Orientadores: Patrícia Maria Bergamo Favaro, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T08:01:05Z (GMT). No. of bitstreams: 1 Pereira_JoaoKleberNovais_M.pdf: 3579832 bytes, checksum: 2f21cc28ac6161522a1ab6a958a04389 (MD5) Previous issue date: 2014 / Resumo: As neoplasias linfoides constituem um grupo heterogêneo de doenças originadas por alterações genéticas das células progenitoras hematopoéticas de origem linfoide, levando à proliferação clonal desordenada de células B ou T, e ao desenvolvimento de leucemias linfoides e linfomas. É notória a participação de diferentes vias de sinalização envolvidas tanto no desenvolvimento como na manutenção das neoplasias hematológicas. A ativação constitutiva da via de sinalização PI3K/AKT/mTOR é bem descrita na leucemia linfoide aguda de células T (LLA-T) e recentemente identificou-se, em modelos animais, que a atividade da PI3K coopera com o desenvolvimento do linfoma de Burkitt (LB). Deste modo, o papel da via PI3K/AKT/mTOR no crescimento e sobrevivência celular, duas características importantes da leucemogênese, transformou-a em um potencial alvo farmacológico. Seguindo essa perspectiva, o presente trabalho procurou avaliar o potencial terapêutico de NVP-BKM120, um pan-inibidor de PI3K de classe I, em linhagens celulares de LLA-T (Jurkat e MOLT-4) e LB (Daudi e NAMALWA). NVP-BKM120 foi capaz de diminuir a viabilidade celular e capacidade clonogênica dessas células. Foi observada uma parada na fase G2/M do ciclo celular, com subsequente diminuição de Ciclina B1 e aumento da apoptose pelas vias intrínseca e extrínseca, nas linhagens Jurkat, MOLT-4 e NAMALWA. Também foi observada diminuição da fosforilação da AKT e dos alvos downstream ao mTORC1, P70S6K e 4EBP1, e aumento da razão BAX:BCL2. Houve um aumento da produção de AVOs nas linhagens celulares após o tratamento com a droga, o que sugere ativação da autofagia. Portanto, este estudo demonstra a capacidade antitumoral de NVP-BKM120 contra linhagens celulares de LLA-T e LB. Nossos resultados sugerem que a diminuição da proliferação celular, após o tratamento com a droga, seja devido à redução da Ciclina B1 e que o aumento da razão BAX:BCL2 seja um dos mecanismos envolvidos na indução da apoptose / Abstract: The lymphoid neoplasms are a heterogeneous group of diseases caused by genetic alterations that were originated from hematopoietic progenitor cells of lymphoid origin, leading to uncontrolled clonal proliferation of B or T cells, and to the development of lymphoid leukemias and lymphomas. These findings emphasize the involvement of different signaling pathways involved in both the development and the maintenance of hematological malignancies. Constitutive activation of the PI3K /AKT/mTOR signaling pathway is well described for acute lymphoblastic leukemia T cells (T-ALL), recently been identified in animal models, that the activity of PI3K cooperates with the development of Burkitt's lymphoma (LB).Thus, the role of the PI3K / AKT / mTOR pathway in cell growth and survival, two important features of leukemogenesis, morphed into a potential drug target. Following this perspective, the present study aimed to evaluate the therapeutic potential of NVP-BKM120, a pan-PI3K inhibitor class I in cell lines of T-ALL (Jurkat and MOLT-4) and LB (Daudi and NAMALWA). NVP-BKM120 was able to decrease cell viability and clonogenic capacity of these cells. A blocked phase was observed in G2/M phase of the cell cycle with subsequent reduction of Cyclin B1 and increased apoptosis by the intrinsic and extrinsic pathways in the lines Jurkat and MOLT-4 NAMALWA. It was also observed, decreased phosphorylation of AKT and of the downstream targets mTORC1, p70S6K and 4EBP1, and an increase of BAX:BCL2 ratio. There was an increase in AVOs production in the cell lines after treatment with the drug, suggesting activation of autophagy. Therefore, this study demonstrates the antitumor ability of NVP-BKM120 against cell lines of T-ALL and LB. Our results suggest that the decrease in cell proliferation after treatment with the drug, is due to the reduction of Cyclin B1 and the increase of the BAX:BCL2 ratio is one of the mechanisms that are involved in the induction of apoptosis / Mestrado / Fisiopatologia Médica / Mestre em Ciências

Student Number : 8044255 - PhD thesis - School of Pathology; Discipline Virology - Faculty of Health Sciences / Knowledge of the timing of mother-to-child transmission (MTCT) of HIV-1 is an important issue in reducing the risk of infant infection. Prior to giving birth therefore an HIV-1 positive mother should be provided with anti-HIV-1 drugs (antiretrovirals) during the shortest time possible to ensure both efficacy and minimal toxicity of the antiretrovirals to the newborn. However, in the absence of timeous administration of nevirapine (NVP) or zidovudine (AZT) to the mother at the onset of labour, infants are given post-exposure prophylaxis (PEP). Despite antiviral prophylaxis some infants still become infected. In an attempt to mimic the in vivo scenario we investigated, in Chapter Three, the replication ability of a primary isolate (M502L) in peripheral blood mononuclear cells (PBMC) isolated from healthy donors exposed to different concentrations of NVP or AZT either prior to or post-infection, but that reflected mean neonatal plasma concentrations measured following maternal dosing. In phytohaemagglutinin (PHA) stimulated cultures M502L exhibited some growth. Maintaining NVP and AZT in the culture medium resulted in decreased viral growth over time. In contrast to that expected certain donors demonstrated elevated p24 antigen levels in the presence of HIV-1 and NVP or AZT. This suggested that cells were more conducive to HIV-1 replication either because of cellular activation or due to cellular production of cytokines/chemokines. The in vitro study highlighted (i) the differential permissiveness of cells from different donors for HIV-1 infection, (ii) different abilities of antiretrovirals (ART) to circumvent infection in different individuals and (iii) immunomodulatory effects of ART in vitro. Commencing in Chapter Four we elected to investigate, in vivo, the immunomodulatory consequences of HIV-1 exposure and infection in two groups of HIV-1-exposed newborns whose mothers either received NVP at the onset of labour or who only received NVP as PEP within 72 hours of birth. Short-course antiretroviral drug regimens are known to reduce the risk of MTCT of HIV-1 but mechanisms affording protection of such interventions remain poorly defined. Since T-cell activation is an important factor in productive HIV-1 infection, we tested the hypothesis that single-dose NVP reduces immune activation, which in turn reduces the likelihood of transmission. We compared concentrations of cord and maternal blood plasma immune activation markers, neopterin, β2-microglobulin (β2-m) and soluble L-selectin (sL-selectin) in the two groups of HIV-1-exposed newborns and among HIV-unexposed controls. In utero exposure of the infant to HIV-1, regardless of NVP exposure, led to demonstrable increases in levels of immune activation markers, this being most notable in the presence of pre-existing infection. Contrary to what was hypothesized, immune activation was increased by pre-birth exposure to single-dose NVP, with this effect being enhanced in infants already infected at birth. Our data suggest that reductions in immune activation do not explain transmission prevention effects of single-dose NVP. Our data also suggest a biological explanation for why HIV-1 infected infants exposed perinatally to antiretroviral drugs might experience hastened disease progression, namely that the immunological mileau in some HIV-1 infected individuals treated with NVP favours increased HIV-1 replication. Cytokines and chemokines function to stimulate, or suppress cellular proliferation and differentiation and have unique immunomodulatory properties. Furthermore, they have the potential to protect against HIV-1 infection or to regulate HIV-1 replication. In Chapter Five we therefore questioned whether exposure to HIV-1 or NVP influences cytokine/chemokine levels of infants born to HIV-1 infected mothers. We compared levels of interleukin (IL)-7, IL-10, stromal cell-derived factor: SDF-1α (CXCL12), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage inflammatory protein-1α: MIP-1α (CCL3), macrophage inflammatory protein-1β: MIP-1β (CCL4) and regulated upon activation, normal T-cell expressed and secreted: RANTES (CCL5) of the two groups of HIV-1-exposed newborns and among the HIV-unexposed controls. HIV-1 exposure in the absence of single-dose NVP was not found to impact significantly on the levels of IL-7, IL-10, GM-CSF, CXCL12, CCL3, CCL4 or CCL5 and single-dose NVP had no appreciable effect on these cytokine/chemokine levels. Cord blood plasma levels of IL-7, CXCL12 and GM-CSF were found to be independent of mothers’ levels. Single-dose NVP reduced the ability of cord blood mononuclear cell (CBMC) to produce GM-CSF spontaneously. Maternal and infant (HIV-1 exposed NVP unexposed) IL-10 levels were significantly correlated. Significantly elevated levels of IL-10 were associated with pre-existing infection in NVP unexposed newborns. CCL3, CCL4 and CCL5 levels in NVP unexposed uninfected infants were not different from those of control infants but correlated significantly with IL-7 levels. HIV-1 specific cellular immune responses are elicited in a proportion of infants born to HIV-1 seropositive mothers and have been associated with protection from maternal HIV-1 transmission. In Chapter Six, levels of the immune activation markers neopterin, β2-m, sL-selectin, the immunomodulatory and haematopoietic factors IL-7, CXCL12, GM-CSF and the immunoregulatory cytokine IL-10 were examined amongst the group of newborns, that received NVP as PEP within 72 hours of birth, of which a proportion had specific cellular responses to HIV-1 envelope (Env) peptides. It was our aim to determine in infants that elicit HIV-1 specific cellular immune responses (Env+) and those that lack the specific responses (Env-), whether these factors could predict transmission and whether the former group of infants exhibit unique immune features that might distinguish them from Env- non-responders. Our data suggested that none of the factors tested were predictive of HIV-1 transmission but confirmed that infants with cellular responses to HIV-1 envelope peptides were associated with lack of subsequent infection. In particular, our data demonstrated an association between HIV-1 specific cellular immune responses, lower maternal viral load and lack of infection suggesting that sustained exposure to antigen (reduced maternal viral load) may be responsible for the strong priming effect. Furthermore, an association between reduced GM-CSF levels and the presence of HIV-1 specific responses was demonstrated, which suggested therefore that newborn infants that elicited HIV-1 specific cellular immune responses exhibited different immune capabilities from those without responses. Finally, in Chapter Seven we looked at how immune activation and priming impact on thymic output of T-cells in newborn infants. Unfortunately, sample volumes of the two groups of HIV-1-exposed newborns used in the previous three Chapters became limited with the result that we chose to address these questions using anonymously collected cord blood samples from infants, some of which were used to supplement the placebo group of the the UNAIDS-sponsored clinical trial of short-course zidovudine-lamivudine (AZT-3TC). At the time the AZT-3TC trial was conducted short-course antiviral prophlyaxis was not the standard of care for the prevention of MTCT of HIV-1. The thymus is known to be essential for establishing diversity of the T-cell pool, and morphological thymic changes and effects on naïve T-cells and T-cell receptor excision circle (TREC) concentrations have been reported in studies of HIV-1 infected children and adults. As it is not known to what extent in utero exposure to HIV-1 and infection affects T-cell division in newborn infants, we elected to determine TREC levels of infants born to HIV-1 seropositive mothers that were not exposed to antiretrovirals. The impact of increased immune activation on TREC levels and the consequence of HIV-1 exposure or infection on circulating levels of IL-7 (raised levels indicative of T-cell depletion) was also investigated. HIV-1 exposure or infection did not result in significant losses of TREC. TREC levels were not affected by immune activation associated with HIV-1 exposure and infection and IL-7 levels were not raised. Infants that elicited HIV-1 specific cellular immune responses exhibit TREC levels that were similar to those of infants without HIV-1 specific responses. These data suggested that newborn infants of HIV-1 seropositive mothers demonstrated no altered thymopoietic ability compared to control infants. Furthermore, HIV-1 specific immune responses, (indicative of post-thymic memory T-cell expansion) did not influence thymic output measured in newborn infants. In conclusion, the in vitro study demonstrated that there is a high degree of variability between PBMC isolated from different donors with respect to viral replication and drug effectivity which suggests that these phenomena are likely to exist within patient (infant as well as adult) populations. While immune activation is considered central to productive infection we demonstrated that immune activation is increased by HIV-1 exposure and by single-dose NVP. Exposure to HIV-1 alone or with NVP did not influence birth levels of IL-7, IL-10, CXCL12, GM-CSF, CCL3, CCL4 and CCL5. Furthermore, levels of these factors did not predict infection outcome in the infant. Immune activation and haematopoietic growth factors are modulated independently of the mother but maternal factors such as IL-10 and exposure to single-dose NVP, which reduces responsiveness of CBMC, could impact on the infant. HIV-1 specific cellular immune responses at birth, which are elicited in a proportion of infants born to HIV-1 positive mothers, are of immunological significance and can predict lack of subsequent infection. Disturbances in thymic output are not readily detectable at birth when using TREC to assess de novo T-cell synthesis, alternatively there is a homeostatic balance between thymic output and peripheral T-cell proliferation in newborns of HIV-1 infected mothers. Overall our data suggests that (i) there are immune consequences of being born to an HIV-1 positive mother, (ii) short-course antiretroviral prophylaxis does impact on the developing immune system of the infant and (iii) while the direct effects of single-dose NVP are not disputed, there are indirect consequences of NVP exposure on immune cells. Despite the consequences of HIV-1 exposure or the result of being born to a HIV-1 seropositive mother or exposure to single-dose NVP, our data proposes that the immune system of newborn infants is capable of responding as demonstrated by the enhanced immune activation. It remains important to determine the correlates of immune protection for the development of novel immuno-therapeutic and vaccine strategies and maternal-infant transmission of HIV-1 provides a model which can address questions of protective immune processes. Understanding the influence of antiretrovirals on immune processes remains an important component of the drug mechanisms, (aside from their direct antiretroviral activity), that may underlie reductions in maternal-infant transmission of HIV-1. Furthermore, how antiretrovirals influence immune processes and immune development (together with exposure to HIV-1/consequences of being born to an HIV-1 seropositive mother), may impact on subsequent immune responsiveness to infectious organisms or childhood vaccines.

Hydrogels are polymeric materials that have three-dimensional polymeric networks, which are able to absorb and retain a large amount of water within their structures without being dissolved. Among the synthetic hydrogel, poly(2-hydroxylethyl methacrylate) (poly(HEMA)) has been of great interest because of its excellent biocompatibility with the three-dimensional networks. Therefore, poly(HEMA) hydrogels have been widely used in many areas, especially in biomedical and pharmaceutical areas, for such applications as packing materials in chromatography, sorbents in controlled release and drug delivery, implanting materials in tissue engineering. However, the applications of poly(HEMA) are still limited because of its weak mechanical strength and network properties. Therefore, in recent decades, the challenge of how to modify and control the polymer properties and how to build highly porous structures in it has received considerable attention because these modifications could significantly improve the performance of poly(HEMA) hydrogels for more favorable applications. Although HEMA and its polymers have been studied for more than 40 years, few reports about the preparation of micro-/nano-porous poly(HEMA) hydrogel particles and the requirements of their applications have risen. Furthermore, how to control the porous structures and the properties of HEMA copolymers have not been well understood. Accordingly, the objectives of this research were to investigate the synthesis of the porous copolymeric particles of HEMA with various comonomers (MMA, St and NVP), to characterize the porous structures and particle morphology, to simulate the synthesis process and porous characteristics, to explore the effects of the polymer compositions and the porous structures on the swelling properties, and to apply the resultant polymeric particles in the controlled release of the hydrophilic model drug. In the present studies, HEMA was copolymerized with three different comonomers, methyl methacrylate (MMA), styrene (St) and N-vinyl-2-pyrrolidone (NVP), respectively, to prepare highly porous particles crosslinked using ethylene glycol dimethacrylate (EGDMA) in the presence of 1-octanol used as a porogen by means of suspension copolymerization in an aqueous phase initiated by 2,2-azobisisobutyronitrile (AIBN). Nano-pores were observed in the present studies. The pore size and the swelling properties of these particles can be successfully controlled by changing comonomers or adjusting the crosslinker and porogen concentration. The results indicate that lower crosslinker or porogen concentration favors generating smaller pores, whereas a higher concentration of a hydrophilic comonomer, higher crosslinker concentration and higher porogen volume ratio promote the generation of larger pores. In addition, the effects of the porous structures and the network properties on the swelling properties were explored. The swelling capacity of the porous particles is reduced with an increase in the EGDMA molar concentration. However, higher porosity in the particles and higher amount of hydrophilic comonomer result in a higher swelling capacity of the particles. The gel formation and the porous characteristics of HEMA/comonomer/EGDMA systems were simulated using the mathematical models combining the reaction kinetics and the thermodynamics. It was found that the model over-predicted the experimental results of the porosity because the pores and the networks are shrunk or collapsed during the porogen removal. Therefore, the model predicts the maximum porosity that the polymeric particles can reach. If the hydrophobic contents are higher, the model gives better prediction of the porosity. It is concluded that the microporous structures of HEMA related hydrogels could be controlled by a properly designed process based on the knowledge gained via this research. The output of this research helps with a better understanding for industrial production of micro-porous hydrogels and their applications.

Hydrogels are polymeric materials that have three-dimensional polymeric networks, which are able to absorb and retain a large amount of water within their structures without being dissolved. Among the synthetic hydrogel, poly(2-hydroxylethyl methacrylate) (poly(HEMA)) has been of great interest because of its excellent biocompatibility with the three-dimensional networks. Therefore, poly(HEMA) hydrogels have been widely used in many areas, especially in biomedical and pharmaceutical areas, for such applications as packing materials in chromatography, sorbents in controlled release and drug delivery, implanting materials in tissue engineering. However, the applications of poly(HEMA) are still limited because of its weak mechanical strength and network properties. Therefore, in recent decades, the challenge of how to modify and control the polymer properties and how to build highly porous structures in it has received considerable attention because these modifications could significantly improve the performance of poly(HEMA) hydrogels for more favorable applications. Although HEMA and its polymers have been studied for more than 40 years, few reports about the preparation of micro-/nano-porous poly(HEMA) hydrogel particles and the requirements of their applications have risen. Furthermore, how to control the porous structures and the properties of HEMA copolymers have not been well understood. Accordingly, the objectives of this research were to investigate the synthesis of the porous copolymeric particles of HEMA with various comonomers (MMA, St and NVP), to characterize the porous structures and particle morphology, to simulate the synthesis process and porous characteristics, to explore the effects of the polymer compositions and the porous structures on the swelling properties, and to apply the resultant polymeric particles in the controlled release of the hydrophilic model drug. In the present studies, HEMA was copolymerized with three different comonomers, methyl methacrylate (MMA), styrene (St) and N-vinyl-2-pyrrolidone (NVP), respectively, to prepare highly porous particles crosslinked using ethylene glycol dimethacrylate (EGDMA) in the presence of 1-octanol used as a porogen by means of suspension copolymerization in an aqueous phase initiated by 2,2-azobisisobutyronitrile (AIBN). Nano-pores were observed in the present studies. The pore size and the swelling properties of these particles can be successfully controlled by changing comonomers or adjusting the crosslinker and porogen concentration. The results indicate that lower crosslinker or porogen concentration favors generating smaller pores, whereas a higher concentration of a hydrophilic comonomer, higher crosslinker concentration and higher porogen volume ratio promote the generation of larger pores. In addition, the effects of the porous structures and the network properties on the swelling properties were explored. The swelling capacity of the porous particles is reduced with an increase in the EGDMA molar concentration. However, higher porosity in the particles and higher amount of hydrophilic comonomer result in a higher swelling capacity of the particles. The gel formation and the porous characteristics of HEMA/comonomer/EGDMA systems were simulated using the mathematical models combining the reaction kinetics and the thermodynamics. It was found that the model over-predicted the experimental results of the porosity because the pores and the networks are shrunk or collapsed during the porogen removal. Therefore, the model predicts the maximum porosity that the polymeric particles can reach. If the hydrophobic contents are higher, the model gives better prediction of the porosity. It is concluded that the microporous structures of HEMA related hydrogels could be controlled by a properly designed process based on the knowledge gained via this research. The output of this research helps with a better understanding for industrial production of micro-porous hydrogels and their applications.

BK virus (BKV) is a ubiquitous polyomavirus known to asymptomatically reside in the renal tissues of up to 90% of the human population. BK virions reactivate during periods of intense immunosuppression and can cause disease in renal transplant recipients, such as BKV-associated nephropathy (BKVAN). BKVAN can lead to loss of the transplanted renal grafts. For this reason, the study of BKV biology is of importance to the transplant community. Previous studies have shown that BKV upregulates the pro-growth mTOR pathway in host cells, thereby increasing BKV replicative efficiency. Downstream effectors of the mTOR pathway, particularly p70S6 kinase, control the basal rate of protein translation, in part through regulation of ribosomal biogenesis. It was hypothesized that viral upregulation of the mTOR pathway is beneficial for viral replication due to an increase in the number of ribosomes available to translate viral proteins. Therefore, inhibition of the mTOR pathway could reduce viral replication. This study investigated whether host cell mTOR inhibition could reduce BK viral replication in an in vitro model. We utilized the dual PI3K/mTOR inhibitor NVP BEZ-235 (Novartis Pharmaceuticals), which potently downregulates expression of both upstream (PI3K) and central (mTOR) effectors of the mTOR pathway. Immortalized renal epithelial cells were exposed to varying concentrations of BEZ-235 for a period of 48 hours, infected with BK virus for three hours, and allowed to grow for a further 48 hours. Cell populations were then assayed via quantitative PCR (qPCR), Western blotting and fluorescent immunohistochemical staining to determine the effect of BEZ-235 on BK viral replication. Western blot experiments confirmed the effectiveness of BEZ-235's inhibition of the mTOR pathway in a renal epithelial cell culture model, as well as downregulation of the mTOR pathway during BK viral infection. Western blotting for the key BK replicative protein Large T antigen showed a dose-dependent decrease in expression, with increasing concentrations of BEZ-235. Fluorescent immunohistochemical staining showed a dose-dependent decrease in expression of Large T antigen staining in host cell nuclei. qPCR results were inconclusive, in that no clear pattern in the number of BKV genomes per cell population could be observed across the range of BEZ-235 concentrations tested. While results from our study indicate that BEZ-235 can reduce BKV replication in vitro, further in vitro experimentation, including repetition of approaches already carried out as well as novel approaches, will be needed to definitively confirm inhibition of the mTOR pathway as a viable antiviral strategy.

In der Stahlindustrie sind strategische Entscheidungen mit hohen Investitionsbedarfen verbunden und aufgrund komplexer Produktionsvorgänge oft nur schwer zu bewerten. Im Rahmen der Arbeit wird ein Modell vorgestellt, das eine Verbindung zwischen der Veränderung technischer Parameter im Produktionsnetzwerk und ihren Auswirkungen auf den Wert der Unternehmung zulässt. Hierfür werden die zukünftigen Freien Cashflows bestimmt, die aus der Betriebstätigkeit (Produktion und Vertrieb) der Unternehmung resultieren und diskontiert zu einem Unternehmenswert aufsummiert (DCF). Entscheidend hierbei ist der Trade-off zwischen der technischen Detailtreue des Modells und der Handhabbarkeit (Modularität) bei der kurzfristigen Bewertung strategischer Fragestellungen. Abschließend wird der Einsatz des Modells anhand von Beispielen demonstriert.

In der Stahlindustrie sind strategische Entscheidungen mit hohen Investitionsbedarfen verbunden und aufgrund komplexer Produktionsvorgänge oft nur schwer zu bewerten. Im Rahmen der Arbeit wird ein Modell vorgestellt, das eine Verbindung zwischen der Veränderung technischer Parameter im Produktionsnetzwerk und ihren Auswirkungen auf den Wert der Unternehmung zulässt. Hierfür werden die zukünftigen Freien Cashflows bestimmt, die aus der Betriebstätigkeit (Produktion und Vertrieb) der Unternehmung resultieren und diskontiert zu einem Unternehmenswert aufsummiert (DCF). Entscheidend hierbei ist der Trade-off zwischen der technischen Detailtreue des Modells und der Handhabbarkeit (Modularität) bei der kurzfristigen Bewertung strategischer Fragestellungen. Abschließend wird der Einsatz des Modells anhand von Beispielen demonstriert.

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system. By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells. Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.

Introduction: Hyperemesis gravidarum (HG) in pregnancy can cause severe and persistent vomiting, nausea, dehydration, vitamin and mineral deficiencies. The symptoms can have varying degrees of impact on women's physical, emotional, social, and marital lives and affect their quality of life. Some women voluntarily terminate their pregnancies due to HG. Despite the severe consequences, the etiology of this disorder remains unknown. HG is thought to be caused by a mixture of hormonal variables and genetics. Various therapies have been documented with varying degrees of efficacy. Nevertheless, there is a lack of research on the actual causes of the disease and effective medications, as well as a lack of research on patients' experiences with HG, its treatments, and its consequences. Aim: This thesis aims to examine women's own experiences of HG, with the following research questions: How do women experience HG in their daily lives? What kind of support do they have in coping? What kind of approach do they receive from the health care system? What medical interventions were offered to them? and how effective were they perceived? Method: The thesis adopts a qualitative approach. Some prominent women's groups on Facebook were used to spread the information about the study, and 20 women in Sweden who had suffered from HG during pregnancy and had given birth no more than four years ago were interviewed individually online via Zoom. The data was analyzed using the thematic analysis method by creating sub-themes and themes through coding. Findings: The analysis revealed two themes: 'Trapped in my own body' and 'Longing for optimal HG care'. The first theme described the women's daily lives, which were significantly affected by HG. Due to severe vomiting and nausea, many of them were bedridden for long periods and unable to take care of their children or do household chores. They had significant problems with eating food, drinking water, odors, and oral hygiene. Many were unable to work, and most were on long-term sick leave. Women relied on their partners and families to cope with HG. The disease was compounded by mental health problems such as isolation, loneliness, sadness, and even depression. The second theme revealed that medications and their perceived effects were experienced differently. No drug could completely cure the disease in every case, and HG treatments were not considered optimal by any woman. Medical facilities often lacked knowledge about the disease and the drugs available, and there were no functioning care plans. Lack of knowledge meant that most women in the early stages of pregnancy were not taken seriously by the health system, and it took a long time for many of them to receive appropriate treatment. Conclusion: HG was perceived by women as a severe disease that caused significant physical and psychological obstacles in life and made women feel imprisoned in their bodies. Their most considerable support in coping with this illness was their partners and family. Despite these severe problems, most received inadequate care at health facilities mainly due to ignorance of the disease. This study has highlighted a great need for clear organizational structures, a working care plan, and more knowledge of HG in all health departments. Evidence-based national and local guidelines are needed to improve the availability and quality of health care. Based on the women’s experiences, the findings of this study may provide essential insights for implementing such guidelines in clinical practice.

Karzinome der Gallenwege sind mit einer schlechten Prognose assoziiert. Eine potentiell kurative chirurgische Resektion ist bei der Mehrzahl der Patienten aufgrund des späten Zeitpunkts der Diagnosestellung nicht möglich, so dass derzeit vorrangig palliative Therapieansätze Anwendung finden. Das nur geringe Ansprechen auf konventionelle Radio- oder Zytostatikatherapie begründet die Notwendigkeit neuer Therapieansätze. Einen möglichen Angriffspunkt stellt hierbei die Polo-like-Kinase 1 (Plk1) dar, da ihre zentrale Rolle in der Regulation des Zellzyklus zunehmend erkannt und eine vermehrte Expression in malignem Tumorgewebe verglichen mit gesundem Gewebe nachgewiesen wurde. Das Dihydropteridinon BI 2536 ist ein poten-ter, niedermolekularer und selektiver Hemmstoff der Plk1 und sollte daher auf seine Wirksamkeit an Gallenwegskarzinomen untersucht werden. In der vorliegenden Arbeit konnte gezeigt werden, dass BI 2536 die untersuchten 14 Zelllinien von Gallenblasen- und Gallengangskarzinomen wirkungsvoll hemmt. Das Ansprechen unterschied sich zwischen den Zelllinien und ordnet sich vergleichbar zu Veröffentlichungen an anderen malignen Tumoren ein. Die Expression von Plk1 und dessen assoziierten Transkriptionsfaktor FoxM1 konnte bei Westernblot-Versuchen bei allen Zelllinien nachgewiesen werden, was eine Bedeutung in der Onkogenese vermuten lässt. Die Behandlung mit BI 2536 beeinflusste jedoch die Proteinmenge beider nicht. An für die folgenden Versuche ausgewählten drei Zelllinien zeigten sich in der reversen Transkription mit anschließender Echtzeit-Polymerase-Kettenreaktion (qRT PCR) ähnliche Ergebnisse in Bezug auf die exprimierte mRNA von Plk1. Westernblot-Analysen ermittelten keine signifikanten Veränderungen der an wichtigen intrazellulären Kaskaden beteiligten Proteine p42/44 und Akt sowie deren phosphorylierten Formen. Obwohl die Proteinmenge des Mitosemarkers Phospho-Histon H3 ebenso unverändert blieb, führte die Behandlung mit BI 2536 – dies zeigen Ergebnisse der Durchflusszytometrie – zu einer signifikanten, dosisabhängigen Zunahme der G2/M Fraktion des Zellzyklus und Zunahme der Apoptose-rate. Der maximale Hemmeffekt in der Behandlung von BI 2536 lag bei einer Inkubations-dauer von vier Tagen. Die Empfehlungen aus den klinischen Studien der Phase II von BI 2536 sowie dem Ziel der Vermeidung von Resistenzen ergibt sich die Notwendigkeit von Kombinationsversuchen mit Zytostatika, die in einer anderen Phase des Zellzyklus angreifen. Die in der Behandlung von Gallenwegskarzinomen etablierten Antimetaboliten 5-Fluorouracil und Gemcitabin wurden hierzu ausgewählt und es zeigten sich für 5 Fluorouracil synergistische, für Gemcitabin hingegen additive Kombinationseffekte. Zusätzlich wurde die Wechselwirkung mit dem IGF 1-Rezeptor-Inhibitor NVP-AEW541 untersucht, der ebenfalls einen neuen Behand-lungsansatz in der Krebstherapie darstellt und bei Gallenwegskarzinomen in vitro wirksam ist. Auch hier zeigen sich synergistische Effekte, die jedoch erst in höheren Behandlungs-dosen auftraten. Die Ergebnisse dieser Arbeit zeigen, dass die Hemmung der Plk1 bei Gallenblasen- und Gallengangskarzinomen einen wirksamen Behandlungsansatz darstellt. Auf der Grundlage der in dieser Arbeit beschriebenen Ergebnisse wird eine weitere präklinische und klinische Testung von selektiven Plk1-Hemmstoffen wie BI 2536 an Gallenwegskarzinomen empfohlen.