Mammalian homolog Tribbles (Trbs) is a newly characterized protein family that includes three different isoforms: Trb1, Trb2, and Trb3. Tribbles are serine/threonine kinases lacking catalytic activity, thus their classification as pseudokinases. Despite their catalytic inactivity, Tribbles can interact with different proteins and regulate different biological functions. The most studied tribble family member, Trb3, was reported to play a major role in Drosophila's ventral furrow formation. Further studies revealed that Trb3 is also involved in diabetes, stress-response, and development. Previously, Trb3 upregulation was detected in certain types of cancer but its function remains unknown. The goal of our study is to gain a better understanding of the biological function of Trb3 in cancer, including the molecular mechanism of action. Using the cohort analysis, we identified higher levels of Trb3 in the lung tumor compared to the normal tissue. Furthermore, higher Trb3 expression in the lung tissue was associated with a poor survival in cancer patients. Silencing of Trb3 in A549 promoted cell growth. On the other hand, overexpression of Trb3 in NCI-H358 inhibited cell growth. The analysis of cell cycle gene profiling revealed a decrease in several genes that are essential for cell cycle progression in S phase in Trb3 overexpressed NCI-H358. The cell proliferation protein, Ki67, was also decreased in Trb3 overexpressed NCI-H358 cells. Moreover, Tb3 overexpressed cells formed higher colony number in soft agar assay and depicted higher migration ability in the Boyden chamber assay. Mesenchymal markers SNAIL, TWIST and N-cadherin were upregulated while epithelial E-cadherin was significantly reduced. Interestingly, prosurvival protein Akt was also reduced post Trb3 overexpression. Trb3 expression was associated with a poor survival. However, we discovered that Trb3 overexpression inhibited cell growth. Thus, we hypothesized that Trb3 expression might contribute to tumorigenesis during cellular metabolic stress. In order to understand the potential role of Trb3 in metabolic stress, NCI-H358 cells were treated with five different cellular stressors to mimic the tumor microenvironment. All stressors used were shown to induce endogenous Trb3 expression. Moreover, stress proteins ATF4, CHOP and ASNS were induced by all stressors. One of the stressors used was rotenone, an inhibitor of the complex I of the electron transport chain. Rotenone treatment induced Trb3 expression. This expression inversely correlated with cytochrome C expression. Furthermore, Trb3 expression positively correlated with the expression of mitophagic genes PINK1, Parkin and p62, which suggest that Trb3 is induced during ROS-mediated oxidative stress to participate in the clearing of damaged mitochondria. This targeted clearing of the mitochondria, a process known as mitophagy is essential for the cell survival of the lung cancer cells. Last, Trb3 overexpression rendered cancer cells resistant to docetaxel and cisplatin, two chemotherapeutic drugs used in lung cancer treatment. On the other hand, Trb3 depleted cells were more sensitive to the drugs. Our results suggest that Trb3 is activated in the primary tumor to promote metabolic adaptation through cell cycle arrest and the inhibition of aerobic glucose metabolism through Akt inhibition. Furthermore, Trb3 is essential during cell survival post ROS-mediated stress and participates in the clearing of damaged mitochondria during mitophagy. Last, stress-mediated activation of Trb3 confers lung cancer cells with chemoresistance and suggest that Trb3 could be a potential target in lung cancer therapy.

Orientadores: José Rodrigo Pauli, Dennys Esper Corrêa Cintra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-26T10:24:59Z (GMT). No. of bitstreams: 1 Rodrigues_BarbaradeAlmeida_M.pdf: 2531599 bytes, checksum: 8ab5a3a29ab7a93465c0db3ffb7c5f4e (MD5) Previous issue date: 2014 / Resumo: A sinalização da insulina no hipotálamo desempenha papel crucial na manutenção da massa corporal. Uma vez ativada, a proteína Akt aumenta a fosforilação do fator de transcrição FoxO1, que é um importante mediador da sinalização insulina no hipotálamo e estimula a transcrição de neuropeptídeos anorexigênicos. Nesse contexto, devido a capacidade de se ligar diretamente a proteína quinase B (Akt), a Mammalian Homolog of Drosophila Tribbles 3 (TRB3) torna-se uma importante molécula de regulação da sinalização da insulina. Em adição, a sinalização do estresse de retículo endoplasmático (estresse de RE) via proteína quinase de retículo endoplasmático (pPERK) e um fator de transcrição mediador de apoptose denominado CHOP, é considerado como mecanismo responsável pela super-expressão de TRB3 em roedores obesos e diabéticos. Por outro lado, o exercício físico é considerado uma das mais importantes estratégias não farmacológicas para a prevenção e/ou tratamento da obesidade. O exercício é capaz de proteger contra a inflamação, o estresse de RE e aumentar a sinalização da insulina no hipotálamo de roedores obesos. Entretanto, os efeitos do exercício agudo na modulação da TRB3 não foram descritos. O objetivo do presente estudo foi investigar o papel do exercício agudo de natação sobre a expressão da TRB3 e associação TRB3/Akt no hipotálamo de roedores obesos. Foram utilizados ratos wistar divididos em três grupos: Lean (controle magro), alimentados com uma ração padrão para roedores, DIO (alimentados com uma dieta rica em gordura), e DIO-EXE (submetidos a um protocolo de natação aguda de exercício). Para reforçar que o aumento da TRB3 está associada com o estresse de RE, e que uma sessão aguda de exercício é capaz de reverter esse aumento, usamos outros três grupos: controle (Lean) recebendo uma infusão de veículo intracerebroventricular (i.c.v.), magros que receberam i.c.v. de thapsigargina (indutor farmaológico de estresse de RE) e magros que receberam i.c.v. de thapsigargina e submetidos a um protocolo de exercício agudo de natação. O protocolo de exercício físico consistiu de uma sessão de natação, composto por dois períodos de três horas, com uma pausa de 45 minutos. Previamente a eutanásia dos animais, foram realizadas as análises da glicemia, insulinemia, sensibilidade à insulina e a mensuração da ingestão alimentar no período de 12h. Quatro horas após a última sessão de exercício, o tecido hipotalâmico foi removido para análise de proteínas de interesse através das técnicas de imunoprecipitação, imunoblot e de PCR em tempo real. Como resultado, verificou-se que o exercício reduziu os níveis de proteína TRB3 e a interação entre a proteína TRB3 e Akt, aumentou a fosforilação de FoxO1 e restaurou os efeitos anorexígenos de insulina no hipotálamo de ratos DIO-EXE. Além disso, nossos resultados revelaram que a i.c.v. de thapsigargina bloqueou os efeitos anorexígenos mediados por insulina em ratos magros, e o exercício físico restaurou a ação da insulina e a fome, assim, os efeitos supressivos do exercício agudo sobre os níveis de proteína TRB3 pode ser relacionada, no mínimo em parte, pela redução do estresse de RE em neurônios hipotalâmicos / Abstract: Hypothalamic insulin signalling plays an important role in the maintenance of body weight. Once activated, Akt increases the phosphorylation of FoxO1, that is an important mediator of insulin signalling in the hypothalamus and stimulates the transcription of the orexigenic neuropeptides. In this context, due to the capacity to bind directly to protein kinase B (Akt), Mammalian Homolog of Drosophila Tribbles 3 (TRB3) is emerging as an important player in the regulation of insulin signalling. In addition, endoplasmatic reticulum (ER) stress signaling (pPERK and CHOP pathway) has been considered the main mechanism responsible by TRB3 overexpression in obese and diabetic rodent. On the other hand, the exercise is considered one of the most important non-pharmacological strategies to prevent and/or treat obesity. The exercise is able to protect against inflammation and ER stress and to increase insulin signaling in the hypothalamus of obese rodents. However, the effects of acute exercise on TRB3 modulation were not described. Thus, the main aim of the present study was to investigate the role of an acute swimming exercise on the hypothalamic TRB3 expression and the TRB3/Akt association in obese rodents. Male Wistar rats were divided randomly into three groups: control (lean) - fed with a standard rodent chow -, DIO - fed with a high fat diet -, and DIO submitted to a swimming acute exercise protocol (DIO-EXE). In order to reinforce that the TRB3 increase is associated with ER stress and that an acute session of exercise is able to reverse this increase, we used other three groups: control (lean) receiving an intracerebroventricular (i.c.v.) infusion of vehicle, lean receiving an i.c.v. infusion of thapsigargin (an ER stress inducer), and lean receiving an i.c.v infusion of thapsigargin and performed a swimming acute exercise protocol. The physical exercise protocol consisted of a swimming acute session, composed of two periods of three-hours with a break of forty-five minutes. Previously the euthanasia of the animals were performed the analyses of the glycaemia and insulinemia serum, insulin sensitivity through of insulin tolerance test and food intake after i.c.v. insulin injection. Four hours after the last exercise session and anesthesia procedures, the hypothalamic tissue was removed for analysis of the interested proteins through of immunoblotting, immunoprecipitation and PCR real time techniques. In this study, we demonstrated that the exercise reduced the TRB3 protein levels and disrupted the interaction between TRB3 and Akt protein, increased the phosphorylation of Foxo1 and restored the anorexigenic effects of insulin in the hypothalamus of DIO rats. Additionally, our results revealed that intrahypothalamic infusion of thapsigargin blocked the anorexigenic effects mediated by insulin in lean rats and physical exercise restored the suppressive action of insulin on appetite, Thus, the suppressive effects of acute exercise on TRB3 protein levels may be related, at least in part, to the decrease of endoplasmatic reticulum stress in the hypothalamic cells / Mestrado / Metabolismo e Biologia Molecular / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

Systemic sclerosis (SSc) is immune-mediated fibrotic disease of unknown aetiology. Among the dominant pathogenic manifestations of SSc belong vascular changes, production of autoantibodies, activation of innate and adaptive immune responses and fibrotic processes. Transforming growth factor beta (TGF-β) has been identified as a central profibrotic factor stimulating fibroblasts to produce collagen. There are, however, a number of other mediators involved in the pathogenesis of SSc. Mutual activation and amplification of these molecules and their cascades may be a central mechanism of the SSc pathogenesis. Hedgehog (Hh) canonical signalling pathway plays an important role in the development and progression of fibrotic diseases. Expression of Hh target genes can be regulated through a canonical or non-canonical signalling cascade. The non-canonical activation of GLI transcription factors by TGF-β has not yet been investigated in SSc. The substantial part of this thesis is focused on the study of the mutual interaction of TGF-β and Hh signalling pathway. In vitro analysis confirmed TGF- β/SMAD3 dependent activation of GLI2 in dermal fibroblasts. Fibroblasts specific knockout of GLI2 prevented the development of experimental fibrosis in vivo. Combined targeting of canonical and non-canonical Hh...