A infecção por tipos de HPV de alto risco oncogênico é o principal fator de risco para o desenvolvimento do carcinoma do colo uterino, uma das neoplasias mais frequentes em mulheres de todo o mundo. Este grupo de vírus também está associados a uma proporção importante de outros cânceres anogenitais e de tumores de cabeça e pescoço. Os HPVs de alto risco expressam duas oncoproteínas, E6 e E7, que agem sobre fatores celulares específicos alterando diferentes vias de vinalização. A oncoproteína E6 é capaz de unir-se à proteína supressora de tumor p53, alterar a sua capacidade funcional e promover sua degradação pela via de proteólise dependente de ubiquitina. Mais ainda, a interação da proteína E6 com várias proteínas celulares por exemplo, E6AP, TNFR1, Bak e caspase 8 confere resistência a apoptose; hADA3, p300, CARM1 e SET7 a remodulação da cromatina; NFX1-91 a ativação da telomerase; STAT-1 e TLR-9 a evasão imune, ATR, BCRA1, MCM7, MGMT. XRCC1 a estabilidade genômica; DLG1, MAGI1-3, SCRIBBLE, paxilina e fibulina a perda de polaridade celular e indução de hiperplasia. No entanto, as consequências de muitas dessas interações não têm sido bem estudadas em queratinócitos primários humanos, a célula alvo natural do HPV. No presente estudo analizamos o efeito da proteína E6 de HPV16 (alto risco) e HPV11 (baixo risco) na expressão e na atividade de fatores envolvidos na regulação/execução de apoptose induzida pelas citocinas TNF e TRAIL e pelo quimioterápico Rapamicina. Através de ensaios de proliferação/viabilidade observamos que as células que expressam E6 de ambos os tipos virais apresentam resistência às citocinas e à rapamicina, quando comparadas a culturas controle. Além disso, observamos que as células que expressam E6 apresentam diferenças no padrão de expressão de proteínas envolvidas na regulação das vias extrínseca e intrínseca da apoptose. / The infection with oncogenic HPV types is the main risk factor for the development of cervical cancer, one of the most common malignancies in women worldwide. This group of viruses is also associated with a significant proportion of other anogenital cancer and head and neck tumor. High-risk HPV express two oncoproteins, E6 and E7, which act on specific cellular factors altering difefferent signaling pathways. For instance E6 oncoprotein is able to bind the p53 tumor suppressor protein and promote its degradation by the ubiquitin dependent proteolysis pathway. Furthermore, the interaction of E6 with various cellular proteins for example, E6AP, TNFR1, caspase 8 Bak and confers resistance to apoptosis; hADA3, p300, and CARM1 SET7 the reshaping of chromatin; NFX1-91 telomerase activation; STAT-1 and TLR-9 immune evasion, ATR, BCRA1, MCM7, MGMT. XRCC1 genomic stability; Dlg1, MAGI1-3, scribble, paxillin and fibulin loss of cell polarity and hyperplasia of induction. In the present study we analyzed the effect of the E6 protein of HPV (highrisk) and HPV11 (low-risk) on the expression and activity of factors involved in the regulation/execution of apoptosis induced by the cytokines TNF and TRAIL and the chemotherapeutic agent Ramaycin. Using proliferation/viability assays we observed that cells expressing E6 from either viral cytokines and Rapamycin when compared with control cells. Besides, we observed that cells expressing E6 exhibit differences in the expression pattern of protein involved in the regulation of apoptosis extrinsic and intrinsic pathways.

Pbp1 is a component of glucose deprivation induced stress granules and is involved in P-body-dependent granule assembly. We have recently shown that Pbp1 plays an important role in the interplay between three sensory protein kinases in yeast: AMP-regulated kinase (Snf1 in yeast), PAS kinase 1 (Psk1 in yeast), and the target of rapamycin complex 1 (TORC1), to regulate glucose allocation during nutrient depletion. This signaling cascade occurs through the SNF1-dependent phosphorylation and activation of Psk1, which phosphorylates and activates poly(A)- binding protein binding protein 1 (Pbp1), which then inhibits TORC1 through sequestration at stress granules. In this study we further characterized the regulation of Pbp1 by PAS kinase through the characterization of the role of the Psk1 homolog (Psk2) in Pbp1 regulation, and the identification of functional Pbp1 binding partners. Human ataxin-2 (ATXN2) is the homolog of yeast Pbp1 and has been shown to play an important role in the development of several ataxias. In this study we have also provided the evidence that human ataxin-2 can complement Pbp1 in yeast, and that human PAS kinase can phosphorylate human ataxin-2. Further characterizing this interplay between PAS kinase and Pbp1/ATXN2 aid in understanding pathways required for proper glucose allocation during nutrient depletion, including reducing cell growth and proliferation when energy is low. In addition, it yields valuable insights into the role of ataxin-2 in the development of devastating ataxias.

<p>Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. </p><p>Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6.</p><p>Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM.</p><p>Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. </p><p>Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. </p><p>In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.</p>

Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6. Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM. Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.

La protéine kinase TOR (Target Of Rapamycin) a été identifiée chez la levure et les mammifères comme participant à deux complexes protéiques qui servent de carrefour entre la perception des facteurs endogènes et exogènes et la stimulation de la croissance cellulaire. Depuis la découverte de la kinase AtTOR chez Arabidopsis thaliana, des études ont été menées afin de mieux caractériser son rôle chez les plantes et l'influence de son niveau d'expression sur la régulation du métabolisme et du développement.Au cours de ce travail, j'ai contribué à l'étude de cette kinase en étudiant l'influence de l'inactivation de TOR sur la composition du ribosome au niveau protéique et sur le niveau de phosphorylation de ces protéines, ainsi que sur l'organisation du méristème au niveau moléculaire et cytologique Au cours de cette étude, j'ai montré que certaines protéines constitutives du ribosome pourraient être des cibles de l'activité TOR au niveau de leur abondance et/ou de leur état de phosphorylation. Ainsi, l'inactivation de TOR entraine une diminution du niveau de phosphorylation des protéines RPS6 et pourrait influencer l'abondance des protéines acides constitutives du stalk ribosomal, une structure importante dans la régulation de la traduction. Les résultats obtenus suggèrent également que l'activité TOR est nécessaire au maintien du méristème à l'état fonctionnel en régulant les voies importantes contrôlant la division et la différentiation au sein de cette structure.

A infecção por tipos de HPV de alto risco oncogênico é o principal fator de risco para o desenvolvimento do carcinoma do colo uterino, uma das neoplasias mais frequentes em mulheres de todo o mundo. Este grupo de vírus também está associados a uma proporção importante de outros cânceres anogenitais e de tumores de cabeça e pescoço. Os HPVs de alto risco expressam duas oncoproteínas, E6 e E7, que agem sobre fatores celulares específicos alterando diferentes vias de vinalização. A oncoproteína E6 é capaz de unir-se à proteína supressora de tumor p53, alterar a sua capacidade funcional e promover sua degradação pela via de proteólise dependente de ubiquitina. Mais ainda, a interação da proteína E6 com várias proteínas celulares por exemplo, E6AP, TNFR1, Bak e caspase 8 confere resistência a apoptose; hADA3, p300, CARM1 e SET7 a remodulação da cromatina; NFX1-91 a ativação da telomerase; STAT-1 e TLR-9 a evasão imune, ATR, BCRA1, MCM7, MGMT. XRCC1 a estabilidade genômica; DLG1, MAGI1-3, SCRIBBLE, paxilina e fibulina a perda de polaridade celular e indução de hiperplasia. No entanto, as consequências de muitas dessas interações não têm sido bem estudadas em queratinócitos primários humanos, a célula alvo natural do HPV. No presente estudo analizamos o efeito da proteína E6 de HPV16 (alto risco) e HPV11 (baixo risco) na expressão e na atividade de fatores envolvidos na regulação/execução de apoptose induzida pelas citocinas TNF e TRAIL e pelo quimioterápico Rapamicina. Através de ensaios de proliferação/viabilidade observamos que as células que expressam E6 de ambos os tipos virais apresentam resistência às citocinas e à rapamicina, quando comparadas a culturas controle. Além disso, observamos que as células que expressam E6 apresentam diferenças no padrão de expressão de proteínas envolvidas na regulação das vias extrínseca e intrínseca da apoptose. / The infection with oncogenic HPV types is the main risk factor for the development of cervical cancer, one of the most common malignancies in women worldwide. This group of viruses is also associated with a significant proportion of other anogenital cancer and head and neck tumor. High-risk HPV express two oncoproteins, E6 and E7, which act on specific cellular factors altering difefferent signaling pathways. For instance E6 oncoprotein is able to bind the p53 tumor suppressor protein and promote its degradation by the ubiquitin dependent proteolysis pathway. Furthermore, the interaction of E6 with various cellular proteins for example, E6AP, TNFR1, caspase 8 Bak and confers resistance to apoptosis; hADA3, p300, and CARM1 SET7 the reshaping of chromatin; NFX1-91 telomerase activation; STAT-1 and TLR-9 immune evasion, ATR, BCRA1, MCM7, MGMT. XRCC1 genomic stability; Dlg1, MAGI1-3, scribble, paxillin and fibulin loss of cell polarity and hyperplasia of induction. In the present study we analyzed the effect of the E6 protein of HPV (highrisk) and HPV11 (low-risk) on the expression and activity of factors involved in the regulation/execution of apoptosis induced by the cytokines TNF and TRAIL and the chemotherapeutic agent Ramaycin. Using proliferation/viability assays we observed that cells expressing E6 from either viral cytokines and Rapamycin when compared with control cells. Besides, we observed that cells expressing E6 exhibit differences in the expression pattern of protein involved in the regulation of apoptosis extrinsic and intrinsic pathways.

Efficient gene transfer has been achieved in several animal models using different vector systems, leading to stable transgene expression. The tight control of this expression is now an important outcome for the field of gene therapy. Such regulation is likely to be required for therapeutic applications and in some instances for safety reasons. For this purpose, several regulatable systems depending on small molecules have been developed. Among these, the tetracycline and the rapamycin dependent systems have been largely used. However, if long-term regulation of the transgene has been obtained in small animal models using these inducible systems, when translational studies were initiated in larger animals, the development of an immune response against proteins involved in transgene regulation were often observed. Such immune response was especially documented when using the TetOn tetracycline regulatable system in nonhuman primates (NHP). Humoral and destructive cellular immune responses against the transactivator involved in this regulation system were documented in a large majority of NHP leading to the complete loss of the transgene regulation and expression. This review will describe the immune responses observed in these different model systems applied for transgene regulation. Focus will be finally given on future directions in which such immune responses might be surmounted, enabling long-term transgene regulation in future clinical developments of gene transfer.

Exercise has been considered a cornerstone of diabetes prevention and treatment for decades, but the benefits of resistance training are less clear. Nineteen non-diabetic subjects (10 metabolic syndrome, 9 sedentary controls) underwent 8 weeks of supervised resistance training. After training, strength and V̇ O2max increased by 10% in both groups. Percent body fat decreased in subjects with the metabolic syndrome. Additionally, lean body mass increased in both groups (p<0.05). Expression of glucose transporter protein-4 (GLUT4), the principle insulin-responsive glucose transporter, increased significantly in both groups. 5-adenosine monophosphateactivated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) expression increased in both groups, indicating increased protein synthesis and mitochondrial biogenesis. Markers of insulin resistance measured by a euglycemic hyperinsulinemic clamp did not improve in subjects with the metabolic syndrome but increased significantly in control subjects (13%). Resistance training upregulates intracellular signaling pathways that may be beneficial for ameliorating the metabolic syndrome.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

Nutrient sensing kinases sense available nutrients and regulate cell activity accordingly. Three of these enzymes are AMP regulated kinase (AMPK, or Snf1 in yeast), PAS kinase, and target of rapamycin (TOR), are conserved from yeast to man and have overlapping function. AMPK and Snf1 are important in sensing when nutrient status in the cell is low and down regulating energy consuming pathways. PAS kinase is required for glucose homeostasis in the cell, and responds to glucose levels. TOR senses nutrients such as amino acids and upregulates cell growth pathways primarily through protein synthesis. Due to the varying nature of these enzymes, cross talk is expected in order for the cell to properly regulate cellular metabolism and growth in response to energy and nutrient availability. Previous studies have shown that activation of yeast PAS kinase under nutrient stress conditions requires the presence of Snf1. The aim of this thesis is to determine whether Snf1 directly phosphorylates and activates PAS kinase through both in vivo and in vitro approaches. PAS kinase was found to require Snf1 for both activation and phosphorylation in vivo. In vitro kinase assays were also performed to confirm a direct phosphorylation event. The results from this study support the direct phosphorylation and activation of PAS kinase by Snf1, linking cellular energy status to glucose allocation.