PHARMACOLOGICAL CHARACTERIZATION OF THE SIGNAL TRANSDUCTION PATHWAYS MODULATED BY A3 RECEPTORS IN CANCER CELLS: POSSIBLE TARGETS FOR THERAPEUTIC INTERVENTION.

Adenosine is an ubiquitous autacoid that modulates a variety of cellular functions through occupancy of four cell surface G-protein-coupled receptors, named A1, A2A, A2B and A3. In particular, adenosine was found to exert its effects on cell proliferation, clone formation ability, UV resistance, and cell death mainly through the A3 subtype, which is highly expressed in tumor cells. Adenosine also plays a role in the promotion of angiogenesis. In this thesis I have characterized the signal transduction pathways modulated in hypoxia by A3 adenosine receptors in two different human tumor models: the colon cancer HT29 and the melanoma A375 cell lines. I have performed the study in hypoxia, present in most solid tumors, which shifts the cellular phenotype toward an increase in adenosine. Furthermore, hypoxic tumor cells are resistant to conventional chemiotherapy and radiotherapy. I have investigated the modulation of hypoxia-inducible factor-1 (HIF-1) through adenosine via its receptor subtypes. HIF-1 is a transcription factor that functions as a master regulator of oxygen homeostasis. HIF-1 is a heterodimer composed of an inducibly expressed HIF-1α subunit and a constitutively expressed HIF-1β one. In normoxia, HIF-1α is rapidly degraded by the ubiquitin proteasome system, whereas exposure to hypoxic conditions prevents its degradation. I have studied the signaling pathways modulated by A3 receptors which involved Akt, MEK, and p38 MAPK. I have demonstrated that adenosine increased in hypoxic colon carcinoma cells HIF-1α and vascular endothelial growth factor (VEGF) through the A3 receptor stimulation. Furthermore, the stimulation of A2B receptor increased interleukin-8 (IL-8) expression. Pretreatment of cells with caffeine, which is a methylxanthine antagonist of adenosine receptors, significantly reduced adenosine-induced VEGF promoter activity and VEGF and IL-8 expression. The kinases have a key role in A3 receptor ability to enhance HIF-1α and VEGF protein expression. Moreover, Akt, ERK 1/2, and p38 MAPK are required for the IL-8 expression increase induced by A2B receptor activation. Then, I have examined the modulation of IL-8, VEGF and HIF-1 by the DNA-damaging agents etoposide and doxorubicin, and I have analyzed the influence of the adenosinergic signaling on the chemotherapeutic drug effects. I have demonstrated that A2B receptor blockade can impair IL-8 production, whereas blocking A3 receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with etoposide and doxorubicin. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. Exposure of melanoma cells to the chemotherapeutic drugs resulted in the increase of p38, Akt and ERK1/2 phosphorylation levels. Moreover, etoposide and doxorubicin strongly inhibited HIF-1α protein expression. The A2B receptor antagonist MRE 2029F20 attenuated the increase in p38, Akt and ERK1/2 phosphorylation levels induced by the chemotherapeutic drugs, and when used alone it reduced p38, Akt and ERK1/2 phosphorylation basal levels. The A3 receptor antagonist MRE 3008F20 alone reduced HIF-1α protein, and in combination with the chemotherapeutic drugs further decreased HIF-1α protein accumulation. Therefore, the results of this thesis provide evidence of how human tumor growth may be influenced through the adenosinergic system and how the adenosine receptors modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively

miRNAs as Influencers of Cell-Cell Communication in Tumor Microenvironment

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level, inducing the degradation of the target mRNA or translational repression. MiRNAs are involved in the control of a multiplicity of biological processes, and their absence or altered expression has been associated with a variety of human diseases, including cancer. Recently, extracellular miRNAs (ECmiRNAs) have been described as mediators of intercellular communication in multiple contexts, including tumor microenvironment. Cancer cells cooperate with stromal cells and elements of the extracellular matrix (ECM) to establish a comfortable niche to grow, to evade the immune system, and to expand. Within the tumor microenvironment, cells release ECmiRNAs and other factors in order to influence and hijack the physiological processes of surrounding cells, fostering tumor progression. Here, we discuss the role of miRNAs in the pathogenesis of multicomplex diseases, such as Alzheimer's disease, obesity, and cancer, focusing on the contribution of both intracellular miRNAs, and of released ECmiRNAs in the establishment and development of cancer niche. We also review growing evidence suggesting the use of miRNAs as novel targets or potential tools for therapeutic applications

Os desafios da graduação da categoria de país menos avançado

Mestrado em Desenvolvimento e Cooperação InternacionalA presente dissertação de mestrado tem como objetivo caraterizar o processo de graduação da categoria de País Menos Avançado (PMA) da Organização das Nações Unidas (ONU) e compreender quais os desafios que os países que já passaram por este processo enfrentaram. Procura esclarecer as consequências inerentes à graduação, quando um país deixa de se qualificar como PMA, especificamente as implicações que o fim dos benefícios exclusivos traz para o país em questão. Através de uma análise dos processos de graduação e dos percursos de desenvolvimento dos cinco países já graduados desta categoria - Botswana, Cabo Verde, Maldivas, Samoa e Guiné Equatorial - procura-se verificar quais os impactos da graduação sobre o crescimento e o desenvolvimento no período pós-graduação e discutir as estratégias utilizadas para mitigar os efeitos negativos da cessação dos benefícios após o período de transição. Conclui-se que apesar da graduação implicar efetivamente o fim de diversos benefícios para os países recém-graduados, esta não ocorre de forma abrupta e, em certas áreas, não corresponde à completa cessação dos mesmos. A adequada preparação para a graduação por parte dos países em questão, em cooperação com os comités especializados da ONU e com os parceiros de desenvolvimento, permite a adoção de estratégias com vista a que o processo de graduação não seja traumático. A análise dos cinco casos de estudo permite concluir que as estratégias de transição de categoria adotadas têm sido benéficas e que, após a graduação, estes países têm de uma forma geral prosseguido trajetórias de crescimento consistentes.This dissertation aims to characterize the graduation process from the United Nations' (UN's) Least Developed Country (LDC) category and to understand the challenges faced by those countries that have gone through this process. It discusses in detail the consequences of graduation, when a country ceases to be a LDC and becomes a Developing Country, particularly the implications of the cessation of certain benefits granted exclusively to LDCs. Based on an analysis of the graduation processes and development paths of the five countries that graduated from the LDC category up until now - Botswana, Cabo Verde, Maldives, Samoa and Equatorial Guinea - we seek to ascertain the impacts of graduation in terms of growth and development in the post-graduation period, and to discuss the strategies used to mitigate the negative effects of the cessation of the benefits after the transition period. We conclude that even though certain benefits effectively end for newly graduated countries, this does not occur abruptly, and, in certain areas, it does not mean a complete cessation. Adequate preparation for graduation by the countries in question, in cooperation with the specialized committees of the UN and the development partners, makes it possible to adopt strategies aimed at ensuring that the graduation process is not a traumatic one. By analysing the five case studies, we find that the transition strategies that have been adopted have been largely beneficial, and that most of these countries have remained on a consistent growth path after graduation.info:eu-repo/semantics/publishedVersio

Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update

Despite considerable progress in treatment protocols, B-lineage acute lymphoblastic leukemia (B-ALL) displays a poor prognosis in about 15–20% of pediatric cases and about 60% of adult patients. In addition, life-long irreversible late effects from chemo- and radiation therapy, including secondary malignancies, are a growing problem for leukemia survivors. Targeted therapy holds promising perspectives for cancer treatment as it may be more effective and have fewer side effects than conventional therapies. The phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway is a key regulatory cascade which controls proliferation, survival and drug-resistance of cancer cells, and it is frequently upregulated in the different subtypes of B-ALL, where it plays important roles in the pathophysiology, maintenance and progression of the disease. Moreover, activation of this signaling cascade portends a poorer prognosis in both pediatric and adult B-ALL patients. Promising preclinical data on PI3K/Akt/mTOR inhibitors have documented their anticancer activity in B-ALL and some of these novel drugs have entered clinical trials as they could lead to a longer event-free survival and reduce therapy-associated toxicity for patients with B-ALL. This review highlights the current status of PI3K/Akt/mTOR inhibitors in B-ALL, with an emphasis on emerging evidence of the superior efficacy of synergistic combinations involving the use of traditional chemotherapeutics or other novel, targeted agents

Microalgae as a Nutraceutical Tool to Antagonize the Impairment of Redox Status Induced by SNPs: Implications on Insulin Resistance

Microalgae represent a growing innovative source of nutraceuticals such as carotenoids and phenolic compound which are naturally present within these single-celled organisms or can be induced in response to specific growth conditions. The presence of the unfavourable allelic variant in genes involved in the control of oxidative stress, due to one or more SNPs in gene encoding protein involved in the regulation of redox balance, can lead to pathological conditions such as insulin resistance, which, in turn, is directly involved in the pathogenesis of type 2 diabetes mellitus. In this review we provide an overview of the main SNPs in antioxidant genes involved in the promotion of insulin resistance with a focus on the potential role of microalgae-derived antioxidant molecules as novel nutritional tools to mitigate oxidative stress and improve insulin sensitivity

Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent Akt reactivation

The PI3K/Akt/mTOR signaling cascade is a key regulatory pathway controlling cell growth and survival, and its dysregulation is a reported feature of B-precursor acute lymphoblastic leukemia (B-pre ALL). Torin-2 is a novel, second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. It has been shown that Torin-2 displayed dramatic antiproliferative activity across a panel of cancer cell lines. To investigate if Torin-2 could represent a new option for the treatment of B-pre ALL, we tested its activity on a panel of B-pre ALL cell lines. In all of them Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range. Torin-2 caused both apoptosis and autophagy, induced cell cycle arrest in G0/G1 phase and affected both mTORC1 and mTORC2 activities as assessed by their specific substrate dephosphorylation. Torin-2 alone suppressed feedback activation of PI3K/Akt, whereas the mTORC1 inhibitor RAD001 required the addition of the Akt inhibitor MK-2206 to achieve the same effect. These pharmacological strategies targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new promising therapeutic strategy for treatment of B-pre ALL patients

The novel dual PI3K/mTOR inhibitor NVP-BGT226 displays cytotoxic activity in both normoxic and hypoxic hepatocarcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common lethal human malignancies worldwide and its advanced status is frequently resistant to conventional chemotherapeutic agents and radiation. We evaluated the cytotoxic effect of the orally bioavailable dual PI3K/mTOR inhibitor, NVP-BGT226, on a panel of HCC cell lines, since hyperactivated PI3K/Akt/mTOR signaling pathway could represent a biomolecular target for Small Inhibitor Molecules in this neoplasia. We analyzed the drug activity in both normoxia and hypoxia conditions, the latter playing often a relevant role in the induction of chemoresistance and angiogenesis. In normoxia NVP-BGT226 caused cell cycle arrest in the G0/G1 phase of the cell cycle, induced apoptosis and autophagy at low concentrations. Interestingly the drug inactivated p-Akt and p-S6 at < 10 nM concentration. In hypoxia NVP-BGT226 maintained its cytotoxic efficacy at the same concentration as documented by MTT assays and Western blot analysis. Moreover, the drug showed in hypoxia inhibitory properties against angiogenesis by lowering the expression of the transcription factor HIF-1α and of VEGF. Our results indicate that NVP-BGT226 has a potent cytotoxic effect on HCC cell lines also in hypoxia condition, thus emerging as a potential candidate for cancer treatment in HCC targeted therap

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway and NUP214-ABL1 fusion protein in human acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a neoplasm of precursor cells committed to the B-cell and T-cell lineages involving bone marrow and blood, with a rapid onset and frequent chemotherapy resistance and refractory relapses (1). Philadelphia chromosome-positive (Ph+) ALL accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes, among which BCR-ABL1 is the most commonly found, are markers of very poor prognosis. Amplification of the NUP214-ABL1 oncogene can be detected only in patients with T-ALL (2). The PI3K/Akt/mTOR signaling pathway is activated in many solid cancers and in leukemias and plays a crucial role in tumorigenesis. Furthermore, the presence of RTKs (Receptor Tyrosine Kinases) by ABL1 fusion proteins may result in activation of the PI3K/Akt/mTOR axis. T cell malignancies bearing the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been found. In this work we analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on two NUP214-ABL1 positive T-ALL cell lines, ALL-SIL and PEER that also displayed Akt hyperactivation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis as deducted by a readout of drug efficacy, four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226 (BGT226), ZSTK474 and Torin-2, showed a relevant cytotoxic efficacy on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of the compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability which was associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL

Narrativas del origen histórico de los derechos humanos en los manuales de derecho

Tomando como corpus de análise manuais sobre direitos humanos citados na bibliografi a dos programas de disciplinas dos cursos de Direito do Estado do Rio Grande do Sul, apresentamos três marcas frequentes no modo de narrar a história dos direitos humanos ali presentes: o estatuto da fonte histórica, a noção de evolução histórica e a pretensão de neutralidade. Estamos apoiados na afi rmativa de que o modo de contar a história dos direitos humanos traz implicações diretas na defi nição do que sejam esses direitos. A narrativa histórica estabelece conexões possíveis entre direitos humanos e determinados temas (por exemplo, direito de família), ao mesmo tempo em que difi culta ou impossibilita conexões dos direitos humanos com outros temas (por exemplo, direito empresarial)Based on the human rights manuals cited in the bibliography of the Law program syllabi of the state of Rio Grande do Sul as the corpus of analysis, we present three characteristics frequently found in the recounting of the history of human rights: the statute of the historic source, the notion of historic evolution and the claim to neutrality. We are supported by the statement that the mode of recounting the history of human rights has direct implication in the defi nition of what these rights are. On the one hand, the historic narrative establishes possible connections between human rights and specifi c themes ( for example, family law). On the other hand, it inhibits or prevents connections between human rights and other themes ( for example, business law).Tomando como corpus de análisis manuales sobre derechos humanos citados en la bibliografía de los programas de disciplinas de los cursos de Derecho del Estado de Rio Grande del Sur, presentamos tres marcas frecuentes en el modo de narrar la historia de los derechos humanos allí presentes: el estatuto de la fuente histórica, la noción de evolución histórica y la pretensión de neutralidad. Estamos apoyados en la afi rmación de que el modo de contar la historia de los derechos humanos trae implicaciones directas en la defi nición de lo que sean esos derechos. La narrativa histórica establece conexiones posibles entre derechos humanos y determinados temas (por ejemplo, derecho de familia), al mismo tiempo que difi culta o imposibilita conexiones de los derechos humanos con otros temas (por ejemplo, derecho empresarial)