The progesterone receptor membrane component 1 (PGRMC1) is a multifunctional protein with a heme-binding domain that promotes cellular signaling via receptor trafficking, and is essential for some elements of tumor growth and metastasis. PGRMC1 is upregulated in breast, colon, lung and thyroid tumors. We expanded the analysis of PGRMC1 in the clinical setting, and report the first analysis of PGRMC1 in human oral cavity and ovarian tumors and found PGRMC1 to correlate with lung and ovarian cancer patient survival. Furthermore, we discovered a specific role for PGRMC1 in cancer stem cell viability. PGRMC1 directly associates with the epidermal growth factor (EGFR) in cancer cells, and we reviewed multiple signaling-associated pathways that are important in trafficking wild-type and mutant EGFR. To better understand the potential of PGRMC1 in receptor tyrosine kinase trafficking, we extended our research to the insulin receptor (IR). Changes in insulin signaling have been linked to multiple diseases, because IR plays a key role in glucose metabolism, cellular survival and proliferation. We found PGRMC1 to co-precipitate with IR in cancer cells and in an adipose model system. PGRMC1 increased IR plasma membrane levels in multiple cancer cell lines, and was also found to increase plasma membrane levels of two glucose transporters. Treatment with a PGRMC1 ligand significantly increased IR levels in human adipocytes. Moreover, we demonstrate that both insulin binding and glucose uptake are dependent on PGRMC1.

Health inequalities take many forms related to race, gender, socioeconomic status, ethnic, language and many other axes throughout communities around the world. Type two diabetes, high blood pressure, and high cholesterol are examples of conditions (among many others) that disproportionately affect Latino@s in the U.S.. The research of this dissertation is based on fieldwork conducted throughout several predominantly Latin@ neighborhoods in Chicago, IL. This dissertation examines how Latin@s in Chicago navigate health and well-being, and how they engage in agentive strategies in the face of chronic disease. I recorded individual life histories and semi-structured interviews, focus groups, and participant observation at various community events and settings. The stories of these Chicago Latin@s are shared here in an effort to de-homogenize the depiction of Latin@s in the U.S. by paying attention to local narratives, and especially to those related to living with chronic disease.

Patients with liver disease have an increased risk for malnutrition because of side effects of the disease. The Nutrition Focused Physical Exam (NFPE) was developed for nutrition professionals to aid physicians in a nutrition-based diagnosis of malnutrition. The purpose of this study was to examine the NFPE for its validity in liver disease patients being evaluated for transplant. In addition, the NFPE was used to assess incidence and severity of malnutrition in end stage liver disease patients and compare these results to already developed malnutrition tools such as the Patient Generated-Subjective Global Assessment (PG-SGA), Triceps Skinfolds (TSF), Mid-Arm Circumference (MAC), Lumbar Index, and Total Psoas Muscle Area (TPA). The NFPE was found to be highly correlated with PG-SGA results. There was a weak correlation between the NFPE and the TSF, MAC, and Lumbar Index/TPA, except when comparing the bottom 25% quartile of the Lumbar Index to severe malnutrition using the NFPE. This resulted in a moderate correlation. The odds-ratio for hospital admission based on malnutrition and severe malnutrition were both extremely high (14.571, 18.857 respectively). These preliminary results reinforce the significance of the NFPE and the need for additional studies using this tool.

In horses, a painful and often debilitating disease known as laminitis can result in impaired function and, in severe cases, euthanasia. Equine Metabolic Syndrome (EMS) is a syndrome in horses that results in development of laminitis and is characterized by the presence of general and/or regional adiposity (“cresty neck”), aberrations in blood lipid concentrations, insulin resistance (IR) and/ or hyperinsulinemia. Therapies have focused on improving the state of obesity and insulin resistance with the goal of diminishing the likelihood of laminitis development. A definitive cause for laminitis has not been established, but hyperinsulinemia and IR are likely candidates as experimental states of hyperinsulinemia have been shown to induce laminitis and improvements in insulin sensitivity and obesity have been associated with a decreased risk of laminitis development. This dissertation discusses associations between obesity and IR, as well as potential therapies for alleviating insulin resistance with the ultimate goal of decreasing the risk of developing laminitis. Therapies evaluated included chromium and magnesium, levothyroxine sodium, and metformin hydrochloride. Horses were treated with each supplement for 10 to 36 weeks, depending on the supplement tested, and physical measurements such as body weight, neck circumference, and body condition score were obtained. Throughout each study, blood concentrations of glucose, insulin, and plasma lipids were analyzed. Chromium and magnesium currently do not appear to have any effect on insulin sensitivity, whereas results of levothyroxine administration indicate therapeutic responses, as does metformin, though results indicate further work are required. Research contained in this dissertation focuses on the potential of identifying animals at risk of developing IR and laminitis through measurement of blood biomarkers such as adiponectin and glucagon-like peptide 1. Assays to measure markers included enzyme-linked immunosorbent assays, western blots, and radioimmunoassays. Glucagon-like peptide 1 currently does not appear to differ between healthy and IR animals, but protein band density of high-molecular weight adiponectin does appear to be lower in horses with IR when compared to healthy animals. There is still much to learn about IR in horses, and therapy appears to be dependent on a case by case basis.

Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Previously, we demonstrated that mice made obese from chronic consumption of a high-fat (HF) diet have marked elevations in systemic concentrations of angiotensin II (AngII). Pancreatic islets have been reported to possess components of the renin-angiotensin system (RAS), including angiotensin type 1a receptors (AT1aR), the primary receptor for AngII, and angiotensin converting-enzyme 2 (ACE2), which negatively regulates the RAS by catabolizing AngII to angiotensin-(1-7) (Ang-(1-7)). These two opposing proteins have been implicated in the regulation of β-cell function. We hypothesized that the RAS contributes to the decline of β-cell function during the development of T2D with obesity. To test this hypothesis we first examined the effects of whole-body deficiency of ACE2 in mice on β-cell function in vivo and in vitro during the development of T2D. Whole-body deficiency of ACE2 resulted in impaired β-cell adaptation to insulin resistance with HF-feeding and a reduction of in vivo glucose-stimulated insulin secretion (GSIS) associated with reduced β- cell mass and proliferation. These results demonstrate that ACE2 plays a role in the adaptive response to hyperinsulinemia with obesity. In islets from HF-fed mice, AngII inhibited GSIS. In mice with pancreatic-specific deletion of AT1aR, AngII-induced inhibition of GSIS in vitro from islets of HF-fed mice was abolished. However, there was no effect of pancreatic AT1aR-deficiency on glucose homeostasis in vivo in HF-fed mice exhibiting pronounced hyperinsulinemia. Notably, pancreatic weight, insulin content and basal and glucose-stimulated insulin secretion from islets were decreased in mice with pancreatic AT1aR deficiency. These results suggest that AT1aR may contribute to pancreatic cell development, and also contribute to AngII-induced reductions in GSIS from islets of HF-fed mice. Overall, these studies suggest a role for the RAS in the regulation of β-cell function in T2D.

Angiotensinogen is the only known precursor in the renin-angiotensin system, a hormonal system best known as an essential regulator of blood pressure and fluid homeostasis. Angiotensinogen is sequentially cleaved by renin and angiotensin- converting enzyme to generate angiotensin II. As the major effector peptide, angiotensin II mainly function through angiotensin type 1 receptor. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and more recently renin inhibitors are widely known as the 3 classic renin-angiotensin system inhibitory drugs against hypertension and atherosclerosis. Here, we developed an array of regents to explore the effects of angiotensinogen inhibition. First, we demonstrated that genetic deficiency of angiotensinogen not only protected against hypercholesterolemia- induced atherosclerosis but also prevented diet-induced obesity. Then we found weekly intraperitoneal injection of antisense oligonucleotides against angiotensinogen remarkably surpressed body weight gain in mice fed a western diet, which was absent from classic renin-angiotensin system inhibition. The suppressed body weight gain was attributable to diminished body fat mass gain and enhanced energy expenditure. More excitingly, angiotensinogen antisense oligonucleotides regressed body weight gain on obese mice. Together, our findings revealed a unique feature of angiotensinogen inhibition beyond classic renin angiotensin inhibition and demonstrated therapeutic potentials of angiotensinogen antisense oligonucleotides against hypertension, atherosclerosis, and obesity. We also developed an in vivo system to explore the functional consequences of disrupting a conserved Cys18-Cys137 disulfide bridge in angiotensinogen. The formation of this disulfide bridge could trigger conformational changes in angiotensinogen, thereby facilitating renin cleavage of angiotensinogen. It was predicted that the redox-sensitive disulfide bridge might change the efficiency of angiotensinogen/renin reaction to release angiotensin II, thus modulate angiotensin II-dependent functions. We determined effects of the presence and absence of the disulfide bridge on angiotensin II concentrations and responses in mice expressing either native angiotensinogen or Cys18Ser, Cys137Ser mutated angiotensinogen in liver via adeno-associated viral vectors. Contrary to the prediction, disruption of Cys18-Cys137 disulfide bridge in angiotensinogen had no discernible effects on angiotensin II production and angiotensin II-dependent functions in mice.

Obesity is associated with insulin resistance, dyslipidemia, and cardiovascular disease. The current obesity epidemic is the result of surplus energy consumption. Excess energy is stored in expanding adipose tissue. Adipose tissue growth entails the enlargement of existing adipocytes, the formation of new fat cells from preexisting progenitors, and the coordinated development of supporting vasculature. Identifying adipocyte progenitors and the mechanism of adipose tissue expansion is crucial for the development of new strategies to combat obesity and its complications. Though important progress has been made towards understanding the developmental origin of adipocytes, the identities of adipocyte progenitors are still not completely known. The main objective of this study is to determine whether endothelial cells of the adipose tissue can give rise to new adipocytes. Our results indicate that murine endothelial cells of adipose tissue are pluripotent and can potentially give rise to preadipocytes. Lineage tracing experiments using the VE-Cadherin-Cre transgenic mouse reveal localization of reporter genes in endothelial cells, preadipocytes and white and brown adipocytes. Moreover, capillary sprouts from human adipose tissue, which have predominantly endothelial cell characteristics, are found to express Zfp423, a preadipocyte determination factor. In response to PPARγ activation, endothelial characteristics of sprouting cells are progressively lost, and cells form structurally and biochemically defined adipocytes. Taken together, our data support an endothelial origin of a population of adipocytes. The ability of the vascular endothelium to give rise to adipocytes may explain how angiogenesis and adipogenesis can be temporally and spatially coordinated. Analysis of BAT and WAT revealed that adipose depots have distinct compositions of adipocyte progenitors. Of the CD45-CD29+Sca1+CD24+ progenitor population, only 17% and 52% express VE-Cadherin in WAT and BAT, respectively. Our data show that the number of these specific progenitors in BAT and WAT are highly variable and suggest that a considerable number of adipocytes progenitors may have a non-endothelial cell origin. Differences in composition and types of adipocyte progenitors may explain the differences in the adipocytes phenotypes that we observe in discrete depots. In brief, we find that the vascular endothelium gives rise to a population of brown and white fat cells, and that the number of endothelial-derived adipocyte progenitors residing in BAT and WAT is highly variable. These results expand our current understanding of adipose tissue growth, and, we hope, will accelerate the development of treatments for obesity-related complications.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-01T13:23:23Z No. of bitstreams: 1 arquivo total.pdf: 1394266 bytes, checksum: a9d347ea65453f0d1739cd46695f619e (MD5) / Made available in DSpace on 2017-02-01T13:23:23Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 1394266 bytes, checksum: a9d347ea65453f0d1739cd46695f619e (MD5) Previous issue date: 2016-02-17 / INTRODUCTION: Adolescence comprises the age 10-19 years. This phase is marked biological and physiological changes, including highlighting the bone mineral growth, which is dependent on the nutritional status of calcium and vitamin D. In recent years, studies of adolescents showed high rates of failure / vitamin D deficiency in several countries. Vitamin D has an important role in bone growth and recent evidence has associated the vitamin D status with diabetes, hypertension, cancer and increased biomarkers of oxidative stress and inflammation in adults and the elderly. In adolescents, studies have shown an association between low serum concentrations of vitamin D and the increase in cardiometabolic risk factors. OBJECTIVE: The aim of this study was to evaluate the insufficiency/deficiency of vitamin D and its association with cardio metabolic risk factors in adolescents. METHODS: A Cross-sectional study including 209 adolescents aged 15 to 19 years old, recruited from public schools in the city of João Pessoa, Brazil. Personal data and sun exposure were collected through questionnaires. The dietary intake was assessed by applying a 24-hours recall. Nutritional status was assessed by body mass index (BMI), waist circumference (WC) and the waist-to-height ratio (WHtR) according to the cut-offs for age criteria. Blood pressure was measured according to the protocol of the Brazilian Society of Hypertension. It was collected blood samples to measure levels of 25-hidrovitmina D, Parathyroid hormone, glucose and lipid profile, C-reactive-protein (CPR), alpha-1-acid-glycoprotein(AGP-A), malondialdehyde (MDA), and total antioxidant capacity (TAC). The research was approved by the Research Ethics Committee of the CCS / UFPB under the Protocol 0139/15. Statistical analysis was performed using the "Statistical Pac age for Social Sciences" (SPSS Inc., Chicago, USA), version 21, adopting significance of p <0.05. RESULTS: The insufficiency/deficiency (≤ 30 ng/mL) of vitamin D was observed in 57, 4 % (CI 95% -50,6-64) of the adolescents, and in 76 % (CI 95%-62,8-86,3) of those were overweight. Dietary intake of vitamin D was inadequate ( 60 ± 18 μg) in 100% of the sample. Low levels of 25 (OH) D were associated with female gender (RP= 2, 3; CI 95% = 1, 8-3, 5; p=0), central adiposity measures by the WC (p = 0.02) and WHR (0.01), lower TAC (p = 0.01) and serum calcium (p = 0.00). The adolescents showed an inverse correlation between the total cholesterol (p = 0.01; r = -0.167) and BMI (p = 0.02; r = -0.166) with the levels of 25 (OH) D. The CPR values, the AGP-A, and MDA were increased in those that were both vitamin D deficient/insufficient and overweight (p = 0.00). CONCLUSION: The insufficiency/deficiency of vitamin D was associated with BMI, WC and WHtR, lower TAC and higher total cholesterol levels, suggesting that inadequate levels of vitamin D promotes the increased risk in the development of diseases associated with overweight, process inflammatory and oxidative stress. / INTRODUÇÃO: A adolescência compreende a idade de 10 a 19 anos. Esta fase é marcada transformações biológicas e fisiológicas, entre elas destacando-se o crescimento mineral ósseo, o qual é dependente do estado nutricional de cálcio e vitamina D. Nos últimos anos, estudos realizados com adolescentes demonstram altas prevalências de insuficiência/deficiência de vitamina D em diversos países. A vitamina D possui papel essencial no crescimento ósseo e recentemente evidências tem associado o status de vitamina D com diabetes, hipertensão, câncer e aumento dos biomarcadores de estresse oxidativo e processo inflamatório na população adulta e idosa. Em adolescentes, estudos já demonstram uma associação entre as baixas concentrações séricas de vitamina D e o aumento dos fatores de riscos cardiometabólicos. OBJETIVO: Avaliar a insuficiência/deficiência da vitamina D e sua associação com fatores de riscos cardiometabólico em adolescentes. METODOLOGIA: Foi realizado estudo transversal com 209 adolescentes de idade entre 15 e 19 anos recrutados em escolas públicas na cidade de João Pessoa-PB, Brasil. Os dados pessoais e de exposição solar foram coletados por meio de questionários. A ingestão dietética foi avaliada por meio da aplicação do recordatórios 24 horas. O estado nutricional foi avaliado pelo índice de massa corporal (IMC), circunferência da cintura (CC) e razão cintura/altura (RCA) seguindo os pontos de corte para faixa etária estabelecida. A pressão arterial foi medida segundo o protocolo da Sociedade Brasileira de Hipertensão. Foram coletadas amostras de sangue a fim de analisar as concentrações séricas de 25-hidroxivitamina D (25(OH)D), Paratormônio (PTH), perfil glicolipídico, Proteína C Reativa (PCR), Alfa 1 Glicoproteína Ácida (AGP-A), Malondialdeído (MDA) e capacidade antioxidante total (CAT). A Pesquisa foi aprovada pelo Comitê de Ética em Pesquisa do CCS/UFPB sob o protocolo nº 0139/15. A Análise estatística foi realizada através do “Statistical Pac age for the Social Sciences” (SPSS Inc., Chicago, USA), versão 21, adotando-se significância de p<0,05. RESULTADOS: A insuficiência/deficiência de vitamina D (≤ 30 ng/mL) foi observada em 57,4 % (IC 95% -50,6-64) dos adolescentes, e em 76 % (IC 95%-62,8-86,3) dos classificados com excesso de peso. O consumo dietético de vitamina D foi inadequado (60 UI ± 18) em 100 % da amostra. Baixos níveis de 25(OH)D estiveram associados ao sexo feminino (RP= 2,3; IC 95 % = 1,8-3,5; p= 0,00), adiposidade central pelas medidas de CC (p=0,02) e RCA (0,01), menor nível de CAT (p= 0,01) e Cálcio sérico (p= 0,00). Os adolescentes apresentaram uma correlação inversa entre Colesterol total (p=0,01; r= -0,167) e IMC (p=0,02; r=-0,166) com os níveis de 25 (OH)D. Os valores de PCR, AGP-A, MDA estiveram aumentados no grupo com hipovitaminose D classificados com excesso de peso (p=0,00). CONCLUSÃO: A insuficiência/deficiência de vitamina D esteve associada ao IMC, CC e RCA, menor CAT e maiores níveis de colesterol total, sugerindo que níveis inadequados de vitamina D promove o aumento do risco no desenvolvimento de doenças associadas ao excesso de peso, processo inflamatório e estresse oxidativo.

Orientador: Egberto Ribeiro Turato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T09:19:39Z (GMT). No. of bitstreams: 1 Vieira_CarlaMaria_D.pdf: 1345949 bytes, checksum: af8dbcb1223eda58565ee2366f98fba3 (MD5) Previous issue date: 2010 / Resumo: O comportamento alimentar é um tema relevante no tratamento da obesidade, dislipidemias, hipertensão, diabetes tipo II e outros agravos metabólicos crônicos. A associação de pelo menos três destes distúrbios tem sido reconhecida na literatura médica como Síndrome Metabólica, considerada um problema de saúde emergente. O objetivo desse estudo foi conhecer significados psicológicos e culturais do comportamento alimentar vinculados ao processo de adoecimento crônico por Síndrome Metabólica, de pessoas com excesso de peso, em situação de cuidado. Método Clínico Qualitativo foi aplicado em ambulatório de hospital universitário, na região sudeste do Brasil. Entrevistas individuais semidirigidas, conduzidas em profundidade com nove sujeitos portadores de Síndrome Metabólica, compuseram a amostra intencional fechada por critério de saturação. O corpus do trabalho composto pelas expressões verbais e não verbais foi analisado a partir do exercício de repetidas leituras flutuantes. Referencial psicodinâmico de base analítica complementado com contribuições da antropologia da alimentação apoiou a interpretação dos resultados organizados em oito categorias: 1- O corpo gordo estigmatizado: corpo sem saúde e sem beleza; 2- O fetiche da cirurgia para curar a obesidade; 3- O desejo contido: comer muito pode ser pouco para aplacar a fome de ordem emocional; 4- A conquista da autonomia: ressignificar a alimentação para a convivência com os sintomas da Síndrome Metabólica; 5- O estado depressivo e paralisante dos sujeitos com dificuldades para estabelecer o autocuidado; 6- Significados da dieta: a perda do prazer e da liberdade de comer; 7- Conquistas no manejo do tratamento a partir do enfrentamento de situações de vulnerabilidade; 8- Autonomia versus prescrição. Da discussão salientamos a rejeição à obesidade revelada pelos sujeitos que proporciona um desvio do foco no manejo do autocuidado, permeado pela cultura lipofóbica de enaltecimento da magreza e juventude. O desejo dos sujeitos por realizar uma cirurgia que seja capaz de curar o excesso de peso vincula-se ao significado da gordura corporal como doença. São reflexos culturais da medicalização da obesidade que dificultam os sujeitos a aceitarem e assumirem o controle sobre o seu próprio corpo. A dissociação das práticas alimentares como determinantes do ganho excessivo de peso, assim como a utilização dosalimentos para preencher lacunas de ordem psíquica, revelaram fragilidade em lidar com conflitos emocionais e dificuldades para assumir a responsabilidade sobre o processo de cuidado. Os significados de perda de prazer e cerceamento da liberdade de comer atribuídos à dieta, igualmente colaboram negativamente para o manejo do processo de adoecimento. No entanto, o saber culinário, a valorização da identidade da cultura alimentar e a comensalidade em família demonstraram potencialidades na reorganização da alimentação no cotidiano. A baixa adesão às prescrições dietéticas encontra-se associada a um padrão ideal de comportamento alimentar e distingue-se da postura de respeito e apoio para o estabelecimento de autonomia na elaboração de recursos pessoais para enfrentamento do processo de adoecimento crônico ao longo da vida. O caráter interdisciplinar deste trabalho proporcionou a valorização do exercício de escuta dos significados psicológicos e culturais do comportamento alimentar com contribuições no âmbito do cuidado nutricional de portadores de Síndrome Metabólica / Abstract: Eating behaviors are relevant issues in the treatment for obesity, dyslipidemias, hypertension, type II diabetes and other chronic metabolism disorders. Literature has established that an association of three or more of those disorders is referred to as Metabolic Syndrome, which is considered a growing health issue. The objective of the present study was to identify the psychological and cultural meanings of eating behavior linked to the process of developing chronic disease due to Metabolic Syndrome, in people receiving care. The Qualitative Clinical Method was applied at the outpatient clinic of a university hospital in Southeast Brazil. Semi-directed individual interviews were conducted in depth with nine Metabolic Syndrome patients, who comprised the purposive sample determined by saturation. The verbal and non-verbal statements composed the corpus of the study, which was analyzed based on repeated in-depth readings. Results were interpreted based on a psychodynamic framework with an analytical grounding, complemented with contributions from the anthropology of eating, thus permitting to organize the results into the following eight categories: 1- The stigmatized fat body: a body without health and without beauty; 2- The fetish of surgery to cure obesity; 3- The restrained desire: eating much could be little to quench their emotional hunger; 4-The conquest of autonomy: reassign meanings to feeding to manage the symptoms of Metabolic Syndrome; 5- The depressive and paralyzing state of subjects with difficulties to establish self-care; 6- Meanings of the diet: the loss of pleasure and the liberty of eating; 7- Achievements in handling the treatment based on dealing with vulnerability situations; 8-Autonomy versus prescription. A rejection towards obesity was revealed by the subjects, which shifts their focus from managing self-care, as it is permeated by a lipophobic culture that exalts thinness and youth. The subjects' desire to undergo a surgery that would cure their excessive weight is associated to their associating a meaning of disease to body fat. The cultural thoughts of medicalizing obesity account for the difficulty that subjects have to accept and take control over their own body. The dissociation of eating habits as determinants of excessive weight gain and using food to fill in emotional gaps revealed fragility of dealing with emotional conflicts, and difficulties of taking responsibility over the care process. The meanings assigned to loosing pleasure and the loss of liberty to eat, both associated with dieting, have a negative effect on the process of managing the disease. On the other hand, culinary knowledge, valuing the identity of eating culture and commensality in the family demonstrate the potential of reorganizing everyday eating. Poor adherence to diet prescriptions is associated to an ideal eating behavior standard and stands apart from a position of respect and support towards establishing autonomy in developing personal resources to deal with the process of chronic illness through life. The interdisciplinary character of the present study promoted the valorization of the practice of listening to the psychological and cultural meanings of eating behaviors, hence offering contributions regarding the nutritional care of individuals with Metabolic Syndrome / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Orientador: Lício Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T06:28:45Z (GMT). No. of bitstreams: 1 Santos_AndressaCoopedos_D.pdf: 9572929 bytes, checksum: ce4c4fb2095c6ffe99868ec77f19d5b3 (MD5) Previous issue date: 2011 / Resumo: A ativação da sinalização através do toll-like receptor-4 (TLR4) e a indução de estresse de retículo endoplasmático (ERE) são importantes mediadores da resistência à insulina na obesidade e em outras situações nas quais há um excesso de ácidos graxos saturados. Em um estudo recente, demonstrou-se que sinalização através de TLR4 pode, per se, induzir a ativação de ERE, sugerindo que a ativação do TLR4 é o evento inicial para a indução do estresse celular que contribui para o aumento da expressão de genes de resposta inflamatória. No entanto, os mecanismos que conectam essas duas vias distintas são desconhecidos. As chaperonas GRP78 e GRP94 exercem uma função importante no processamento das moléculas recém-traduzidas do TLR4. Além disso, a chaperona GRP94 é responsável por sua translocação e conteúdo na superfície celular. Durante uma ativação prolongada da via de sinalização do TLR4, a demanda por novas moléculas sintetizadas aumenta, e consequentemente, a demanda por novas chaperonas. Por esta razão, nós aventamos a hipótese de que sob uma ativação extrema da via do TLR4, a síntese de proteínas sobrepujaria a expressão de chaperonas, dessa forma induzindo ERE. Para testar essa hipótese, monócitos da linhagem THP-1 foram incubados com LPS e foi avaliada a expressão e ativação de proteínas responsivas ao ERE por real-time PCR, citometria de fluxo, imunoprecipitado e western blot. Em alguns experimentos, as células foram privadas de glicose ou tratadas com siRNA para aumentar ou diminuir, respectivamente, a expressão das chaperonas. Experimentos de time-course revelaram que o LPS induz um aumento de 2,5 vezes na expressão do TLR4, iniciando após 8 h, com um pico após as 24 h e permanecendo significantemente aumentado após 48 h. A expressão de GRP78 foi aumentada em três vezes com um aumento acentuado após 24 h sem aumento às 8 h, enquanto o GRP94 aumenta apenas 1,5 vezes com um pico após 2 h que retorna aos valores basais após 8 h do estímulo. Não houve aumento da expressão protéica das chaperonas após 48 h. A indução de ERE por LPS foi detectada antes de 4 h do estímulo observado pela avaliação da via da PERK/eIF2a, IRE1 e ATF6 e se mantém ativado após 48 h. Adicionalmente, a privação de glicose em células THP-1 aumenta a expressão de GRP94 e GRP78 em 2,5 e 11 vezes, respectivamente. Na ausência de glicose, o tratamento com LPS não induz ERE. A inibição da expressão das chaperonas por siRNA anula o efeito da privação de glicose em proteger as células do desenvolvimento de ERE induzido por LPS. Portanto, a hiperexpressão das chaperonas GRP78 e GRP94 protegem as células do ERE induzido por LPS. Assim, defeito na expressão das chaperonas induzido por TLR4 é um mecanismo envolvido na integração da sinalização do TLR4 e ERE / Abstract: TLR4 activation and the induction of endoplasmic reticulum stress (ERS) are two of the most important mechanisms connecting excessive fat with insulin resistance. Recently, it was shown that activation of TLR4 can, per se, induce ERS, suggesting that TLR4 is a primary event in the induction of the cellular stress that contributes to increased inflammatory gene expression. However, the mechanisms linking these molecular events are unknown. The chaperones GRP78 and GRP94 play an important role during the assembly of newly translated TLR4 molecules. In addition, the chaperone GRP94 escorts the protein to the cell membrane. Under prolonged activation, the demand for newly synthesized TLR4 molecules increases, and thus, the demand for new chaperones. Therefore, we hypothesized that under increased activation of TLR4, the synthesis of the protein would not be matched by the expression of chaperones, thus, triggering ERS. To test this hypothesis, the monocyte cell line THP-1 was incubated with LPS and the expression/activation of proteins involved in ERS was determined by real-time PCR, flow-cytometry, immunoprecipitation and immunoblot. In some experiments, cells were deprived of glucose or treated with siRNA to increase or decrease, respectively, the expression of the chaperones. Time-course experiments revealed that LPS led to a 2.5-fold increase of TLR4 expression starting as early as 8h, peaking after 24h and remaining significantly increased after 48h. The expression of GRP78 underwent a 3-fold increase with a sharp rise at 24h (no increase at 8h), while GRP94 increased by only 1.5-fold with a peak at 2h and an early return to base-line levels. None of the chaperones were increased after 48h. LPS-induced ERS was detected as early as 4h after stimulus as detected by the evaluation of PERK/eIF2?, IRE1 and ATF6 pathways. Strong signals of ERS were still present after 48h. The pre-incubation of THP-1 in glucose-deprived medium produced 2.5 and 11-fold increases of GRP94 and GRP78, respectively. Upon glucosedeprivation, LPS could no longer induce ERS. Inhibition of chaperone expression by siRNA completely abrogated the effect of glucose deprivation to protect cells from LPS-induced ERS. Thus, the hyperexpression of GRP78 and GRP94 protect cells from LPS-induced ERS. Defective TLR4-induced chaperone expression is a mechanism involved in the integration of TLR4 signaling and ERS / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica