PART1, TITLE: A p53-inducible long non-coding RNA PICART1 mediating cancer cell proliferation and migration. Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs are characterized thus far. A novel lncRNA transcript with 2.53 kb in length was identified by a transcriptome sequencing analysis, named p53-inducible cancer-associated RNA transcript 1 (PICART1). This PICART1 is upregulated by p53 through a p53-binding site at -1808 to -1783bp. In breast and colorectal cancer cells and tissues, PICART1 expression was decreased. Ectopic expression of the PICART1 suppressed growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated the cell growth and migration. In these cells, the expression of PICART1 lowered down the levels of p-AKT (Thr308 & Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, but p21cip1/Waf1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade. PART2, TITLE: The novel long non-coding RNA PANCR is a tumor suppressor gene in breast cancer. Long non-coding RNAs (lncRNAs) function as oncogenes or tumor suppressors in development and progression of cancer. Chromosome 16q22.1 region is frequently deleted in breast cancer, which may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new lncRNA tumor suppressor, named p53 activating non-coding RNA (PANCR), located in this Chromosome 16q22.1 region. This PANCR lncRNA consists of 1.5kb in length. Our data showed that PANCR was downregulated in breast cancer tissues and cell lines. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31 (62.0%) of 50 cases when compared to adjacent normal breast tissues. In breast cancer cells MCF7 cells, ectopic expression of PANCR suppressed cell proliferation in culture, but in contrast, shRNA–mediated silencing of PANCR promoted cell growth and proliferation.

Orientador: Jayme Augusto de Souza Neto / Resumo: Aedes aegypti é a espécie de mosquito emergente em áreas urbanas com maior impacto na saúde pública, sendo o principal vetor dos arbovírus dengue, Zika e chikungunya. Por esse motivo, se faz indispensável a compreensão de mecanismos moleculares associados a processos fisiológicos em A. aegypti, como resposta imune, comportamento e homeostase intestinal. Conforme observado em outros organismos, a via PI3K/AKT tem papéis importantes no metabolismo, na reprodução, na tolerância ao estresse e na imunidade. A quinase AKT atua como um regulador negativo da via PI3K/AKT, fosforilando o fator de transcrição FOXO e impedindo sua translocação nuclear. Nosso objetivo foi avaliar o perfil transcricional em A. aegypti com o gene akt silenciado e avaliar as consequências deste silenciamento sobre a microbiota bacteriana. Além disso, investigamos uma provável ativação mitocondrial quando do silenciamento de akt. Mostramos que o silenciamento de akt resultou na ativação de genes essenciais para a manutenção da homeostase intestinal do mosquito, como peptídeos antimicrobianos (AMPs) e genes codificadores de enzimas associadas à produção de espécies reativas de oxigênio (ROS). Notavelmente, observou-se uma forte repressão de 11 profenoloxidases (PPOs), além de uma potente indução de genes codificadores de subunidades da enzima NADH desidrogenase, em comparação com o respectivo grupo controle, sugerindo que tal indução esteja associada a um provável aumento da atividade mitocondrial. No context... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aedes aegypti is the emerging mosquito species in urban areas with the greatest impact on public health, being the main vector of arboviruses dengue, Zika and chikungunya. For this reason, it is essential to understand the molecular mechanisms associated with physiological processes in A. aegypti, such as immune response, behavior and intestinal homeostasis. As observed in other organisms, the PI3K / AKT pathway has important roles in metabolism, reproduction, stress tolerance and immunity. AKT kinase acts as a negative regulator of the PI3K / AKT pathway, phosphorylating the FOXO transcription factor and preventing its nuclear translocation. Our objective was to evaluate the transcriptional profile in A. aegypti with the silenced akt gene and to evaluate the consequences of this silencing on the bacterial microbiota. In addition, we investigated a possible mitochondrial activation during the akt silencing. We have shown that akt silencing resulted in the activation of essential genes for the maintenance of mosquito intestinal homeostasis, such as antimicrobial peptides (AMPs) and genes encoding enzymes associated with the production of reactive oxygen species (ROS). Notably, a strong repression of 11 profenoloxidases (PPOs) was observed, as well as a potent induction of genes encoding subunits of the NADH dehydrogenase enzyme, in comparison with the respective control group, suggesting that such induction is associated with a possible increase in mitochondrial activity. In t... (Complete abstract click electronic access below) / Doutor

Submitted by BRUNO TINOCO NUNES (croookes@gmail.com) on 2018-09-25T16:26:37Z No. of bitstreams: 1 Tese_Bruno_Tinoco_25_09_2018.pdf: 4179397 bytes, checksum: eb771bc06b406e51cb4c11fd78aa1814 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-09-25T16:43:00Z (GMT) No. of bitstreams: 1 nunes_bt_dr_bot.pdf: 4179397 bytes, checksum: eb771bc06b406e51cb4c11fd78aa1814 (MD5) / Made available in DSpace on 2018-09-25T16:43:00Z (GMT). No. of bitstreams: 1 nunes_bt_dr_bot.pdf: 4179397 bytes, checksum: eb771bc06b406e51cb4c11fd78aa1814 (MD5) Previous issue date: 2018-07-23 / Outra / Aedes aegypti é a espécie de mosquito emergente em áreas urbanas com maior impacto na saúde pública, sendo o principal vetor dos arbovírus dengue, Zika e chikungunya. Por esse motivo, se faz indispensável a compreensão de mecanismos moleculares associados a processos fisiológicos em A. aegypti, como resposta imune, comportamento e homeostase intestinal. Conforme observado em outros organismos, a via PI3K/AKT tem papéis importantes no metabolismo, na reprodução, na tolerância ao estresse e na imunidade. A quinase AKT atua como um regulador negativo da via PI3K/AKT, fosforilando o fator de transcrição FOXO e impedindo sua translocação nuclear. Nosso objetivo foi avaliar o perfil transcricional em A. aegypti com o gene akt silenciado e avaliar as consequências deste silenciamento sobre a microbiota bacteriana. Além disso, investigamos uma provável ativação mitocondrial quando do silenciamento de akt. Mostramos que o silenciamento de akt resultou na ativação de genes essenciais para a manutenção da homeostase intestinal do mosquito, como peptídeos antimicrobianos (AMPs) e genes codificadores de enzimas associadas à produção de espécies reativas de oxigênio (ROS). Notavelmente, observou-se uma forte repressão de 11 profenoloxidases (PPOs), além de uma potente indução de genes codificadores de subunidades da enzima NADH desidrogenase, em comparação com o respectivo grupo controle, sugerindo que tal indução esteja associada a um provável aumento da atividade mitocondrial. No contexto comportamental, o transcriptoma de A. aegypti com o gene akt silenciado revelou a modulação de 5 odorant binding proteins (OBPs), das quais todas foram reprimidas no quarto dia após o silenciamento de akt. Além das OBPs, identificamos duas long wavelengh sensitive opsins, ambas reprimidas após dois e quatro dias. Interessantemente, observamos que, dos 345 genes modulados, a grande maioria foi regulada negativamente. O silenciamento de akt, além de modular a carga da microbiota bacteriana de A. aegypti, também modulou táxons específicos após quatro dias de silenciamento, como as Classes Gammaproteobacteria e Betaproteobacteria. Nosso conjunto de dados coloca a via PI3K/AKT no cerce de importantes processos fisiológicos, contribuindo para elucidar mecanismos moleculares até então pouco compreendidos do mosquito. / Aedes aegypti is the emerging mosquito species in urban areas with the greatest impact on public health, being the main vector of arboviruses dengue, Zika and chikungunya. For this reason, it is essential to understand the molecular mechanisms associated with physiological processes in A. aegypti, such as immune response, behavior and intestinal homeostasis. As observed in other organisms, the PI3K / AKT pathway has important roles in metabolism, reproduction, stress tolerance and immunity. AKT kinase acts as a negative regulator of the PI3K / AKT pathway, phosphorylating the FOXO transcription factor and preventing its nuclear translocation. Our objective was to evaluate the transcriptional profile in A. aegypti with the silenced akt gene and to evaluate the consequences of this silencing on the bacterial microbiota. In addition, we investigated a possible mitochondrial activation during the akt silencing. We have shown that akt silencing resulted in the activation of essential genes for the maintenance of mosquito intestinal homeostasis, such as antimicrobial peptides (AMPs) and genes encoding enzymes associated with the production of reactive oxygen species (ROS). Notably, a strong repression of 11 profenoloxidases (PPOs) was observed, as well as a potent induction of genes encoding subunits of the NADH dehydrogenase enzyme, in comparison with the respective control group, suggesting that such induction is associated with a possible increase in mitochondrial activity. In the behavioral context, the A. aegypti transcriptome with the mutated akt gene revealed the modulation of 5 odorant binding proteins (OBPs), all of which were repressed on the fourth day after akt silencing. In addition to the OBPs, we identified two long wavelengh sensitive opsins, both repressed after two and four days. Interestingly, we observed that of the 345 modulated genes, most were down-regulated. The silencing of akt, besides modulating the bacterial microbiota load of A. aegypti, also modulated specific taxa after four days of silencing, such as the Gammaproteobacteria and Betaproteobacteria classes. Our data set puts the PI3K / AKT pathway in the vicinity of important physiological processes, contributing to elucidate the mosquito's poorly understood molecular mechanisms.

Ischemic stroke causes irreversible damage to the brain. The hippocampus is a vulnerable region and plays an important role in cognition and locomotor activity. Puerarin is a phytoestrogen that has beneficial effects in treating neurological disorders. How puerarin protects against hippocampal injury and its molecular mechanisms remain to be elucidated. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with puerarin alone or together with LY294002 (an PI3K inhibitor) before ischemia/ reperfusion (I/R). The open-and closed-field tasks and Morris water maze (MWM) test were used to assess the effects of puerarin on anxiety-like behavioral and cognitive impairment following I/R. Hematoxylin-eosin staining(HE) was used to examine the survival of hippocampal CA1 pyramidal neurons, and immunoblotting was performed to examine the expression of the related proteins. By using the rat model for transient I/R, we demonstrated that puerarin pretreatment significantly increased the travelling distance and number of crossings in the open-and closedfield tests, reduced latency and increased the proportion of distance and time in zone IV in the MWM. The number of live cells in the hippocampus is sharply increased by puerarin pretreatment. We further observed that the levels of phosphorylated Akt1, GSK-3 beta and MCL-1were elevated and those of cleaved-caspase-3 were reduced in the puerarin-treatment group. Notably, the PI3K inhibitor LY294002 counteracted all of the effects of puerarin. Our findings suggest that puerarin protects the hippocampus from I/R damage by activating the PI3K/Akt1/GSK-3 beta/MCL-1 signaling pathway.

Cancer and neurodegeneration are detrimental conditions associated with an inappropriate regulation of cell survival and cell death, causing compromised cells to evade death or excessive death of healthy neurons. The mammalian target of rapamycin complex 2 (mTORC2) has been implicated in the regulation of cell survival by phosphorylating the protein kinase Akt. This is dependent upon the scaffold protein Sin1, a core component of mTORC2. The requirement of Sin1 in cell survival, and in particular in neuronal survival, has not been established due to the early embryonic lethality of mice with a targeted deletion of the Sin1 gene. To circumvent this issue, a novel conditional mouse knockout model was established. The role of Sin1 in regulating cell survival was evaluated in fibroblasts and cortical neurons. The loss of Sin1 significantly affected the phosphorylation and activity of Akt in fibroblasts and caused a reduction in cell survival by potentially inducing premature senescence. In contrast, the loss of Sin1 caused an increase in caspase-independent cell death in cortical neurons. Gene-expression analysis of Sin1 knockout cortical neurons demonstrated an important down-regulation of transcription factors, cytoskeletal proteins and components of signalling pathways involved in neuronal survival, aiding to uncover the mechanism by which Sin1 promotes neuronal survival. Taken together, the results presented in this study show a key role of the scaffold protein Sin1 in regulating neuronal survival.

Multiple signaling pathways control cellular response to environmental cues such as nutrients, growth factors and stress. Interpretation of these cues requires coordinated regulation of intracellular signaling pathways. Our attempt to understand how cells coordinate different signaling pathways led to the discovery of two crosstalk mechanisms between different signaling cascades. We found that PI3K-AKT signaling reduces EGFR signaling to the parallel ERK-MAPK pathway by enhancing EGF induced EGFR degradation. At the molecular level AKT activates PIKfyve to facilitate EGFR trafficking from early endosomes to the lysosomes. Using a mass spectrometry based approach we also found growth factor signaling by EGF inhibits stress response. In particular, inhibiting RSK signaling downstream of EGF increased the activity of stress activated kinases p38, MSK2 and ERK5. We propose that when growth factors are present active RSK phosphorylates and inhibits a master regulator of stress response MEKK3, which leads to termination of MEKK3 signaling to downstream kinases. Our unbiased phosphoproteomic approach also lead to identification of many ERK and RSK substrates that will help us explain how growth factor signaling regulates a wide variety of biological processes.

It is well established that the Wnt pathway is associated with tumorigenesis in a wide range of human cancers, including a majority of breast cancers. However, due to diverse roles of Wnt signalling, therapeutic targeting has not yielded consistent results and underlying mechanisms remain unclear. Here, I show that breast cancer cell lines with high E-cadherin expression are resistant to TCF4 inhibitors and develop cancer stem cell characteristics. Conversely, cells with low levels of E-cadherin are very susceptible to cell death with the same treatment. My results suggest that breast cancer cells in an epithelial-like state, but not mesenchymal-like state, will be more responsive to therapeutic targeting of the Wnt/TCF pathway. Importantly, E-cadherin high cells show robust Akt activation, whereas E-cadherin low cells do not. Thus, combinational inhibition of both Wnt and Akt signalling is needed to effectively target breast cancer cells in both the epithelial and mesenchymal states.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13386号 / 論医博第2218号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Macrophage apoptosis is an important process in the pathophysiology of atherosclerosis. Oxidized low-density lipoproteins (OxLDL) are a major component of lesions and potently induce macrophage apoptosis. Cannabinoid receptor 2 (CB2), the predominant macrophage cannabinoid receptor, modulates several macrophage processes associated with ongoing atherosclerosis; however, the role of CB2 in macrophage apoptosis is unknown. To determine if CB2 influences a macrophage apoptotic pathway relevant to atherosclerosis, we examined the effect of CB2 deficiency on OxLDL-induced macrophage apoptosis. In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) analysis of resident peritoneal macrophages detected significantly fewer apoptotic CB2-/- macrophages than CB2+/+ macrophages after incubation with OxLDL (27.9 ± 4.7% vs. 61.9 ± 8.5%, P < 0.001) or 7-ketocholesterol (7KC) (18.9 ± 10.5% vs. 54.1 ± 6.9%, P < 0.001), an oxysterol component of OxLDL. Caspase-3 activity; proteolytic conversion of procaspase-3; and cleavage of a caspase-3 substrate, PARP, were also diminished in 7KC-treated CB2-/-macrophages. Furthermore, the deactivation of the prosurvival kinase, Akt, in response to 7KC was impaired in CB2-/-macrophages. These results suggest that CB2 expression increases the susceptibility of macrophages to OxLDL-induced apoptosis, in part, by modulating the effect of oxysterols on the Akt survival pathway and that CB2 may influence atherosclerosis by modulating lesional macrophage apoptosis.