreams: 1 tese_6281_Dissertação_Roberta Bitencourt Moreira_2013.pdf: 2949871 bytes, checksum: 0c46a98a4735b38e93978376b508fac6 (MD5) Previous issue date: 2013-03-06 / A Leucemia mielóide crônica (LMC) é caracterizada por uma alteração citogenética conhecida, o cromossomo Philadelphia (Ph),resultado da translocação recíproca t(9;22)(q34;q11).O gene de fusão bcr-abl codifica uma proteína de fusão com atividade tirosina cinase aumentada que desregula vias de transdução de sinais ligadas a proliferação, apoptose e diferenciação celular. A evolução natural da LMC quando não tratada é trifásica, mas atualmente, esta dinâmica foi alterada a partir do desenvolvimento dos inibidores de tirosina cinase (ITC). O imatinibe (Glivec®, Novartis) foi o primeiro ITC aprovado para o tratamento da LMC. A atualização de oito anos do estudo internacional randomizado do interferon e imatinibe (IRIS), ratificou a eficácia e a segurança do uso de imatinibea longo prazo com uma sobrevida global de 85% e uma sobrevida livre de evento de 81%.Entretanto, algumas mutações no domínio cinasedo gene bcr-ablconferem resistência elevada a um ou mais ITC influenciando a escolha da terapia subsequente como no caso de uma mutação T315I, que é altamente resistente ao imatinibe.Apesar de múltiplos fatores contribuírem para a resistência ao imatinibe, a presença de mutação é mais prevalente e tem sido a mais investigada. Diante disso, foram selecionados pacientes de dois hospitais da Região da Grande Vitória que realizam atendimento aos pacientes portadores de LMC pelo Sistema Único de Saúde (SUS) que são encaminhados para o Laboratório de Biologia Molecular do Centro de Transplante de Medula Óssea (CEMO) do Instituto Nacional de Câncer (INCA) para avaliação da resposta molecular aos ITCs, para análise de mutação. Também foi desenvolvido um banco de dados no Microsoft Access® para LMCque permite relacionar informações clínicas e laboratoriais de citogenética e biologia molecular, facilitando o acompanhamento de pacientes com LMC, a compreensão da evolução da doença e o desenvolvimento de pesquisas biotecnológicas. / The chronic myeloid leukemia (CML) is characterized by a cytogenetic alteration known as Philadelphia chromosome (Ph), a result of reciprocal translocation t (9; 22) (q34; q11). The resulting fusion bcr-abl gene encodes a protein with tyrosine kinase constitutive activity that deregulates signal transduction inducing proliferation, apoptosis, and cellular differentiation. The natural evolution of CML is currently changing with the development of tyrosine kinase inhibitors (TKI). Imatinib (Gleevec® , Novartis) was the first TKI approved for the treatment of CML. The eight-year update of the international randomized study of interferon and imatinib (IRIS), confirmed the efficacy and safety of imatinib in the long term with an overall survival of 85% and an event-free survival of 81%. However, some mutations in the kinase domain BCR-ABL confer resistance to one or more TKI, influencing the choice of therapy, as in the case of a T315I mutation, which is highly resistant to imatinib. Although many factors contribute to the resistance to imatinib, the presence of mutations is more prevalent and has been further investigated. Therefore, were aimed to perform mutation analysis in patients with a resistant phenotype of two cancer reference hospitals in Vitoria, ES, Brazil. We also developed a CML database relating clinical information and laboratory cytogenetics and molecular biology results, facilitating the monitoring of CML patients, as well as the understanding of disease progression.

A Leucemia Mielóide Crônica (LMC) é uma doença rara. No entanto, os avanços nas pesquisas básica e clínica nos últimos anos, colocaram a LMC em evidência sendo hoje uma neoplasia maligna potencialmente curável. O diagnóstico e tratamento desta doença são, no entanto, extremamente caros. Não havendo dados sistemáticos nem registros de incidência da LMC no Rio Grande do Sul ou no Brasil, o levantamento de dados baseado em registros dos centros de referência se justifica também para planejar ações em saúde. Entre 1996 e 2000, 276 casos foram diagnosticados. A estimativa de casos novos anuais foi de aproximadamente 0,6:100.000 habitantes, e a idade média no momento do diagnóstico foi 42 anos e 4 meses (±16 anos e 2 meses). Quanto ao tratamento e evolução destes pacientes, dos 257 avaliados, 56 (21,8%) foram submetidos ao transplante alogênico de medula óssea, com taxa de sobrevida em 5 anos de 75% e 27% para as fases crônica e acelerada/blástica, respectivamente. O tempo médio de sobrevida para os 257 pacientes foi de 47,7 meses (IC 43,3 - 52,1). Comparando ao relatado na literatura, encontramos um menor número anual de novos casos e também uma média de idade no diagnóstico mais baixa. Isto poderia ser explicado pela menor referência de idosos a serviços terciários de saúde. Para os pacientes transplantados, os resultados foram semelhantes aos relatados na literatura. / Although rare, the advances made in basic and clinical research throughout the last years have thrown a spotlight on CML. Diagnosis and treatment of CML is of high cost. Since there is no systematic data or information about the incidence of CML in Rio Grande do Sul or Brazil, the data obtained from reference centers serve to estimate the number of CML cases in our state to better plan health actions. Between 1996 and 2000, 276 cases were diagnosed. The annual estimate of new cases was approximately of 0,6:100,000 inhabitants, and the median age at diagnosis was 42 years and 4 months (±16 years and 2 months). The mean overall survival time for the 257 patients was 47,7 months (CI 43,3-52,1). That could be explained by the lack of referral for older patients. Regarding treatment and evolution, of the 257 valuable patients, 56 (21,8%) were submitted to allogeneic BMT with a five-year survival of 75% and 27% for chronic and accelerated/blastic phases, respectively. In conclusion, we found a lower estimated incidence and a lower median age at diagnosis. For the transplanted patients the results were similar to those reported in the literature.

A Leucemia Mielóide Crônica (LMC) é uma doença rara. No entanto, os avanços nas pesquisas básica e clínica nos últimos anos, colocaram a LMC em evidência sendo hoje uma neoplasia maligna potencialmente curável. O diagnóstico e tratamento desta doença são, no entanto, extremamente caros. Não havendo dados sistemáticos nem registros de incidência da LMC no Rio Grande do Sul ou no Brasil, o levantamento de dados baseado em registros dos centros de referência se justifica também para planejar ações em saúde. Entre 1996 e 2000, 276 casos foram diagnosticados. A estimativa de casos novos anuais foi de aproximadamente 0,6:100.000 habitantes, e a idade média no momento do diagnóstico foi 42 anos e 4 meses (±16 anos e 2 meses). Quanto ao tratamento e evolução destes pacientes, dos 257 avaliados, 56 (21,8%) foram submetidos ao transplante alogênico de medula óssea, com taxa de sobrevida em 5 anos de 75% e 27% para as fases crônica e acelerada/blástica, respectivamente. O tempo médio de sobrevida para os 257 pacientes foi de 47,7 meses (IC 43,3 - 52,1). Comparando ao relatado na literatura, encontramos um menor número anual de novos casos e também uma média de idade no diagnóstico mais baixa. Isto poderia ser explicado pela menor referência de idosos a serviços terciários de saúde. Para os pacientes transplantados, os resultados foram semelhantes aos relatados na literatura. / Although rare, the advances made in basic and clinical research throughout the last years have thrown a spotlight on CML. Diagnosis and treatment of CML is of high cost. Since there is no systematic data or information about the incidence of CML in Rio Grande do Sul or Brazil, the data obtained from reference centers serve to estimate the number of CML cases in our state to better plan health actions. Between 1996 and 2000, 276 cases were diagnosed. The annual estimate of new cases was approximately of 0,6:100,000 inhabitants, and the median age at diagnosis was 42 years and 4 months (±16 years and 2 months). The mean overall survival time for the 257 patients was 47,7 months (CI 43,3-52,1). That could be explained by the lack of referral for older patients. Regarding treatment and evolution, of the 257 valuable patients, 56 (21,8%) were submitted to allogeneic BMT with a five-year survival of 75% and 27% for chronic and accelerated/blastic phases, respectively. In conclusion, we found a lower estimated incidence and a lower median age at diagnosis. For the transplanted patients the results were similar to those reported in the literature.

A leucemia mielóide aguda (LMA) acomete pacientes de todas as faixas etárias com freqüência aumentada em idosos. Nosso estado não apresenta registros sistemáticos da patologia, justificando-se desta forma o levantamento através dos centros de diagnóstico e tratamento, os quais concentram a maioria dos casos. Foram incluídos todos os pacientes com diagnóstico de LMA “de novo” levantados a partir dos registros dos centros de diagnóstico e tratamento da patologia no estado no período entre 1996 e 2000. Foram computados no período 532 pacientes (taxa estimada de 1,04 casos / 100.000 habitantes / ano). A idade média ao diagnóstico foi 41,0 anos e 46,6 % dos casos eram do sexo masculino. Houve distribuição semelhante nas 7 mesorregiões geográficas do estado. Em relação ao número de casos por faixa etária, observou-se uma taxa estimada em 0,5 – 1 caso / 100.000 habitantes até a faixa etária dos 45 anos, atingindo 3,5 casos / 100.000 habitantes na faixa etária igual ou superior a 70 anos. A sobrevida média considerando todos os casos foi de 17%, aumentando para 25% considerando a população abaixo de 60 anos e alcançando 40% na população entre 10 e 20 anos. Tais dados evidenciam uma distribuição semelhante por região, estimativa de incidência na faixa infantil semelhante a países desenvolvidos e bastante menor na população idosa. A mortalidade geral foi semelhante àquela relatada na literatura. / Acute myeloid leukemia (AML) affects patients in all age groups, but it is more frequent among elderly people. The state of Rio Grande do Sul does not have systematic data on AML; thus these data have to be collected from hospitals or centers where the disease is diagnosed and treated. Patients with de novo acute myeloid leukemia were included based on the information retrieved from hospital registries between 1996 and 2000. At the time, 532 patients had been registered (estimated rate of 1.04 cases per 100,000 inhabitants / year). Mean age at diagnosis was 41.0 years, and 46.6% of the patients were male. Cases were similarly distributed across the seven meso-regions of Rio Grande do Sul. There was an estimated incidence of 0.5 to 1 case per 100,000 inhabitants up to the age of 45 years, and of 3.5 cases per 100,000 inhabitants for individuals aged 70 years and older. The mean five-year survival rate was 17% for all cases, 25% for patients under the age of 60 years, and 40% for those aged between 10 and 20 years. There was a similar distribution of AML cases per region, an incidence rate in the pediatric population similar to that of developed countries, and a much lower incidence among elderly patients. Overall mortality was comparable to that reported in the literature.

abstract: Acute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes in myeloid blood cells. These changes cause them to resume an undifferentiated state, proliferate, and maintain growth throughout the body. AML is commonly treated with chemotherapy, but recent efforts to reduce therapy toxicity have focused on drugs that specifically target and inhibit protein products of the cancer’s aberrantly expressed genes. This method has proved difficult for some proteins because of structural challenges or mutations that confer resistance to therapy. One potential method of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoter region of many potential oncogenes and have regulatory roles in their transcription. This study analyzes the therapeutic potential of the compound GQC-05 in AML. This compound was shown in vitro to bind and stabilize the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Through qPCR and western blot analysis, a GQC-05 mediated downregulation of MYC mRNA and protein was observed in AML cell lines with high MYC expression. In addition, GQC-05 is able to reduce cell viability through induction of apoptosis in sensitive AML cell lines. Concurrent treatment of AML cell lines with GQC-05 and the MYC inhibitor (+)JQ1 showed an antagonistic effect, indicating potential competition in the silencing of MYC. However, GQC-05 is not able to reduce MYC expression significantly enough to induce apoptosis in less sensitive AML cell lines. This resistance may be due to the cells’ lack of dependence on other potential GQC-05 targets that may help upregulate MYC or stabilize its protein product. Three such genes identified by RNA-seq analysis of GQC-05 treated cells are NOTCH1, PIM1, and RHOU. These results indicate that the use of small molecules to target the MYC promoter G-quadruplex is a viable potential therapy for AML. They also support a novel mechanism for targeting other potentially key genetic drivers in AML and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2017