Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian cancers. Previous studies indicate that these genes can exhibit cooperative oncogenicity by cross-regulating one another and facilitating cancer development. Our earlier studies demonstrated that treatment of ovarian cancer cells with arsenic trioxide (As2O3) promotes cytoprotective autophagy regulated by induction of SnoN to antagonize the cytotoxic effects of As2O3. Since exact mechanisms underlying As2O3-induced SnoN expression and cytoprotective responses were unclear, we hypothesized that SnoN may be regulated by signaling pathways involving genes amplified at the 3q26 locus. Phospholipid scramblase 1 (PLSCR1) is located at 3q23 proximal to the amplified 3q26 region. It had been implicated in disruption of plasma membrane asymmetry by mediating phospholipid scrambling, a process critical for cellular events such as blood coagulation and apoptosis. However, recent findings have led to more investigations on the role and regulation of PLSCR1 in cancer development and immune responses. PLSCR1 expression is regulated by various stimuli including growth factors (EGF, G-CSF, and SCF), cytokines (IFN), and differentiation-inducing agents (ATRA). Despite these studies, transcriptional regulation of PLSCR1 remains incompletely understood. Numerous studies have suggested a critical role for PLSCR1 in the pathophysiology of various cancers including leukemia, ovarian cancer, colorectal cancer, and metastatic liver cancer. However, the precise contribution of PLSCR1 and its regulation in ovarian cancer development is unclear. Since PLSCR1 (at 3q23) is located in close proximity to SnoN/SkiL (at 3q26.2), we hypothesized that PLSCR1 expression in ovarian cancer cells could be regulated by SnoN. Herein, we present studies that primarily focus on understanding the role and regulation of SnoN/SkiL (a TGFβ pathway regulator) and PLSCR1 (an interferon-regulated gene), which are located at 3q26.2 and 3q23, respectively, in epithelial ovarian cancer. In Chapter 3, we determined that activation of the PI3K signaling pathway mediates SnoN expression and cytoprotective responses upon stimulation of ovarian cancer cells with As2O3. We first identified that As2O3 stimulation leads to activation of EGFR and its downstream signaling mediators as well as modulates its interaction with the adaptor proteins, ShcA and Grb2. Interestingly, while treatment with a general SFK inhibitor (PP2), reduced the As2O3-induced EGFR activation and SnoN induction, a more specific inhibitor SU6656 did not alter SnoN expression. Further, via studies utilizing specific inhibitors and siRNA targeting PI3K, we determined that inhibition of PI3K signaling pathway decreases SnoN induction and increases apoptosis in ovarian cancer cells in response to As2O3. This suggests that PI3K (PIK3CA) activity is required for the As2O3-mediated SnoN induction and the cell survival responses in ovarian cancer cells. Finally, we determined by siRNA-mediated knockdown that EGFR and MAPK1 alter As2O3-induced cell death response independently of SnoN induction. In Chapter 4, via bioinformatic analyses, we identified that PLSCR1 DNA copy number and mRNA expression is elevated in ovarian cancer patients and cell lines relative to immortalized (Tag/hTERT) normal ovarian surface epithelial (OSE) cells. Interestingly, altered PLSCR1 DNA and mRNA levels were correlated with SnoN in ovarian cancers. We next identified that SnoN knockdown leads to a significant (~35%, P2O3 transcriptionally downregulates PLSCR1 in a ROS-independent mechanism. Furthermore, PLSCR1 knockdown, similar to SnoN knockdown increases ovarian cancer cell sensitivity to As2O3. PLSCR1 knockdown increases cleaved PARP (marker of apoptosis) with a consequent reduction in LC3-II levels (marker of autophagosomes). Collectively, these studies implicate PLSCR1 in the pathophysiology of ovarian cancers and in altering the chemotherapeutic responses in ovarian cancer cells. PLSCR1 is an IFN-regulated gene and mediates antiviral/immune responses. More recent studies in plasmacytoid dendritic cells have implicated PLSCR1 in regulating TLR9 signaling upon stimulation with CpG ODN. However, whether PLSCR1 could mediate the innate immune responses upon stimulation with dsDNA remained unclear. In Chapter 5, we identified that stimulation of normal ovarian and mammary epithelial cells with dsDNA (empty plasmid) markedly induces PLSCR1 consequent with activation of IRF3, a downstream mediator of TLR signaling that transcriptionally regulates the expression of type 1 IFNs. Interestingly, IRF3 knockdown ablates the dsDNA-induced PLSCR1 expression suggesting that PLSCR1 induction in response to dsDNA could be mediated by IRF3. Additionally, we have determined that dsDNA stimulation induces nucleic acid sensing TLRs, TLR9 and TLR4 as well as IFN-α and IFN-β mRNAs. Interestingly, dsDNA stimulation did not induce PLSCR1 or IRF3 activation in ovarian cancer cells suggesting that the mechanisms of IRF3 activation and PLSCR1 induction in response to dsDNA might be dysregulated in ovarian cancers. Collectively, our studies demonstrate a possible synergistic role of SnoN and PLSCR1 in ovarian cancer pathophysiology and suggest a potentially dysregulated role of PLSCR1 in the dsDNA-induced immune responses of malignant epithelial cells relative to normal epithelial cells. These studies could potentially lead to development of a novel combinatorial therapeutic strategy that targets both these molecules for improving treatment of patients with ovarian carcinoma.

<p>When bacterial pathogens infect their hosts, they illicit responses intended on containing and eliminating these invaders. This happens not only on the organismal level, but also on the cellular level. When a cell detects that it has been infected by an intracellular pathogen, it triggers a set of internal signaling events intended to contain the intruder. These events may allow the cell to produce antimicrobial agents or may help recruit members of the immune system to help fight the infection. In the case of closely evolved pathogens, such cell signaling events can be co-opted by the invading bacteria to its advantage. One example of this is infection with the gram-negative bacteria Chlamydia trachomatis. Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase Ras and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses during chlamydial infection. In this study we tested if activation of ERK and cPLA2 occurred as a result of Ras signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. we provide genetic and pharmacological evidence that Ras, ERK and, to a lesser extent, cPLA2 activation are uncoupled during infection, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells cPLA2, and to a lesser extent ERK, signaling played a significant protective role against C. trachomatis. we determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of &#946; interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of autonomous innate immune responses to Chlamydia infections.</p> / Dissertation

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Partial embargo. Vita. Bibliography: 135-163.

Introdução: Este estudo propõe explorar a relação entre judicialização da saúde e equidade no acesso ao tratamento no SUS. Métodos: Utilizou-se comparação de médias e proporções para avaliar a presença ou não de diferenças socioeconômicas (SE) entre 1) três grupos de pacientes com hepatite C crônica - requerentes de Interferon Convencional pela via administrativa (ICA), requerentes de Interferon Peguilado pela via judicial (IPJ) e demandantes de Interferon Peguilado pela via administrativa (IPA) 2) dos pacientes com a população da região metropolitana de Porto Alegre. Resultados: Entre os grupos, a diferença SE não foi estatisticamente significativa, mas comparados à população, nos três grupos havia maior proporção de pacientes nas classes A e B (mais altas) do que na D e E (mais baixas) da classificação da ABEP (Associação Brasileira de Empresas de Pesquisa): no grupo IPJ, 50% das classes A e B e 0% das classes D e E; no IPA, 49,3% das classes A e B e 8,9% das D e E; e no ICA, 44,9% das classes A e B e 6,1% das D e E, enquanto na população como um todo 34,3% estariam nas classes A e B, e, 20,2% nas classes D e E (p<0,001). Diferenças na proporção de pacientes com seguros de saúde privados pertencentes aos três grupos e a população total também foram estatisticamente significativas. Conclusões: Nossos dados permitem inferir que há iniqüidade no acesso a tratamento com medicamento de alto custo entre usuários portadores de hepatite C, porém este fato não é explicado por diferença no acesso a diferentes regimes de tratamento no SUS ou mesmo pela judicialização. Os determinantes atuam antes, no acesso diferenciado ao próprio sistema público conforme a inserção sócioeconômico- cultural dos beneficiários. / Introduction: This study aimed to explore the relationship between Judicialization of Health and equity in the access of treatment via SUS (Brazilian’s Unified Health System). Methods: Means and proportion comparisons were used to evaluate socioeconomic (SE) differences 1) among three groups of patients with chronic Hepatitis C: a) receiving conventional Interferon through an administrative process (ICA), b) receiving Pegylated Interferon through an administrative process (IPA) and c) receiving Pegylated Interferon through a judicial process (IPJ); and 2) between those grups and the whole population of the metropolitan area of Porto Alegre, Brazil. Results: No statistically significant SE differences were found among the patients belonging to each of the three groups, but significant differences were found between each groups and the whole population (P<0,001). While in the whole population 34,3% of the subjects were classified as belonging to classes A and B and 20,2% to classes D and E, in the IPJ group, 50% of the subjects belonged to classes A and B and no one to classes D and E; in the IPA group, 49,3% belonged to classes AB and 8,9% to the DE; and in the ICA group, the proportions were respectively 44,9% AB and 6,1% DE. Proportions covered by private health insurance were also significantly different between the three groups and the population. Conclusions: Considering that hepatites C is not expected to disproportionally affect the highest SE strata, we may say that there is inequity in the access of patients to treatment in the SUS. This fact, however, is not explained by differences in the type or way of access to the medication, including judicialization. The iniquity seems to be previously determined, by differentiated access to specialized care at the public health system, according to socioeconomical and cultural status of the beneficiary.

Introdução: Este estudo propõe explorar a relação entre judicialização da saúde e equidade no acesso ao tratamento no SUS. Métodos: Utilizou-se comparação de médias e proporções para avaliar a presença ou não de diferenças socioeconômicas (SE) entre 1) três grupos de pacientes com hepatite C crônica - requerentes de Interferon Convencional pela via administrativa (ICA), requerentes de Interferon Peguilado pela via judicial (IPJ) e demandantes de Interferon Peguilado pela via administrativa (IPA) 2) dos pacientes com a população da região metropolitana de Porto Alegre. Resultados: Entre os grupos, a diferença SE não foi estatisticamente significativa, mas comparados à população, nos três grupos havia maior proporção de pacientes nas classes A e B (mais altas) do que na D e E (mais baixas) da classificação da ABEP (Associação Brasileira de Empresas de Pesquisa): no grupo IPJ, 50% das classes A e B e 0% das classes D e E; no IPA, 49,3% das classes A e B e 8,9% das D e E; e no ICA, 44,9% das classes A e B e 6,1% das D e E, enquanto na população como um todo 34,3% estariam nas classes A e B, e, 20,2% nas classes D e E (p<0,001). Diferenças na proporção de pacientes com seguros de saúde privados pertencentes aos três grupos e a população total também foram estatisticamente significativas. Conclusões: Nossos dados permitem inferir que há iniqüidade no acesso a tratamento com medicamento de alto custo entre usuários portadores de hepatite C, porém este fato não é explicado por diferença no acesso a diferentes regimes de tratamento no SUS ou mesmo pela judicialização. Os determinantes atuam antes, no acesso diferenciado ao próprio sistema público conforme a inserção sócioeconômico- cultural dos beneficiários. / Introduction: This study aimed to explore the relationship between Judicialization of Health and equity in the access of treatment via SUS (Brazilian’s Unified Health System). Methods: Means and proportion comparisons were used to evaluate socioeconomic (SE) differences 1) among three groups of patients with chronic Hepatitis C: a) receiving conventional Interferon through an administrative process (ICA), b) receiving Pegylated Interferon through an administrative process (IPA) and c) receiving Pegylated Interferon through a judicial process (IPJ); and 2) between those grups and the whole population of the metropolitan area of Porto Alegre, Brazil. Results: No statistically significant SE differences were found among the patients belonging to each of the three groups, but significant differences were found between each groups and the whole population (P<0,001). While in the whole population 34,3% of the subjects were classified as belonging to classes A and B and 20,2% to classes D and E, in the IPJ group, 50% of the subjects belonged to classes A and B and no one to classes D and E; in the IPA group, 49,3% belonged to classes AB and 8,9% to the DE; and in the ICA group, the proportions were respectively 44,9% AB and 6,1% DE. Proportions covered by private health insurance were also significantly different between the three groups and the population. Conclusions: Considering that hepatites C is not expected to disproportionally affect the highest SE strata, we may say that there is inequity in the access of patients to treatment in the SUS. This fact, however, is not explained by differences in the type or way of access to the medication, including judicialization. The iniquity seems to be previously determined, by differentiated access to specialized care at the public health system, according to socioeconomical and cultural status of the beneficiary.

Introdução: Este estudo propõe explorar a relação entre judicialização da saúde e equidade no acesso ao tratamento no SUS. Métodos: Utilizou-se comparação de médias e proporções para avaliar a presença ou não de diferenças socioeconômicas (SE) entre 1) três grupos de pacientes com hepatite C crônica - requerentes de Interferon Convencional pela via administrativa (ICA), requerentes de Interferon Peguilado pela via judicial (IPJ) e demandantes de Interferon Peguilado pela via administrativa (IPA) 2) dos pacientes com a população da região metropolitana de Porto Alegre. Resultados: Entre os grupos, a diferença SE não foi estatisticamente significativa, mas comparados à população, nos três grupos havia maior proporção de pacientes nas classes A e B (mais altas) do que na D e E (mais baixas) da classificação da ABEP (Associação Brasileira de Empresas de Pesquisa): no grupo IPJ, 50% das classes A e B e 0% das classes D e E; no IPA, 49,3% das classes A e B e 8,9% das D e E; e no ICA, 44,9% das classes A e B e 6,1% das D e E, enquanto na população como um todo 34,3% estariam nas classes A e B, e, 20,2% nas classes D e E (p<0,001). Diferenças na proporção de pacientes com seguros de saúde privados pertencentes aos três grupos e a população total também foram estatisticamente significativas. Conclusões: Nossos dados permitem inferir que há iniqüidade no acesso a tratamento com medicamento de alto custo entre usuários portadores de hepatite C, porém este fato não é explicado por diferença no acesso a diferentes regimes de tratamento no SUS ou mesmo pela judicialização. Os determinantes atuam antes, no acesso diferenciado ao próprio sistema público conforme a inserção sócioeconômico- cultural dos beneficiários. / Introduction: This study aimed to explore the relationship between Judicialization of Health and equity in the access of treatment via SUS (Brazilian’s Unified Health System). Methods: Means and proportion comparisons were used to evaluate socioeconomic (SE) differences 1) among three groups of patients with chronic Hepatitis C: a) receiving conventional Interferon through an administrative process (ICA), b) receiving Pegylated Interferon through an administrative process (IPA) and c) receiving Pegylated Interferon through a judicial process (IPJ); and 2) between those grups and the whole population of the metropolitan area of Porto Alegre, Brazil. Results: No statistically significant SE differences were found among the patients belonging to each of the three groups, but significant differences were found between each groups and the whole population (P<0,001). While in the whole population 34,3% of the subjects were classified as belonging to classes A and B and 20,2% to classes D and E, in the IPJ group, 50% of the subjects belonged to classes A and B and no one to classes D and E; in the IPA group, 49,3% belonged to classes AB and 8,9% to the DE; and in the ICA group, the proportions were respectively 44,9% AB and 6,1% DE. Proportions covered by private health insurance were also significantly different between the three groups and the population. Conclusions: Considering that hepatites C is not expected to disproportionally affect the highest SE strata, we may say that there is inequity in the access of patients to treatment in the SUS. This fact, however, is not explained by differences in the type or way of access to the medication, including judicialization. The iniquity seems to be previously determined, by differentiated access to specialized care at the public health system, according to socioeconomical and cultural status of the beneficiary.

Metastasizing is responsible for 90% of death in cancer patients. Metastatic tumour cells have several strategies that they use to invade surrounding tissues - they can migrate together or individually. When individual cells migrate, tumour cells adopt two different morphologies. They are either elongated and migrate using the proteolytically active mesenchymal mode, or they are rounded and migrate in the amoeboid mode. Metastatic tumour cells can switch between these modes, which complicates the development of effective migrastatics. In this work, we focused on the effect of inflammatory signalling on metastatic cell migration. We worked with cell lines of malignant human melanoma, which adopt a mixed morphology and show both amoeboid and mesenchymal phenotype during migration. Upon stimulation of melanoma human cells with interferon beta, a mesenchymal to amoeboid transition occurs. Interferon beta appears to induce amoeboid morphology by maintaining high levels of the ISGF3 complex, which is composed of the heterodimer of STAT 1 and STAT 2 proteins and the IRF9 protein. Upon blocking of Jak / Stat signalling pathway by negative regulators, human melanoma cells return to mesenchymal morphology. Key words - invasiveness, mesenchymal-ameboid transition, interferons, inflammation, migration, metastases

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