Tesi sul tema "IRS-1"

Orientador: Jose Barreto Campello Carvalheira
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A regulação adequada da via de sinalização PI 3-quinase-Akt é essencial para a prevenção da carcinogênese. Dados recentes caracterizaram uma alça de retroalimentação negativa na qual a mammalian target of rapamycin (mTOR), bloqueia a ativação adicional da via Akt/mTOR por meio da inibição da função do substrato 1 do receptor de insulina (IRS-1). Entretanto, a inibição potencial do IRS-1 durante o tratamento com rapamicina não foi estudado. No presente estudo, demonstramos que um oligonucleotídeo anti-sense direcionado ao IRS-1 e a rapamicina antagonizam sinergicamente a ativação da mTOR in vivo e induzem supressão tumoral, por meio de inibição da proliferação e indução de apoptose, em enxertos de células de câncer de próstata. Estes dados demonstram que a inclusão de agentes que bloqueiam o IRS-1 potencializam o efeito da inibição da mTOR no crescimento de enxertos de células de câncer de próstata.
Abstract: Proper activation of phosphoinositide 3-kinase-Akt pathway is critical for the prevention of tumorigenesis. Recent data have characterized a negative feedback loop where in mammalian target of rapamycin (mTOR), blocks additional activation of the Akt/mTOR pathway through inhibition insulin receptor substrate 1 (IRS-1) function. However, the potential of IRS-1 inhibition during rapamycin treatment has not been examined. Herein, we show that IRS-1 antisense oligonucleotide and rapamycin synergistically antagonize the activation of mTOR in vivo and induced tumor suppression, through inhibition of proliferation and induction of apoptosis, in prostate cancer cell xenografts. These data demonstrate that the addition of agents that blocks IRS-1 potentiate the effect of mTOR inhibition in the growth of prostate cancer cell xenografts.
Doutorado
Clinica Medica
Doutor em Clínica Médica

The Insulin Receptor Substrate (IRS) proteins IRS-1 and IRS-2 are cytoplasmic adaptor proteins that organize and propagate intracellular signaling downstream of specific growth factor receptors, including the Insulin and Insulin-Like Growth Factor-1 Receptors (IR and IGF-1R, respectively). Despite sharing a high level of homology and the ability to stimulate Phosphotidylinositol-3-Kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signaling, IRS-1 and IRS-2 play distinct roles in mammary tumor progression. Specifically, IRS-1 promotes growth and proliferation, whereas IRS- 2 promotes motility, invasion, survival, aerobic glycolyis, and metastasis. To further understand the differences between IRS-1 and IRS-2, I investigated the mechanistic basis of IRS-2-mediated PI3K activation. I identified tyrosines in IRS-2 that mediate its recruitment and activation of PI3K in response to insulin and IGF-1 stimulation. Using a PI3K-binding deficient IRS-2 mutant, I demonstrated that IRS-2-dependent PI3K signaling promotes aerobic glycolysis through its ability to selectively regulate the phosphorylation of the Akt effector Glycogen Synthase Kinase-3β (Gsk-3β). I also performed a rigorous comparison of IRS-1 and IRS-2 signal transduction and their ability to regulate functions associated with tumor progression. These studies required the generation of a novel model system where IRS-1 and IRS-2 function could be compared in a genetically identical background. Using this model, I confirmed a role for IRS-1 in growth regulation and IRS-2 in tumor cell invasion, as well as expanded the understanding of differential IRS protein function by showing that IRS-2 more vi effectively promotes Akt activation. The model system I have established can be used for further characterization of IRS-1 and IRS-2-specific functions.

The Insulin Receptor Substrate (IRS) proteins IRS-1 and IRS-2 are cytoplasmic adaptor proteins that organize and propagate intracellular signaling downstream of specific growth factor receptors, including the Insulin and Insulin-Like Growth Factor-1 Receptors (IR and IGF-1R, respectively). Despite sharing a high level of homology and the ability to stimulate Phosphotidylinositol-3-Kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signaling, IRS-1 and IRS-2 play distinct roles in mammary tumor progression. Specifically, IRS-1 promotes growth and proliferation, whereas IRS- 2 promotes motility, invasion, survival, aerobic glycolyis, and metastasis. To further understand the differences between IRS-1 and IRS-2, I investigated the mechanistic basis of IRS-2-mediated PI3K activation. I identified tyrosines in IRS-2 that mediate its recruitment and activation of PI3K in response to insulin and IGF-1 stimulation. Using a PI3K-binding deficient IRS-2 mutant, I demonstrated that IRS-2-dependent PI3K signaling promotes aerobic glycolysis through its ability to selectively regulate the phosphorylation of the Akt effector Glycogen Synthase Kinase-3β (Gsk-3β). I also performed a rigorous comparison of IRS-1 and IRS-2 signal transduction and their ability to regulate functions associated with tumor progression. These studies required the generation of a novel model system where IRS-1 and IRS-2 function could be compared in a genetically identical background. Using this model, I confirmed a role for IRS-1 in growth regulation and IRS-2 in tumor cell invasion, as well as expanded the understanding of differential IRS protein function by showing that IRS-2 more vi effectively promotes Akt activation. The model system I have established can be used for further characterization of IRS-1 and IRS-2-specific functions.

Orientador: Mario Jose Abdalla Saad
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Apesar dos mecanismos da longevidade não serem totalmente conhecidos, sabe-se que mutações em genes que reduzem a sinalização intracelular de insulina/IGF-1 são capazes de aumentar a sobrevida de animais invertebrados e mamíferos Na mosca Drosophila Melanogaster, mutações no receptor de insulina (similar ao gene do receptor de insulina humano) e no gene chico (similar ao gene do IRS-1 humano) levam ao aumento da sobrevida do animal. Em roedores, estudos sobre restrição calórica e diversas mutações tem relacionado a via de sinalização insulina/IGF-1 com longevidade. Em humanos, a sensibilidade à insulina normalmente declina com o envelhecimento, porém existem evidências que idosos longevos apresentam ação da insulina preservada e nível de IGF-1 sérico reduzido, o que sugere que a resposta à insulina tem impacto na longevidade humana. Evidências recentes que reforçam esta teoria demonstram que polimorfismos de genes da via de sinalização de insulina/IGF-1( IGF-1R e PI3K), afetam o IGF-1 sérico e a longevidade humana, sugerindo que este mecanismo esteja conservado através da evolução das espécies. Baseada nestas evidências, nosso estudo investigou a prevalência do polimorfismo mais comum do IRS-1, a substituição de glicina por arginina no códon 972 (Gly972Arg), numa amostra da população longeva brasileira, e a sua associação com diversos fenótipos e com a longevidade. Foram incluídos no estudo 94 idosos, de ambos os sexos, com idades entre 80 e 100 anos. Como grupo controle foram incluídos 194 recém-nascidos da mesma região brasileira. A freqüência genotípica encontrada nos idosos foi: Gly/GIy 80,85%, Gly/Arg 19,15%, e Arg/Arg 0%. Não foi encontrada diferença estatística entre a prevalência do polimorfismo em longevos e em neonatos brasileiros. Também não houve associação deste polimorfismo com resistência à insulina, hipertensão, diabetes, ou com alterações no IMC, circunferência da cintura, e lípides nessa população. Nossos achados indicam que não há associação do polimorfismo Gly972Arg do IRS-1 com longevidade em humanos. E possível que esta falta de relação esteja relacionada ao seu efeito neutro tanto na sensibilidade à insulina quanto no nível de IGF-1.
Abstract: Although the underlying mechanisms of longevity are not fully understood, it is known that mutations in genes that decrease the insulin/IGF-1 signal response pathway are capable of extending life span in a variety of model systems from invertebrates to mammals. In Drosophila Melanogaster, mutations of either the insulin receptor gene (similar of human insulin receptor gene) or the Chico (similar of human IRS-1 gene) led to enhanced longevity. In humans, insulin sensitivity normally declines during aging. Although there are evidences that long-lived subjects have decreased plasma IGF-1 levels and preserved insulin action, thus indicating that insulin responsiveness impacts on human longevity. Recent findings also provide novel and intriguing indications for the involvement of insulin and IGF-1 in the control of aging and longevity in humans. In particular, it has been demonstrated that polymorphic variants of IGF-1 receptor (IGF-IR) and phosphatidylinositol 3-kinase (PI3K) genes affect IGF-1 plasma levels and human longevity, indicating that the impact of these genes on species longevity is an evolutionary conserved property. On the basis of the above-mentioned literature, we investigate the prevalence of a most common IRS-1 variant, a Gly --> Arg substitution at codon 972 (GIy972Arg) in a population-based sample of Brazilian elderly, its association with phenotypes and longevity. Ninety-four Brazilian subjects (men and women), from 80-100 yr. of age, volunteered for this study. As a control population we studied 194 newborn children from our University Hospital. Genotype frequencies were Gly/Gly 80,85%, Gly/Arg 19,15%, and Arg/Arg 0%, which are in Hardy-Weinberg equilibrium In addition, no significant differences among allele and genotype distribution were found between Brazilian elderly and Brazilian newborns Also, the Arg variant was not associated with insulin resistance, hypertension, diabetes, detrimental values for body mass index, waist circumference, and lipids in the Brazilian elderly. Our findings indicate that the IRS-1 Gly972Arg variant is not related with longevity. It is possible that the lack of this association might be related to its neutral effect on insulin sensitivity and on IGF-1 levels.
Mestrado
Clinica Medica
Mestre em Clinica Medica

Orientador: Mario Jose Abdalla Saad
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A insulina, ao se ligar à subunidade a de seu receptor heterotetramérico, dá início a uma série de ações imediatas e tardias, metabólicas e promotoras de crescimento. Tais eventos ocorrem através da estimulação da subunidade 13 transmembrana do receptor, que se autofosforila e ativa a fosforilação de substratos endógenos intracelulares, dos quais o mais estudado é o IRS-l. Esta proteína de peso molecular _160 kDa pode ser bem caracterizada como um substrato direto do receptor de insulina e quando se fosforila associa-se a proteínas com porção SH2, PI-3 quinase e SHP2 ativando-as. Uma etapa distal a estas associações/ativações é a fosforilação em serina da AKT/PKB. Utilizando-se técnicas de "immunoblotting" com anticorpos anti-IRS-l, antifosfotirosina, anti-SHP2 e antiAKT/PKB é possível analisar o grau de fosforilação do IRS-l e AKT/PKB, a concentração protéica da SHP2 e a associação do IRS-l com SHP2, ou seja, as ações iniciais da insulina. Neste estudo investigamos a quantidade protéica da SHP2, o grau de fosforilação do IRS-l e associação com SHP2 e a fosforilação do AKT /PKB no tecido muscular e hepático de ratos normais e em cinco modelos de resistência à insulina: o jejum prolongado, o envelhecimento, o uso agudo de adrenalina, o uso crônico de dexametasona e ratos com diabetes induzido por STZ. Em experimentos com ratos normais para avaliação do efeito tempo após infusão de insulina no grau de fosforilação do IRS-l e associação com SHP2, verificou-se que o pico de fosforilação do IRS-l e associação IRS-l/SHP2 foi aos 30" para o tecido hepático e aos 90" para o tecido muscular, após a infusão de insulina, na veia porta. Nos animais que receberam estreptozotocina, em tecido hepático não houve alteração no nível protéico da SHP2. Observamos um aumento significativo no grau de fosforilação do IRS-l para 141 :!: 12%, p < 0,05, quando comparados aos animais controle. Não houve alteração na associação IRS-l/SHP2 quando comparados com o controle. Houve uma redução para 65 :!: 6%, p< 0,035, no grau de fosforilação do AKT/PKB. Em tecido muscular destes animais tratados com estreptozotocina, não houve alteração no nível protéico da SHP2, ocorrendo um aumento para 191 :!: 14%, p< 0,036 no grau de fosforilação do IRS-l quando comparados com o controle, sem alteração na associação IRS-l/SHP2 quando comparados com o controle. O grau de fosforilação do AKT IPKB foi reduzido para 70 :t 7%, p< 0,04, nos animais tratados com STZ quando comparados com o controle. Animais submetidos ao tratamento agudo de adrenalina não apresentaram alteração no nível protéico da SHP2 tanto para o tecido hepático como para o muscular. Houve uma redução significativa no grau de fosforilação do IRS-l para 37 :t 3%, (p< 0,019), e para 37 :t 7%, (p< 0,003) em tecido hepático e muscular respectivamente, acompanhado de uma diminuição da associação IRS/SHP2 para 52 :t 3%, (p< 0,002), e para 20 :t 7%, (p< 0,041), em tecido hepático e muscular, respectivamente. A fosforilação do AKTIPKB foi reduzida para 60 :t 5%, (p< 0,045), e para 70 :t 6%, (p< 0,030), em tecido hepático e muscular, respectivamente O envelhecimento não alterou o nível protéico da SHP2 em tecido hepático. A fosforilação do IRS-l foi reduzida para 64 :t 7%, (p< 0,029), ocorrendo um aumento na associação IRS-lISHP2 para 155 :t 9%, (p< 0,012) em tecido hepático dos animais senis. O grau de fosforilação do AKTIPKB dos animais senis não apresentou alteração. Em tecido muscular dos animais com 20 meses de idade observamos que não houve alteração do nível protéico da SHP2. O grau de fosforilacão do IRS-l foi reduzido para 50 :t 7%, (p<0,007), acompanhado de uma redução na associação do IRS-lISHP2 para 48 :t 12%, (p< 0,034), como também uma redução no grau de fosforilação do AKTIPKB para 70 :t 6%, (p< 0,05), em tecido muscular do animais senis quando comparados com o controle. Os animais mantidos em jejum prolongado não apresentaram alteração no nível protéico da SHP2 tanto para tecido hepático como muscular. Houve um aumento no grau de fosforilação do IRS-l para 152 :t 13%, (p< 0,007), e para 155 :t 2%, (p< 0,004), em tecido hepático e muscular, respectivamente, em relação aos animais alimentados. Observamos um aumento na associação do IRS-lISHP2 para 136 :t 7%, (p< 0,035), e para 162 :t 7%, (p< 0,019), em tecido hepático e muscular, respectivamente, quando comparados com os animais alimentados. Os animais submetidos ao tratamento crônico com dexametasona tanto para o tecido hepático como para o muscular não apresentaram alteração no nível protéico da SHP2. Houve uma redução significativa no grau de fosforilação do IRS-l para 48 :t 5%, (p< 0,040), e para 36 :t 5%, (p< 0,035), em tecido hepático e muscular, respectivamente, quando comparados com os controles. Não houve alteração na associação IRS-lISHP2 em tecido hepático e muscular do animais que receberam tratamento crônico com dexametasona quando comparados com o controle. A análise in_egrada dos 5 modelos animais sugere que a regulação do grau de fosforilação do IRS-l, bem como da interação deste com a PI 3-quinase depende dos níveis insulinêmicos do animal: animais hiperinsulinêmicos apresentam menor fosforilação e menor interação IRS-l/PI 3-quinase, e animais hipoinsulinêmicos mostram o oposto. Por outro lado, a regulação da interação IRS-lISHP2 não parece manter nenhuma relação com níveis insulinêmicos. Parece que a interação IRS-lISHP2 contribui para a modulação da transmissão do sinal insulínico, influenciando a fosforilação/ativação da AKT/PKB. Em situações com aumento da fosforilação do IRS-I sem aumento na associação IRS-l/SHP2, o efeito final do AKT/PKB é atenuado, como demonstrado em figado e músculo de animais tratados com STZ. Por outro lado, a diminuição da fosforilação do IRS1, sem uma diminuição da associação IRS-lISHP2, protege a fosforilação do AKT /PKB, como demonstrado em figado dos animais senis
Abstract: Insulin stimulates the tyrosine kinase activity of insulin receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-l). After activation on by insulin, IRS-l associates with several proteins, including phosphatidylinositol (PI) 3-Kinase, phosphotyrosine phosphatase Syp, and adapter molecules like Nck, Grb2 and Fyn. U sing antipeptide antibodies to SHP2, to IRS-l, to antiphosphotyrosine and to AKT /PKB it is possible to study insulin-stimulated IRS-l phosphorylation and the association between IRS-l and SHP2; and the of phosphorylation AKT/PKB. It's also possible to quantify the leveI of SHP2. In the present study we have examined early steps of insulin action in muscle and liver in tive animal models of insulin resistance: 72 hours fasting and diabetes (STZ) (hypoinsulinemia), aging and effect of chronic treatment with dexametasone (hyperinsulinemia) and the effect of acute epinephrine treatment (normoinsulinemia). We used immunoprecipitation and immunoblotting technices with specitic antibodies. ln liver of diabetic rats (STZ) there was an increased IRS-1 phosphorylation o 141 :t 12% compared to the control value (p <0.05). The IRS-l/SHP2 association did not change, but the phosphorylation leveI of AKT/PKB was decreased in the liver of rats treated with STZ. In muscle samples there was an increase in IRS-l phosphorylation to 191:t 14% (p< 0.036), without change in the association ofIRS-l with SHP2. There was also no change in SHP2 protein levei, and the AKT/PKB phosphorylation was reduced to 70:t 7%(p< 0.04) of in muscle of diabetic rats compared to contrais. In fasting there was an increase to 152 :t 13% (p< 0.007) in IRS-l phosphorylation. There was also an increase of IRS-lISHP2 association to 136 :t 7% (p< 0.035) in liver tissue of fasting rats, after insulin stimulation compared to contraIs. In muscle samples from fasting rats the results were similar to that in liver. There was an increase to 155 :t 2% (p<0.019) IRS-l in phosphorylation in tissue of diabetic rats compared to contrais after insulin-stimulation. There was an increase of IRS-lISHP2 association of 162:t 7% (p< 0.019) in tissue of diabetic rats compared to contrais. In rats treated acutely with epinephrine there was a decrease in insulin stimulated IRS-1 phosphorylation to 37 :t 3% of control value (p< 0.019), accompained by reduced to 52 :t 3% (p< 0.002) in IRS-l/SHP2 association in liver. The leveI of SHP2 protein was found be unchanged and the AKT!PKB phosphorylation induced by insulin was reduced to 60 :t 5% (p< 0.045) in the liver of rats treated acutely with epinephrine. In muscle of epinephrine treated rats, there was a marked reduction to 57 :t 9% (p< 0.003) in insulin-stimulated IRS-1 phosphorylation compared to controI. The association of IRS-l/SHP2 was reduced to 20 :t 7% (p< 0.041) in muscle of epinephrine treated rats. There was no change in the SHP2 protein leveI, and the phosphorylation of AKT!PKB was reduced to 70:t 6% (p<0.030) in muscle ofrats treated with epinephrine. There was a decrease in IRS-1 phosphorylation to 64 :t 7% in liver of 20-month-old rats compared to 2-month-old rats. When the same blots were subsequently incubated with anti-SHP2 antibody there was an increase to 155 :t 9% (p<0.012) in insulin-induced IRS-l/SHP2 association in liver of 20-month-old rats compared to 2-months-old rats. Aging did not change the leveI of SHP2 protein and the phosphorylation leveI of AKT!PKB in liver. In muscle there was a decrease in insulin-stimulated IRS-1 phosphorylation to 50 :t 7% (p< 0.007) in 20-months-old rats. There was a simultaneous decrease to 48 :t 6% (p< 0.034) in insulin-induced IRS-l/SHP2 association in muscle of 20-months-old rats. The AKT!PKB phosphorylation was reduced to 70 :t 6% (p< 0.05) in muscle of the 20-month-old rats when compared 2-month-old rats. Finaly, dexamethasone treatment for 5 days induced a decrease in insulin-stimulated IRS-1 phosphorylation levels to 48:t 5% (p< 0.040), without changes in insulin-induced IRS-l/SHP2 association in liver of rats. Immunoprecipitation and immunoblotting with anti-SHP2 antibody showed that the leveI of this protein did not change in liver of rats treated with dexamethasone. In muscle tissue the results were similar to that in liver. There was a reduced in insulin-induced IRS-1 phosphorylation to 36 :t 5% (p< 0.035), and there was no change in the association of IRS-1 with SHP2 in muscle of rats treated with dexametasone afier stimulation with insulin. There was also no change in SHP2 protein leveI in muscle oftreated rats compared to controls. In summary, the results demonstrated that in hiperinsulinemia animaIs (i.e.; aging and dexamethasone treatment) there is a increased IRS-I phosphorylation induced by insulin compared to controls and in hipoinsulinemia models (i.e.; fasting and STZ treatment) the IRS-l phosphorylation induced by insulin is reduced. This phenomenon influence the downstream signal transduction through the IRS-l pathway as indicated by reduced insulin-induced AKT/PKB phosphorylation in liver and muscle of diabetic rats; increased insulin-induced IRS-l/SHP2 association in liver and muscle of fasting rats and liver of aging animaIs. Our results suggest that modulation in insulin signal transduction through IRS-I may occur in response to insulin circulatory levels and for metabolic changes
Doutorado
Fisiologia
Doutor em Biologia Funcional e Molecular

La gravidanza è da considerarsi una condizione pro-inflammatoria dove si osserva un’attivazione dei neutrofili circolanti. Con l'avanzare della gravidanza la concentrazione nel sangue di nucleosomi e mieloperossidasi aumenta e riflette la produzione delle trappole extracellulari dei neutrofili (NETs). Abbiamo dimostrato che in corso di diabete mellito gestazionale (GDM) tale produzione è aumentata in confronto ad una gravidanza fisiologica. Elevati livelli di glucosio e TNF-a, segni tipici presenti in corso di GDM, in vitro agiscono in modo sinergico e sono in grado di pre-attivare i neutrofili ed indurre il rilascio delle NETs. Abbiamo ipotizzato che, lo stato di iperattivazione osservato a livello sistemico possa essere associato ad una aumentata attività leucocitaria a livello placentare. A sostegno della nostra ipotesi, si osserva nei villi coriali isolati da placente GDM un’aumentata infiltrazione di PMNs pro-NETotici in associazione ad un accumulo della neutrofili elastasi (NE). Per valutare un possibile effetto pro-infiammatorio del glucosio a livello placentare, abbiamo incubato cellule di trofoblasto (BeWo) in presenza di un’elevata concentrazione di glucosio e successivamente valutato la produzione di TNF-a. È stato interessante rilevare un aumento nel rilascio di TNF- a tale da indurre un effetto pro-NETotico sui PMN con consequente rilascio della NE. Da recenti ricerche è emerso come in corso di diabete e tumore la NE possa essere internalizzata dalle cellule ed alterare la trasduzione del segnale insulinico attraverso la degradazione del substrato 1 del recettore insulinico (IRS1). Esperimenti in vitro hanno dimostrato che in presenza della NE si osserva una riduzione di IRS1 nelle cellule BeWo ed una diminuzione dell’internalizzazione del glucosio. Poichè l’espressione di IRS1 risulta ridotta nelle placente GDM é verosimile ipotizzare che la massiva presenza di NE ne può essere la causa. In conclusione, i nostri dati suggeriscono che in corso di GDM si verifica un’elevata produzione di NETs ed una massiva infiltrazione di pro-NETotici PMN nella placenta. Queste scoperte dimostrano come la NETosi abbia una significativa utilità diagnostica in corso di GDM e la NE un nuovo potenziale bersaglio terapeutico.
Human pregnancy is associated with a mild pro-inflammatory state characterized by activation of circulatory neutrophils (PMNs). Skewing of PMNs responses toward to neutrophil extracellular traps generation (NETs) is reflected in an increased of circulating nucleosomes and myeloperoxidase with advancing gestational age. Our data indicated that this pro-NETotic profile is enhanced in women with gestational diabetes mellitus (GDM). Maternal hyperglycemia and increased levels of TNF-a are a hallmark of GDM and we show a synergistic effect of both factors on the priming and release of NETs. Moreover, we hypothesized that systemic activation was associated with activated PMN in placenta. Indeed, we observed a massive infiltration of pro-NETotic PMNs and neutrophil elastase (NE) accumulation along chorionic villi of GDM placentas. To further explore whether hyperglycemia predisposes to exaggerated inflammatory response in placenta we incubated trophoblast BeWo cells in high glucose conditions and we next tested the TNF-a production capacity. Interestingly, TNF-a level was incresed and exert a pro-NETotic effect on PMN with consequent NE release. Recent studies in cancer tissues and diabetes models have described that released NE induce profound changes in the surrounding cells, altering the signal transducing cascade and promoting insulin resistance via degradation of insulin receptor substrate 1 (IRS1). Our in-vitro data indicate that addition of NE to trophoblast cell line BeWo causes degradation of IRS1 with consequent glucose uptake impairment. IRS1 is reduced in GDM placentas when compared to control placentas, suggesting that the presence of NE might be the causal factor. Taken together, our data showed that GDM is characterized by excessive NET formation and by a massive influx of pro-NETotic PMN into placentas. These findings underline the competence of NETs as a highly relevant diagnostic biomarker for GDM and NE as a new potential therapeutic target.

Obesity-induced chronic inflammation plays a key role in the pathogenesis of insulin resistance and the metabolic syndrome. Proinflammatory cytokines can cause insulin resistance in adipose tissue, skeletal muscle and liver by inhibiting insulin signaling transduction. Interleukin-1 receptor-associated kinase-1 (IRAK-1) is a serine/threonine kinase functioning in Toll-like Receptor signaling pathways, and plays an important role in inflammation and immune response. In our studies, we demonstrated that IRAK-1 is involved with the negative regulation of PI3K-Akt dependent signaling pathway induced by insulin and TLR 2&4 agonists. Out data also indicate that IRAK-1 can interact with IRS-1 protein both in vivo and in vitro. The binding sites for the IRAK1-IRS1 biochemical interaction are IRS-1â s PH domain and IRAK-1â s proline-rich LWPPPP motif. Our studies also indicate that IRAK-1 is involved with the negative regulation of glycogen synthesis through inhibiting PI3K-Akt signaling pathway and thus releasing GSK3βâ s inhibitory effect on glycogen synthase. Moreover, our studies also suggest that IRAK-1 is involved in the activation of transcription factors CREB and ATF-1 by stimulating CREB-Ser133 and ATF-1 phosphorylation. CREB transcription factor family induces genes involved in cellular metabolism, gene transcription, cell cycle regulation, cell survival, as well as growth factor and cytokine genes. That may partially explain our finding that IRAK-1 may be also involved with cell proliferation and survival pathway.
Master of Science

Objetivos: determinar a atividade de tirosina quinase do receptor de insulina- fosforilação de substrato exógeno poly (Glu 4: Tyr 1)- e expressão do susbtrato do receptor de insulina 1 em miométrio normal e mioma. Delineamento: estudo experimental. Pacientes: mulheres com miomas submetidas a histerectomia. Intervenção: frações de membrana plasmática células de miométrio e mioma foram preparadas e a seguir as amostras foram estimuladas com e sem insulina e incubadas com o substrato exógeno poly (Glu 4: Tyr 1). Para estudar a expressão de IRS-1, lisato total dos tecidos foi utilizado. Western blots utilizando anticorpos específicos foram realizados. Principais medidas: medida da incorporação de radioatividade (32P-ATP) nas frações de membrana plasmática incubadas com e sem insulina. Quimioluminescência seguida por densitometria foi usada para avaliar expressão de IRS-1. Resultados: níveis de IRS-1 em miométrio (0,190 ± 0,022) e mioma (0,226 ± 0,022) não foram diferentes (p > 0,05). A fosforilação do substrato exógeno poly (Glu 4: Tyr 1) também não foi diferente entre miométrio (1,566 ± 0,177) e em mioma (1,98 ± 0,612) (p > 0,05). Conclusão: a expressão protéica de IRS-1 não foi diferente entre miométrios e miomas. A capacidade de fosforilar substratos exógenos com resíduos de tirosina foi semelhante em miomas e miométrios. Outras etapas da via de transdução do sinal da insulina podem estar envolvidas na proliferação alterada dos miomas.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell and Molecular Physiology - School of Medicine." Discipline: Cell and Molecular Physiology; Department/School: Medicine.

L’osteosarcoma (OS) è il tumore primitivo dell’osso più comune in età pediatrica e adolescenziale. L’OS è stato recentemente riconsiderato come una patologia da de-differenziamento, legata all’interruzione del processo cui vanno incontro i precursori osteoblastici, quali le cellule staminali mesenchimali (MSCs), per trasformarsi in osteoblasti maturi. Il sistema IGF è coinvolto nella regolazione della proliferazione e del differenziamento di cellule di OS. IRS-1 è un mediatore critico di tale via di segnalazione e il suo livello di espressione modula il differenziamento di cellule ematopoietiche. Lo scopo di questa tesi è stato quello di definire il ruolo di IRS-1 nel differenziamento osteoblastico di MSCs e cellule di OS. Il potenziale differenziativo di cellule di OS umano e murino e di MSCs derivate da midollo osseo è stato valutato tramite Alizarin Red staining e Real Time-PCR. Dai dati ottenuti è emerso come i livelli di espressione di IRS-1 diminuiscano durante il differenziamento osteoblastico. Conseguentemente, i livelli di espressione di IRS-1 sono stati manipolati utilizzando shRNA per down-regolare l’espressione della proteina o un plasmide per sovra-esprimerla. Sia la down-regolazione sia la sovra-espressione di IRS-1 hanno inibito il differenziamento osteoblastico delle linee cellulari considerate. Allo scopo di valutare il contributo di IRS-1 nella via di segnalazione di IGF-1R è stato utilizzato l’inibitore di tale recettore, αIR-3. Anche in questo caso è stata osservata una riduzione della capacità differenziativa. L’inibitore del proteasoma MG-132 ha portato ad un aumento dei livelli di IRS-1, portando nuovamente all’inibizione del differenziamento osteoblastico e suggerendo che l’ubiquitinazione di questa proteina potrebbe avere un ruolo importante nel mantenimento di appropriati livelli di espressione di IRS-1. I risultati ottenuti indicano la criticità dei livelli di espressione di IRS-1 nella determinazione della capacità differenziativa sia di cellule di OS umano e murino, sia delle MSCs.
Osteosarcoma (OS) is the most common primary malignant bone tumor affecting children and adolescents. OS has recently been re-considered as a differentiation disease, caused by genetic and epigenetic alterations which may impair normal bone development by blocking multipotent mesenchymal stem cell (MSCs) differentiation into osteoblasts. The IGF-system is involved in regulating OS cell proliferation and differentiation. IRS-1 is a critical mediator of IGF-1R signaling and its expression level modulates hematopoietic cell differentiation. The aim of this study is to define the role of IRS-1 in the osteoblastic differentiation of MSCs and OS cells. Differentiating potential of human and murine OS cell lines and bone marrow-derived mouse MSCs was evaluated by Alizarin Red staining and real-time PCR. We found that IRS-1 expression level decreased during differentiation. Consequently, IRS-1 expression levels were manipulated using shRNAs to knock-down, or a plasmid to over-express the protein. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. To understand the contribution of IRS-1 in the IGF-1R pathway we used the αIR-3 IGF-1R blocking antibody, which inhibited the differentiation process. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that again inhibited osteoblastic differentiation, suggesting ubiquitination may play a role in maintaining the appropriate expression level of IRS-1. Taken together, these results indicate that IRS-1 expression level is critical for determining the differentiating capacity of MSCs as well as human and mouse OS cells and that precise regulation of IRS-1 expression by cells is required for this commitment to osteoblastic differentiation.

L’osteosarcoma (OS) è il tumore primitivo dell’osso più comune in età pediatrica e adolescenziale. L’OS è stato recentemente riconsiderato come una patologia da de-differenziamento, legata all’interruzione del processo cui vanno incontro i precursori osteoblastici, quali le cellule staminali mesenchimali (MSCs), per trasformarsi in osteoblasti maturi. Il sistema IGF è coinvolto nella regolazione della proliferazione e del differenziamento di cellule di OS. IRS-1 è un mediatore critico di tale via di segnalazione e il suo livello di espressione modula il differenziamento di cellule ematopoietiche. Lo scopo di questa tesi è stato quello di definire il ruolo di IRS-1 nel differenziamento osteoblastico di MSCs e cellule di OS. Il potenziale differenziativo di cellule di OS umano e murino e di MSCs derivate da midollo osseo è stato valutato tramite Alizarin Red staining e Real Time-PCR. Dai dati ottenuti è emerso come i livelli di espressione di IRS-1 diminuiscano durante il differenziamento osteoblastico. Conseguentemente, i livelli di espressione di IRS-1 sono stati manipolati utilizzando shRNA per down-regolare l’espressione della proteina o un plasmide per sovra-esprimerla. Sia la down-regolazione sia la sovra-espressione di IRS-1 hanno inibito il differenziamento osteoblastico delle linee cellulari considerate. Allo scopo di valutare il contributo di IRS-1 nella via di segnalazione di IGF-1R è stato utilizzato l’inibitore di tale recettore, αIR-3. Anche in questo caso è stata osservata una riduzione della capacità differenziativa. L’inibitore del proteasoma MG-132 ha portato ad un aumento dei livelli di IRS-1, portando nuovamente all’inibizione del differenziamento osteoblastico e suggerendo che l’ubiquitinazione di questa proteina potrebbe avere un ruolo importante nel mantenimento di appropriati livelli di espressione di IRS-1. I risultati ottenuti indicano la criticità dei livelli di espressione di IRS-1 nella determinazione della capacità differenziativa sia di cellule di OS umano e murino, sia delle MSCs.
Osteosarcoma (OS) is the most common primary malignant bone tumor affecting children and adolescents. OS has recently been re-considered as a differentiation disease, caused by genetic and epigenetic alterations which may impair normal bone development by blocking multipotent mesenchymal stem cell (MSCs) differentiation into osteoblasts. The IGF-system is involved in regulating OS cell proliferation and differentiation. IRS-1 is a critical mediator of IGF-1R signaling and its expression level modulates hematopoietic cell differentiation. The aim of this study is to define the role of IRS-1 in the osteoblastic differentiation of MSCs and OS cells. Differentiating potential of human and murine OS cell lines and bone marrow-derived mouse MSCs was evaluated by Alizarin Red staining and real-time PCR. We found that IRS-1 expression level decreased during differentiation. Consequently, IRS-1 expression levels were manipulated using shRNAs to knock-down, or a plasmid to over-express the protein. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. To understand the contribution of IRS-1 in the IGF-1R pathway we used the αIR-3 IGF-1R blocking antibody, which inhibited the differentiation process. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that again inhibited osteoblastic differentiation, suggesting ubiquitination may play a role in maintaining the appropriate expression level of IRS-1. Taken together, these results indicate that IRS-1 expression level is critical for determining the differentiating capacity of MSCs as well as human and mouse OS cells and that precise regulation of IRS-1 expression by cells is required for this commitment to osteoblastic differentiation.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.
Vita.
Includes bibliographical references.
Excessive fat synthesis and the subsequent dysregulation of lipid metabolism constitute the major pathological factors of obesity and type 2 diabetes through triggering insulin resistance. Thus, controlling fat synthesis by identifying key sites for regulation of lipogenesis and modulating the lipogenic fluxes may provide novel approaches to intervention of the diseases. As a first step to quantitative investigation of lipogenic fluxes from various carbon sources as related to insulin signaling, relative contribution of glucose, glutamine, and acetoacetate to fat biosynthesis in wild-type (WT) and insulin receptor substrate-i knockout (IRS-1 KO) brown adipocytes were analyzed by stable-isotope labeling, GC/MS, and flux estimation. Glutamine contributed more to fatty acid synthesis than glucose in WT cells while glucose's contribution was heavier in IRS-1 KO cells. Unlike the straightforward pathway for lipogenesis from glucose, two possibilities for glutamine's route to fatty acid synthesis have been proposed: glutaminolysis pathway through conventional tricarboxylic acid cycle and a pathway via reductive carboxylation of a-ketoglutarate to isocitrate. These pathways were integrated into a metabolic network model for quantitative estimation of individual lipogenic fluxes. Incubation of the cells with [U-13C] glutamine for 6 hrs led to metabolic and isotopic steady state where individual fluxes of the model were estimated with 95% confidence by least-square fit method.
(cont.) Dose dependent repression of estimated net flux of reductive carboxylation by specific inhibition of NADP+-dependent isocitrate dehydrogenase and the subsequent reduction in glutamine's contribution to fatty acid synthesis in WT cells strongly indicated that reductive carboxylation is an important site of regulating glutamine's lipogenic flux. Abolition of this net flux, reduction in glutamine's lipogenic contribution, and concurrent rise in glucose's lipogenic contribution in IRS-1 KO cells were consistent with the importance of reductive carboxylation. Differential effects of lack of insulin signaling on individual lipogenic fluxes suggested that there might be specific sites at which insulin signaling regulates lipogenic utilization of carbon sources. These results revealed the importance of other carbon sources such as glutamine in fat synthesis and the means by which the flux of these carbon sources to fat synthesis can be controlled.
by Hun-Tae Yoo.
Ph.D.

Orientadores: Mario Jose Abdalla Saad, Licio Augusto Velloso
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-04T23:40:11Z (GMT). No. of bitstreams: 1 Araujo_ElianaPereirade_D.pdf: 1282393 bytes, checksum: 3f0af814a8ad75f3abbde4e9d917c3c1 (MD5) Previous issue date: 2005
Resumo: O substrato-1 do receptor de insulina (IRS-1) desempenha função importante na conexão entre os receptores de insulina (IR) e do fator de crescimento insulina-like-1 (IGF-1R), e as moléculas que participam das etapas subseqüentes da transdução dos sinais gerados pelos respectivos ligantes. No presente estudo, utilizou-se um oligonucleotídeo antisense para o IRS-1 (AS IRS-1), com a finalidade de inibir a expressão dessa proteína in vivo e avaliar as conseqüências desse bloqueio em diferentes tecidos de ratos Wistar. O tratamento com AS IRS-1 durante três dias reduziu a expressão desta proteína em cerca de 80%, 75% e 65% (p<0,05) respectivamente em fígado, músculo esquelético e tecido adiposo, sendo tal efeito acompanhado por uma redução de 40% (p<0,05) na constante de decaimento de glicose durante o teste de tolerância à insulina (ITT), por uma redução de 78% (p<0,05) no consumo de glicose durante o clamp euglicêmico-hiperinsulinêmico e por um aumento de 90% (p<0,05) nos níveis basais de insulina plasmática. Os efeitos metabólicos produzidos pelo AS IRS-1 foram acompanhados pela redução significativa da fosforilação induzida por insulina da Akt [Ser473] em fígado (85%, p<0,05), músculo esquelético (40% p<0,05) e tecido adiposo (85%, p<0,05), e uma redução significativa da fosforilação em tirosina da ERK induzida por insulina em fígado (20%, p<0,05) e músculo esquelético (30%, p<0,05). Entretanto, o tratamento com AS IRS-1 promoveu aumento (60%, p<0,05) da fosforilação da ERK em tirosina induzida por insulina em tecido adiposo. Por fim, em ratos tratados com AS IRS-1 por oito dias, observou-se um aumento de 100% (p<0,05) da massa relativa de tecido adiposo epididimal e um aumento de 120% (p<0,05) da expressão do receptor nuclear PPAR?. Portanto, a inibição da expressão de IRS-1 em ratos leva resistência à insulina acompanhada pela ativação de uma via de sinalização relacionada ao crescimento celular, exclusivamente em tecido adiposo branco
Abstract: Insulin receptor substrate-1 (IRS-1) has an important role as an early intermediary between the insulin and IGF-1 receptors and downstream molecules that participate in the insulin and IGF-1 signal transduction. We employed an antisense oligonucleotide (IRS-1 AS) to inhibit whole-body expression of IRS-1 in vivo and evaluate the consequences of short-term inhibition of IRS-1 in Wistar rats. Four days treatment with IRS-1 AS reduced the expression of IRS-1 by 80, 75 and 65% (P<0.05) in liver, skeletal muscle, and adipose tissue, respectively. This was accompanied by a 40% (P<0.05) reduction in the constant of glucose decay during an insulin tolerance test, a 78% (P<0.05) reduction in glucose consumption during a hyperinsulinemic-euglicemic clamp, and a 90% (P<0.05) increase in basal plasma insulin levels. The metabolic effects produced by IRS-1AS were accompanied by a significant reduction in insulin-induced [Ser473] Akt phosphorylation in liver (85%, P<0.05), skeletal muscle (40%, P<0.05), and adipose tissue (85%, P<0.05) and a significant reduction in insulin-induced tyrosine phosphorylation of ERK in liver (20%, P<0.05) and skeletal muscle (30%, P<0.05). However, insulin-induced tyrosine phosphorylation of ERK was significantly increased (60%, P<0.05) in adipose tissue of IRS-1 AS treated rats. Moreover, in rats treated with IRS-1 AS for 8 days, a 100% increase (P<0.05) in relative epididymal fat weight and a 120% (P<0.05) increase in nuclear expresssion of peroxisome proliferator-activated receptor-? were observed. Thus, acute inhibition of IRS-1 expression in rats leads to insulin resistance accompanied by activation of a gowth-related pathway exclusively in white adipose tissue
Doutorado
Medicina Experimental
Doutor em Fisiopatologia Medica

Emerging evidence suggests that an important mechanism for the negative feedback control of insulin signalling involves the inhibition of tyrosine phosphorylation of IRS-1 by its prior serine/threonine (ser/thr) phosphorylation. IRS-1 ser/thr phosphorylation has been linked to the dissociation of IRS-1 from the insulin receptor and PI3K, and its degradation via a proteasome-dependent pathway. Studies in animal models have shown that increases in plasma free fatty acids (FFAs) are associated with reduced IRS-1-signalling, and so it has been postulated that elevated FFA cause insulin resistance by activating pathways that negatively regulate insulin action, including hyper-phosphorylation of ser/thr residues in IRS-1. We have shown that in the case of linoleate-induced insulin resistance in L6 rat skeletal muscle cells, the inhibition of IRS-1-dependent signalling arises via effects on both the phosphorylation status and degradation of IRS-1, which are mediated, in part, by IKKb. In addition, the reduction of IRS-1 mRNA levels allude to transcriptional effects of linoleate treatment that also contribute to the observed reduction in the total levels of this protein. PtdOH, particularly dilinoleoyl PtdOH, was found to be significantly increased in linoleate treated L6 cells, and sufficient to induce at least some of the effects on insulin-signalling that are observed upon linoleate treatment. It is unlikely, however, that IKKb and PtdOH are components of the same inhibitory pathway, since inhibiting IKKb activity did not alleviate the effects of PtdOH on IRS-1 tyrosine (tyr) phosphorylation. Moreover, although an integral component of the mechanism by which linoleate induces insulin-resistance in L6 cells, it appears that restoring IRS-1 function in linoleate treated cells is not sufficient to reverse insulin resistance. Hence, we hypothesise that linoleate induces multiple inhibitory pathways in L6 cells, with at last two of these involving IKKb- and PtdOH-dependent inhibition of IRS-1 signalling, which act in parallel to reduce glucose disposal and cause insulin resistance in this model.

L'élaboration de nouvelles thérapies contre le diabète nécessite la compréhension des bases moléculaires de la résistance à l'insuline. Au cours de ma thèse, je me suis intéressée à la Myotubularine, une phosphatase mutée dans la myopathie myotubulaire liée au chromosome X. Nous avons montré que cette enzyme déphosphoryle spécifiquement la position D3 des phosphatidylinositol (3) et (3,5)-phosphate [PI(3)P et PI(3,5)P2]. De plus, nous avons observé que la surexpression de la Myotubularine dans les cellules musculaires L6 inhibe la translocation du transporteur de glucose GLUT4 à la membrane plasmique. Dans une deuxième partie, j'ai étudié l'effet d'une exposition prolongée à l'insuline sur le niveau des protéines Insulin Receptor Substrate-1/2 (IRS-1/2). Cette étude, réalisée dans des cellules musculaires et adipocytaires, a permis de mettre en évidence des différences de régulation entre IRS-1 et IRS-2 mais également une spécificité cellulaire de ces mécanismes. Notamment, dans les adipocytes 3T3L1, l'inhibition des histones déacétylases (HDACs) empêche en partie la baisse de la quantité de protéine IRS-1 (dégradation), probablement via une acétylation d'IRS-1. En revanche, dans les cellules musculaires L6, l'inhibition des HDACs empêche la baisse de la quantité d'ARNm d'IRS-1, mais pas d'IRS-2, vraisemblablement via acétylation de la chromatine au niveau de ce gène. L'ensemble de nos résultats démontre que la transduction du signal de l'insuline est régulée de façon importante au niveau des D3-phosphoinositides et des protéines IRS. Cette étude a en outre permis de mettre en évidence, pour la première fois que les HDACs participent à la régulation des composants de la voie de signalisation de l'insuline par l'hormone elle-même
The research that I have performed for my thesis may be divided into two major sections. Firstly, I have studied the function of myotubularin, a phosphoinositide phosphatase mutated in X-linked myotubular myopathy. We demonstrated that myotubularine dephosphorylates PI(3)P and PI(3,5)P2 at the position D3'. In addition, we have shown that overexpression of myotubularine leads to a significant decrease in insulin-stimulated GLUT4 translocation to the plasma membrane, suggesting that these phosphoinositides are required for normal glucose uptake. Secondly, to gain insight into the deleterious effects of hyperinsulinemia, we studied the effect of prolonged-insulin treatment on “Insulin Receptor Substrates-1/2” (IRS-1/2). This study, performed in 3T3L1 adipocytes and L6 muscle cells, allowed us to demonstrate differences in the regulation between IRS-1 and IRS-2 and also between adipose and muscle cells. In particular, we found that histone deacetylase (HDAC) inhibition protects IRS-1 from downregulation, at the protein level in adipocytes and at the mRNA level in muscle cells. Finally, we demonstrated that IRS-2 participates in PDGF-R signalling and contributes to the regulation of the PDGF-induced signalling pathway and to the control of the actin cytoskeleton. Taken together, our results demonstrate that insulin signalling is regulated importantly at the level of D3-phosphoinositides and IRS proteins. In addition, the present studies provide, for the first time, evidence for a deacetylation-based mechanism by which prolonged in

La protéine IRS-1 (Insulin Receptor Substrate-1) est un des substrats majeurs des récepteurs de l'insuline et de l'IGF-I. En présence d'insuline, IRS-1 est phosphorylé sur tyrosine et peut interagir avec les intégrines. Les intégrines jouent un rôle important dans l'adhésion des cellules entre elles et dans leur attachement à la matrice extracellulaire. Après adhésion cellulaire, la tyrosine kinase FAK est phosphorylée sur tyrosine et activée. Nous avons mis en évidence que l'engagement des intégrines induit la phosphorylation sur tyrosine d'IRS-1 et que FAK interagit avec IRS-1. Cette association est dépendante du domaine C-terminal de FAK, mais ne requiert pas son activité kinase. La surexpression d'IRS-1 avec FAK induit une importante phosphorylation sur tyrosine d'IRS-1 et une activation de la PI 3-kinase associée à IRS-1. Nous avons ensuite montré que les intégrines contrôlent l'expression d'IRS-1. Des fibroblastes en suspension expriment très faiblement l'ARNm d'IRS-1 et la ré-adhésion de ces fibroblastes sur fibronectine ou vitronectine restaure le niveau de l'ARNm. Nous nous sommes également intéresés au rôle de FAK dans la régulation de l'expression d'IRS-1. (. . . ). Le contrôle de l'expression d'IRS-1 par les intégrines permettrait d'expliquer une potentialisation à long terme par l'adhésion cellulaire du signal généré par l'insuline ou l'IGF-I. En revanche, la formation du complexe FAK/IRS-1, ainsi que la phosphorylation d'IRS-1 induite par FAK permettrait d'expliquer une potentialisation à court terme de l'action de l'insuline et de l'IGF-I.

The results of a number of studies have shown that in arterial hypertension (AH), G/T and T/T genotypes of the adiponectin gene (ADIPOQ) and Gly/Arg and Arg/Arg genotypes of the insulin receptor substrate 1 gene (IRS-1) are associated with a greater severity of metabolic disorders and hemodynamic parameters compared with G/G and Gly/Gly genotypes of these genes. The aim of the study: to evaluate the severity of cardiovascular remodeling and metabolic disorders in hypertensive obese patients in the simultaneous presence of two unfavorable genotypes of the ADIPOQ and IRS-1 genes. Methods: We examined 300 AH patients: 200 patients with AH and obesity, 50 patients with AH and normal body weight, 50 patients with AH and overweight, 40 patients with AH, obesity and type 2 diabetes mellitus (DM2), 30 healthy individuals. The polymorphisms of the ADIPOQ and IRS-1 was assessed by molecular genetic method. Results: It was found that in all groups of hypertensive patients, regardless of body weight and the presence of DM2, the simultaneous presence of two unfavorable genotypes of the ADIPOQ and IRS-1 genes occurred significantly more often than in healthy individuals: in 41% of AH patients with obesity, 30% of AH patients with normal weight, 40% of AH with overweight, 57.5% of AH with obesity and DM2 vs. 13.3% of healthy individuals. In hypertensive patients, in the presence of overweight and obesity, the frequency of combination of the two unfavorable genotypes of these genes was significantly higher than in AH patients with normal body weight. Conducting comparative evaluation of AH patients with obesity depending on the presence of two unfavorable genotypes or two protective genotypes of the ADIPOQ and IRS-1 genes showed that carriers of the combination of the G/T + T/T genotype of the ADIPOQ and the Gly/Arg + Arg/Arg genotype of the IRS-1 had a higher body mass index, more pronounced insulin resistance, cardiovascular remodeling, adipokine imbalance, impaired carbohydrate and lipid metabolism. Conclusions: In AH patients, the frequency of the simultaneous presence of two unfavorable polymorphisms of ADIPOQ and IRS-1 genes was higher than in healthy individuals. In AH patients with overweight and obesity, the frequency of combination of the two unfavorable genotypes of the ADIPOQ and IRS-1 genes was significantly higher than in normal body weight. The presence of a combination of two unfavorable genotypes of the ADIPOQ and IRS-1 genes in patients with AH and obesity was associated with a greater severity of cardiovascular remodeling and metabolic disorders compared with the combination of two protective genotypes of these genes.

The aim of the present thesis was to further increase our understanding of mechanisms contributing to and maintaining cellular insulin resistance in type 2 diabetes (T2D). For this reason, the effects of high glucose and insulin levels on glucose transport capacity and insulin signaling, with emphasis on insulin receptor substrate 1 (IRS-1) were assessed in fat cells. Altered levels of IRS-1 have previously been observed in adipose tissue from insulin-resistant and T2D subjects. A high glucose level (≥15 mM) for 24 h exerted only a minor impairment on glucose transport capacity in human adipocytes, as opposed to rat adipocytes. However, when combined with a high insulin level (104 µU/ml), basal and insulin-stimulated glucose transport was significantly impaired in both human and rat adipocytes. This was associated with a depletion of IRS-1 and IRS-2 protein levels in rat adipocytes, as a result of post-translational changes and altered gene transcription, respectively. In human adipocytes was only IRS-1 protein levels reduced. The high glucose/high insulin setting achieved maximal impairment of glucose transport within 6 h. Subsequent incubations of rat adipocytes under physiological conditions could partially restore insulin sensitivity. Interestingly, in both human and rat fat cells, decreased levels of IRSs occurred after the establishment of impaired glucose transport, suggesting that the observed depletion of IRSs is a consequence rather than a cause of insulin resistance. Nonetheless, IRS depletion is likely to further aggravate insulin resistance. Tyrosine phosphorylation of IRS-1 upon insulin stimulation activates the signaling pathway that mediates glucose transport. Pre-treatment of human adipocytes with high glucose and insulin levels was not associated with any alterations in the total IRS-1 Tyr612 phosphorylation following 10 min insulin stimulation. However, a significant increase in basal Tyr612 phosphorylation was observed. Furthermore, a rise in basal IRS-1 Ser312 phosphorylation was found. This is associated with reduced IRS-1 function and is considered to target IRS-1 to degradation pathways, and thus could potentially explain the observed decrease in IRS-1 protein levels. Our results imply an enhanced activation of insulin’s negative-feedback control mechanism that inhibit IRS-1 function. This could potentially have contributed to the observed impairment of insulin action on glucose transport in these cells. Accordingly, we have also shown that the downstream activation of protein kinase B upon insulin-stimulation is significantly impaired in human adipocytes exposed to the high glucose/high insulin setting, indicating a defect in the signaling pathway mediating glucose transport. We also investigated whether there are humoral factors in the circulation of T2D patients that contribute to peripheral insulin resistance. Human adipocytes cultured for 24 h in medium supplemented with 25% serum from T2D subjects, as compared to serum from non-diabetic subjects, displayed significantly reduced insulin-stimulated glucose uptake capacity. The effect could neither be attributed to glucose, insulin, FFA, TNF-α or IL-6 levels in the serum, but other circulating factor(s) seem to be of importance. In conclusion, chronic conditions of elevated glucose and/or insulin levels all impair insulin action on glucose turnover, but to different extents. A clear distinction between rat and human fat cells in the response to these different milieus was also observed. Alterations in the function of the key insulin signaling protein IRS-1 might be involved in the mechanisms underlying the impaired glucose uptake capacity. IRS-1 reduction however, occurs after but probably aggravates the existing insulin resistance. The effects of high glucose and/or insulin levels may be of importance in T2D, but additional novel factors present in the circulation of T2D patients seem to contribute to cellular insulin resistance.

Interferon regulatory factor 1 (IRF-1) is a tumour suppressor protein and transcription factor. It has been shown to modulate target gene expression in response to stimuli, which include viral infection and DNA damage, and to be down-regulated in several forms of cancer. This thesis details the development of an intrabody, an intracellular antibody, that binds specifically to endogenous IRF-1. The binding of the intrabody to IRF-1 enhanced transcription from IRF-1-responsive reporter gene constructs and endogenous promoters, thus it was shown to activate IRF-1. Intrabody binding also increased the rate at which IRF-1 was degraded, suggesting that the intrabody epitope may be regulating both IRF-1 activity and turnover. These results were supported point mutation within the intrabody epitope (P325 to A) as the resultant mutant also displayed both a higher transcriptional activity and increased rate of degradation. In an effort to understand the mechanisms which regulate IRF-1 activity a search for novel IRF-1-interacting proteins was carried out using phage peptide display. This in vitro technique enables the identification of peptides able to bind a specific target protein. The sequence of these peptides can then be used to search protein databases for homologous, full-length proteins that could also bind the target protein. This led to the identification of an IRF-1-binding peptide that held sequence similar to a region of Zinc Finger 350 (ZNF350), a transcription factor involved in regulating the DNA damage response. Subsequently, endogenous ZNF350 and IRF-1 were co-immunoprecipitated from a human cancer cell line. The extreme C-terminus of IRF-1 was shown to be sufficient for an interaction with ZNF350, although a second, more N-terminal site was also shown to be essential for a stable intracellular interaction. This data sheds new light on the role of the extreme C-terminus of IRF-1 in modulating the protein‟s activity. This study also provides new and IRF-1-specific molecular tools, in the form of intrabodies and IRF-1-binding peptides, which could be used in the future to further characterise the activity and regulation of this tumour suppressor protein.

One of the aims of this study was to locate regions in interferon regulatory factor (IRF)-1 and IRF-2 that were responsible for transactivation and DNA binding. Through the use of PCR mutagenesis, progressive deletions from the C-terminal end of IRF-1 and IRF-2 were generated; 6 IRF-1 and 8 IRF-2 deletion mutants were created to study the structure-function relationship of these two proteins. The deletion mutants were named according to the number of amino acids remaining from their N-terminal end: IRF-1 (120), IRF-1 (150), IRF-1 (170), IRF-1 (200), IRF-1 (250), IRF-1 (300); IRF-2 (100), IRF-2 (125), IRF-2 (160), IRF-2 (200), IRF-2 (240), IRF-2 (270), IRF-2 (300), IRF-2 (320). Using the Th CAT and SV$ sb{ rm O} beta$ CAT constructs as reporter genes, the transactivation potential of each deletion mutant was assayed through co-transfection studies in 293 cells. Also, transactivation potential was correlated to the ability of the various deletion mutants to bind DNA using the PRDI sequence as a probe in electrophoretic mobility shift assay (EMSA). Much of the transactivation potential in IRF-1 lies past the first 200 amino acids. Within this C-terminal region between amino acids 226 to 230, is a stretch of 5 consecutive acidic residues (Asp-Glu-Asp-Glu-Glu) which may be critical for transcriptional activation. Also, there are numerous potential phosphorylation sites in the C-terminal region of IRF-1 which may contribute to IRF-1 activity as well. Transcriptional repression by IRF-2 is contained within the first 125 N-terminal amino acids and correlates with the ability of IRF-2 to bind DNA. (Abstract shortened by UMI.)

The highly unstructured interferon regulated transcription factor IRF-1 is a tumour suppressor protein that plays vital roles in the antiviral and DNA damage response pathways. To identify interacting factors that regulate IRF-1 function and expand on the available limited information on its interactome, an in vitro screen was developed using peptide-aptamer affinity chromatography coupled with mass spectrometry. Discrete identified which bind to a number of potential transcriptional regulators including NPM1, YB-1 and TRIM28. The screen also proved useful in identifying binding proteins to the C-terminal Mf1 domain, which is vital for IRF-1-mediated growth suppression and Cdk2 repression, and additionally regulates IRF-1 stability. Thus, an LXXLL motif in the MF1 domain was found to be required for the binding of Hsp70 family members and cooperation with Hsp90 to regulated IRF-1 turnover and activity. These conclusions were supproted by the finding the Hsp90 inhibitors suppressed IRF-1-dependent transcription shortly after treatment, whilst at later time points inhibition of Hsp90 led to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 was increase by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear, but not cytoplasmic, IRF-1. Additionally, a stress specific interaction between IRF-1 and the Hsp70-associated ubiquitin E3 ligase CHIP, that targets Hsp70/Hsp90 clients for proteasomal degradation, was demonstrated. Consequently, decreases in IRF-1 protein levels in cells exposed to heat stress or heavy metal ions were accompanied by the formation of IRF-1:CHIP complexes. Based on observations that CHIP ubiquitination of IRF-1 occurred both in the presence and absence of Hsp70, a model was proposed wherein Hsp70 serves as a factor that recruits CHIP to its substrates and its dissociation from the complex allosterically activates CHIP-dependent substrate ubiquitination. In support of this model, in vitro and biophysical evidence is presented, showing that CHIP in complex with Hsp70 is less flexible and less effective as an E3 ligase that CHIP alone. Thus, in agreement with recent studies, the work done in this thesis highlights the importance of conformational flexibility and of direct binding or 'docking' of CHIP to its substrate(s) in its mechanism of action.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 27-29).
The endoplasmic reticulum (ER) is responsible for the folding and processing of approximately one third of proteins in eukaryotic cells, and homeostasis in this compartment is tightly regulated. The Unfolded Protein Response, or UPR, is activated in response to perturbations in protein folding in the ER, collectively termed ER stress. This compensatory mechanism, mediated by IRE-1, PERK- 1/PEK-1 and ATF-6 in metazoans, resolves an overcrowded ER lumen in part through the increase of protein degradation and folding. Typical studies focus on activation of the UPR in response to characterized chemical agents that potently alter ER function or protein stability and folding, leaving physiological or native roles for the UPR relatively uncharacterized. Richardson et al previously demonstrated a role for the UPR in innate immunity in C elegans. Here, in an effort to understand this role, we demonstrate that intestinal expression of XBP-1 is sufficient to overcome PMK-1 dependent larval lethality on a lawn of pathogenic Pseudomonas aeruginosa. Further, we demonstrate that XBP-1 deficiency results in constitutive ER stress even in the absence of pathogenic infection. This elevated ER stress is reflected by increased activities of both IRE- 1 and PEK-1 under physiological conditions. Our data suggest that negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles not only at the extremes of ER stress but also in the maintenance of ER homeostasis under physiological conditions.
by Stephanie A. Kinkel.
S.M.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A insuficiência cardíaca (IC) é a evolução final das várias formas de doenças cardiovascular, sendo resultado de modificações estruturais, metabólicas e de contratilidade miocárdica. A fim de compreender o papel na dinâmica do metabolismo cardíaco no estado basal e na sobrecarga de pressão, utilizamos os modelos de cre-lox com deleção específica no coração para substrato do receptor de insulina (IRS) e co-ativador do PPAR (PGC-1β) e analisamos a estrutura cardíaca (histologia e estereologia), função cardíaca (ecocardiograma e técnica de Working heart), o metabolismo (isolamento de cardiomiócito e captação de glicose), ação hormonal (Western Blotting), expressão gênica (PCR-RT) de enzimas do metabolismo (lipídico, glicídico, da cadeia respiratória fatores transcricionais e hipertróficos) e a função mitocondrial. Verificamos, nos CIRS12KO, disfunção cardíaca grave, disfunção mitocondrial e prejuízo na expressão gênica das enzimas do metabolismo energético. Nos PGC-1βKO observamos disfunção mitocondrial e alteração de expressão gênica das enzimas do metabolismo energético quando submetidos à sobrecarga de pressão. Através do estudo do metabolismo cardíaco e da expressão gênica nestes diferentes modelos conseguimos explorar as vias metabólicas que levam a hipertrofia compensada à IC. Sugerimos que o mecanismo responsável pela descompensação seja a disfunção mitocondrial em conseqüência à alteração da expressão gênica. E que IRS e o PGC-1β são fatores chaves da dinâmica cardíaca, e que são indispensáveis para a estrutura e funcionamento cardíaco. Além de representar alvo promissor para limitar a transição da hipertrofia cardíaca compensada a insuficiência cardíaca.
Heart failure (HF) is the end stage of different types of cardiovascular diseases and it is characterized by changes in the metabolic and myocardial contractility. We use the models cre-lox with specific knockout for insulin receptor substrate (IRS) and co-activator of PPAR (PGC-1b) (basal and pressure overload). The objective was understood the role in the dynamics of cardiac metabolism. We analyzed cardiac structure (histology and stereology), cardiac function (echocardiography and the working heart technique), metabolism (glucose uptake), hormonal action (Western Blotting), gene expression (RT-PCR) from enzyme metabolism (lipid, carbohydrates, respiratory chain, transcriptional and hypertrophic factors) and mitochondrial function. We found in CIRS12KO, severe cardiac dysfunction, mitochondrial dysfunction and reduction of gene expression. And in the PGC-1bKO when subjected to pressure overload, the progression to heart failure, with mitochondrial dysfunction, and alteration of gene expression from enzyme metabolism. The data show that changes on cardiac metabolism and gene expression in both models explain the metabolic pathways that lead to compensated hypertrophy to HF. We suggest that the mitochondrial dysfunction and the gene expression was possible mechanisms for HF. We conclude that IRS and PGC-1b are key factors of cardiac dynamics, which are essential to the structure and heart function. IRS and PGC-1b represent a promising target for limiting the transition from compensated cardiac hypertrophy to heart failure.

Interferon regulatory factor 1 (IRF-1), the founding member of IRF family, is a nuclear transcription factor first described as a transcription factor that binds to the upstream region of interferon induced genes following viral infection. In addition, IRF-1 has been reported to be involved in cell growth regulation, induction of apoptosis, immune responses, post-transcriptional modification, and cell transformation by oncogenes. Thus, IRF-1 shows accumulative evidence supporting the theory that IRF-1 functions as a tumour suppressor. However, we still lack the knowledge in the regulation and function behind IRF-1 and many other tumour suppressors due to the lack of synthetic tools that can aid in understanding the mechanism of cancer biology. Here we described the creation of synthetic tools that can be applied to study the role of a transcription factor(s) in cancer biology. Firstly, we described the creation, using recombineering technology, of universal bacterial artificial chromosome (BAC) targeting vector. This targeting vector, carry a cre-conditioned STOP cassette that can be targeted at a desired specific area. The resulted targeting vector can aid the generation of mice models with a conditioned knock-in subtle mutation(s). The resulted cre-conditioned mice models are an essential tool for any outstanding research project in cancer biology. Secondly, we described the development of Flp-In System™ from Invitrogen; the system can ease the generation of isogenic stable mammalian expression cell lines. Using this system, we created two isogenic stable cell lines expressing wild-type IRF-1 and a mutant that abolish IRF-1 DNA binding ability (W11R). Both cell lines were investigated using microarray analysis revealing new IRF-1 target genes. We reported the up-regulation of expected standard interferon regulatory genes such as, interleukin-24 (IL-24) and interferon regulatory factor-2 binding protein-2 (IRF2BP2) and the up-regulation of standard apoptotic genes such as, early growth response-1 (EGR-1) and prostate transmembrane protein, androgen induced-1 (PMEPA1) confirming the role of IRF-1 as a tumour suppressor. However, we also reported the up-regulation of secreted phosphoprotein-1 (SPP1) and SH3 and PX domains-2A (SH3PXD2A) which are matricellular protein produced by cancer cells playing a role in cellular adhesion, invasion, tumour growth progression and metastasis. Thus, we proposed a new biological role of IRF-1 in cellular movement. Thirdly, we described the development of a synthetic stable reporter cell line which can report IRF-1 transcriptional activity; such reporter cell line can be used once large scale screening is needed. The created stable reporter cell line was used to screen a kinase inhibitor library which has revealed C3 as an IRF-1 modifier. The newly identified IRF-1 modifier regulates IRF-1 transcriptional activity by inhibiting platelet-derived growth factor receptor (PDGFR) and/or vascular endothelial growth factor receptor (VEGFR) tyrosine kinase. Finally, we validated the synthetic Flp-In System™ by testing the system using a novel oncoprotein model. We have developed a stable cell line that overexpresses an oncoprotein named Anterior Gradient 2 (AGR-2). We have found that AGR-2 can attenuate IRF-1 protein levels dependent of p53. In addition, AGR-2 has been identified as a cellular survivor factor during unfolding protein response. In conclusion, this study descried the creation and the validation of synthetic tools: synthetic cassette for cre-conditioned mice creation, the Flp-In System™ for isogenic stable cell line creation, and IRF-1 reporter cell line for high throughput screening. All synthetic tools were validated and used to investigate IRF-1, a transcription factor that plays a role in cancer and immune system.

IRF-1 (Interferon Regulatory Factor 1) is a transcription factor first identified as a regulator of Interferon expression. Two decades after its discovery, IRF-1 has been shown to be involved in numerous other pathways including apoptosis, cell cycle regulation, DNA damage/repair, immune cell development and inflammation. Transcriptional regulation of IRF-1 by a number of external agents has been extensively studied, however almost nothing is known about the posttranslational regulation of IRF-1 activity. In this study IRF-1 is shown to be phosphorylated at Thr180 by GSK3β (Glycogen Synthase Kinase 3β). Phosphorylated Thr180 promotes interaction with the ubiquitin E3 ligase SCFFbxw7u, (Skp1-Cu11-Fbxw7α) which increases turnover of IRF-1 protein. Phosphorylation dependent ubiquitination of IRF-1 was confirmed, as substitution of Thr180 to alanine reduced IRF-1 ubiquitination and increased stability. Enhanced phosphorylation of IRF-1 (by increasing GSK3β expression) promotes increased ubiquitination/degradation. Transactivation of the TRAIL (TNFα Related Apoptosis Inducing Ligand) promoter by IRF-1 was found to be dependent on GSK3β phosphorylation of Thr180 by use of reporter assays and inducible expression of IRF-1 in breast cancer cell lines. Importantly IRF-1 activity on the TRAIL promoter is dependent on proper turnover by the UPS (Ubiquitin Proteasome System), as chemical inhibition of the proteasome, or reduction in IRF-1 ubiquitination reduced activity in reporter assays. This suggests that phosphorylation of IRF-1 by GSK3β acts as a destruction signal through association with SCFFbxw7a. This signal dependent turnover of IRF-1 is required for proper transcriptional activation of the TRAIL promoter.

Protein ubiquitination is a key regulator of both protein stability and activity, and is involved in the regulation of a vast variety of cellular pathways. The ubiquitination system therefore provides an exciting target for drug development aiming to regulate the function of specific proteins. Our understanding of ubiquitin signalling is far from complete; and if we are to exploit this system for the benefit of human health, it is important to gain a better understanding of this complex posttranslational modification system as well as the effect of ubiquitination on the target protein. The E3 ligases MDM2 and CHIP were implicated in the control of the two transcriptional activators (TAs) IRF-1 and p53, that normally function to maintain health at the cellular and organismal level. Research carried out as part of my PhD has focused on gaining a mechanistic understanding of the ubiquitination process in particular the relationship between the E3 ligase and its substrate. Broadly, the mechanisms of E3 ligase regulation have been linked to substrate specificity and then to the physiological outcome of site-specific ubiquitination of the DNA binding domain of the TAs IRF-1 and p53. More specifically I have; (i) identified a mechanism by which the E3 ligase activity of the CHIP U-box can be allosterically regulated by ligand binding to its TPR domain. (ii) Residues on IRF-1 that are targeted by MDM2 and CHIP have been mapped, revealing that both ligases modify sites exclusively in IRF-1's DNA binding domain (DBD). Furthermore, I showed that, in its DNA bound conformation, IRF-1 is neither bound nor ubiquitinated by the ligases, suggesting a mechanism by which IRF-1 ubiquitination and possibly degradation can be regulated through its DNA binding state. And lastly, (iii) I have shown that both IRF-1 and p53, which have ubiquitin acceptor lysines in their DBD, bind DNA more stably when ubiquitinated. Modelling suggests that interactions between a positively charged surface area of ubiquitin and the negatively charged DNA can stabilises the TA-ubiquitin complex. DBD ubiquitination sites are required for full transactivation potential of both TAs, supporting a role of ubiquitin in their activation. p53 is ubiquitinated in response to activation by IR or Nutlin-3 and these ubiquitinated forms of p53 are localised in the cell nucleus associated with chromatin and do not lead to protein degradation. Taken together, the data imply that p53 and IRF-1 DNA binding ability, and thereby activity, can be modulated by ubiquitin modification.

Les iron regulatory proteins controlent l'homeostasie du fer en modulant l'expression de la ferritine et du recepteur de la transferrine. Cette regulation s'effectue par une interaction des irps avec un ou (plusieurs) element(s) de reponse au fer nomme(s) ire (iron responsive element) localise(s) dans les regions non traduites en 5' de l'arnm de la ferritine et en 3' de l'arnm du recepteur de la transferrine. L'interaction irp/ire en 5' bloque la traduction alors que celle en 3' stabilise l'arnm. En plus de son activite trans-regulatrice, l'irp-1 presente une activite aconitase dans le cytosol convertissant le citrate en isocitrate. Ces deux activites s'excluent mutuellement par un changement conformationnel de la proteine due a la presence ou non d'un centre 4fe-4s. Ce dernier est present au site actif de l'irp-1 lorsqu'elle se trouve sous la forme aconitase. Les modulateurs des activites de l'irp-1 sont la concentration de fer intracellulaire et l'activite no synthase. Nous avons en premier lieu, approfondi la nature de la (ou des) espece(s) oxydante(s) capable(s) de convertir l'irp-1 de sa forme aconitase vers sa forme trans-regulatrice. Nous avons montre que ni le peroxynitrite (onoo), ni les especes reactives oxygenees etaient capables de convertir l'irp-1 vers la forme trans-regulatrices bien qu'ils inhibent l'activite aconitase. Seul le no permet la conversion rapide de l'irp-1 en ciblant son centre fe-s. Afin d'expliquer l'effet curieusement discret du onoo, nous avons precise son mecanisme d'action in vitro. Ce dernier altere le centre fe-s de l'irp-1 jusqu'a sa destruction totale. A forte dose, son effet oxydant se prolonge en etablissant des ponts disulfure avec la cysteine 437 et d'autres cysteines proches du site actif formant ainsi une apo-irp-1 oxydee sans activite. Un environnement faiblement reducteur rend cette nouvelle forme d'irp-1 capable de lier les sequences ires. La seconde irp (irp-2) est denuee de centre fe-s et presente une activite trans-regulatrice constitutive qui est inhibee en presence de onoo et restauree par un agent reducteur. Nous avons egalement montre que son activite pouvait etre inhibee independemment du no dans les macrophages stimules avec l'ifn-.

Résumé : L’autophagie est un mécanisme constitutif et inductible de dégradation des composants cytoplasmiques afin de maintenir l’homéostasie cellulaire. Elle est souvent modulée par les virus car il s’agit également d’un mécanisme de défense antiviral. Elle peut avoir un rôle proviral quand elle est détournée et régulée par les virus. Nous avons précédemment observé au laboratoire que le cytomégalovirus humain (HCMV) stimule la formation des autophagosomes de manière précoce indépendamment de l’expression des protéines virales, puis qu’il entraine un blocage de l’autophagie aux temps tardifs. Dans ce travail, nous avons montré que ce virus a développé des stratégies impliquant la synthèse de deux protéines virales, IRS1 et TRS1, pour inhiber l’autophagie. De façon surprenante, nous avons également mis en évidence un rôle proviral de l’autophagie aux temps tardifs de l’infection par le HCMV. Nous avons pu montrer par des techniques de biochimie et d’imagerie cellulaire que l’expression aussi bien de TRS1 que d’IRS1 est capable de bloquer la formation des autophagosomes dans les cellules. Nous avons identifié le mécanisme d’action de ces protéines. Il est indépendant de la protéine kinase PKR mais nécessite une interaction avec Beclin 1, une protéine de la machinerie autophagique. Nous avons localisé le site d'interaction de Beclin 1 avec IRS1 et TRS1 (BBD pour Beclin 1 binding domain) au niveau de leur région N-terminale. Ce domaine, conservé entre les deux protéines, est nécessaire pour l’inhibition de l’autophagie. Le site d’interaction d’IRS1 a été identifié dans le domaine en superhélice (coiled-coil domain) CCD de Beclin 1. Nous avons caractérisé le rôle de TRS1 et IRS1 dans la modulation de l’autophagie dans le contexte de l’infection virale, en utilisant différents virus mutants : des virus dans lesquels on a supprimé soit le gène IRS1, soit le gène TRS1 et un virus dans lequel il manque les deux gènes IRS1 et TRS1. Les résultats obtenus suggèrent qu’IRS1 et TRS1 sont effectivement toutes les deux impliquées dans ce processus. Afin de mieux comprendre le rôle de l’interaction de ces protéines avec Beclin 1, nous avons étudié le phénotype d’un virus mutant qui n’exprime pas IRS1 et qui contient une délétion de la région BBD de TRS1. Nous avons montré que ce virus mutant ne se lie pas à Beclin 1 et qu’il ne bloque pas l’autophagie. De manière surprenante, il n’a pas de défaut de production virale, suggérant que l’inhibition de l'autophagie ne serait pas essentielle pour la réplication virale. Nous avons développé d’autres approches, comme l’utilisation de modulateurs pharmacologiques de l’autophagie ou de lentivirus hébergeant des shRNA, qui montrent que l’inhibition de l’autophagie est capable de diminuer la production virale et au contraire que sa stimulation l’augmente. Ces derniers résultats suggèrent que l’autophagie pourrait être bénéfique au HCMV dans certaines conditions
Abstract: Autophagy is a constitutive and inducible mechanism of degradation of cytoplasmic components, in order to maintain the cellular homeostasis. Autophagy is often modulated by viruses, because it is also considered as an antiviral defense mechanism. It can have a beneficial role, when it is hijacked and regulated by viruses. We have previously observed in our laboratory that the human cytomegalovirus (HCMV) stimulates autophagosome formation, at the early stage of infection, independently of viral protein expression then, later on, it blocks autophagy. In this work, we showed that this virus has developed strategies involving the synthesis of several viral proteins, such as IRS1 and TRS1, to inhibit autophagy. Surprisingly, we also demonstrated a proviral role of autophagy at late stages of infection with HCMV. We showed, through biochemical and cellular imaging technologies, that expression of both TRS1 and IRS1 is able to block the formation of autophagosomes. We identified the mechanism of action of these proteins. It is independent of the protein kinase PKR but requires interaction with Beclin 1, a protein of the autophagic machinery. We mapped the interaction site of Beclin 1 with IRS1 and TRS1 in their N-terminal region and called it BBD for Beclin 1-binding domain. This domain (BBD)is conserved between the two proteins and essential to inhibit autophagy. We also identified the site of interaction of IRS1 in the coiled-coil domain (CCD) of Beclin 1. We characterized the role of IRS1 and TRS1 in the modulation of autophagy, in the context of viral infection, using different mutant viruses: viruses in which either the IRS1 or the TRS1 gene has been removed and a mutant virus lacking both IRS1 and TRS1 genes. Our results suggest that both IRS1 and TRS1 are involved in the regulation of this process. To better understand the role of the interaction of these proteins with Beclin 1, we studied the phenotype of a mutant virus that does not express IRS1 and which contains a deletion of the N-terminal region of TRS1. We showed that this mutant does not bind to Beclin 1 and is not able to block autophagy. Surprisingly, it has no defects in viral production, suggesting that inhibition of autophagy is not essential for viral replication. We developed other approaches, including the use of pharmacological modulators of autophagy or shRNA knockdown, which show that the inhibition of autophagy is able to reduce viral production and, on the contrary, that its stimulation increases it. These results suggest that autophagy may be beneficial to HCMV in certain conditions

Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumour suppressor, involved in many diverse cellular processes including immune responses and growth regulation. An interesting feature of IRF-1 is that it can both activate and repress gene expression, possibly by acting with co-activator or co-repressor proteins. In a previous phage display assay, a homologous peptide to the known repressor protein, zinc finger 350 (ZNF350), was found to bind to the C-terminus of IRF-1. ZNF350, also known as ZBRK1 (Zinc finger and BRCA1-interacting protein with KRAB domain-1), is a member of the Krüppel-associated box (KRAB)-containing zinc finger (KZF) proteins, which is a group of the widely distributed transcriptional repression proteins in mammals. ZNF350 has previously been shown to repress the expression of a number of genes including ANG1 and GADD45A, often in complex with other proteins. This study confirms the direct interaction between IRF-1 and ZNF350 and identifies key residues, including the LXXLL repression motif within the C-terminus of IRF-1, necessary for the binding interface. The two proteins have additionally been shown to interact within a cellular environment, shown by using techniques including immunoprecipitation and a proximity ligation assay. In addition, the ZNF350/IRF-1 complex formation appears to occur in the basal state of the cell, as opposed to in response to cellular stress such as viral infection or DNA damage. On the basis of ZNF350 being a negative regulator of transcription, a novel technique was developed to identify putative targets of both ZNF350 and IRF-1. This involved an initial bioinformatics screen using candidate IRF-1 binding site data obtained from CENTIPEDE, an algorithm that combines genome sequence information, with cell-specific experimental data to map bound TF binding sites. This allowed for the identification of novel target genes that contained the ZNF350 consensus binding site, GGGxxCAGxxxTTT, within close proximity to an IRF-1 consensus site, such as the immune response gene IL-12A. Lastly, a peptide phage display screen was combined with high-throughput sequencing to identify other potential binding partners of ZNF350 and perhaps help to understand the mechanism by which transcriptional repression is controlled by complex formation.

This study analyses how Stargate SG-1 perpetuates dominant representations of whiteness, and how whiteness is used as a marker of racial identity in American popular culture. The popular science fiction television show Stargate SG-1 continually uses the nonwhite alien to juxtapose the seeming superiority of the white human, with white Americans acting as trusted gatekeepers for the entire planet. Whiteness becomes almost invisible and normative as the alien “other” requires assistance or containment enacted through SG-1’s adventures “off-world”. I also examine the representation of superior white aliens as an extension of these dominant white discourses. It is through the study of the constructed nature of “race” that whiteness is made visible. As represented in Stargate SG-1 whiteness discourses contribute to and reflect “common sense” constructions of race within U.S. society. This examination of Stargate SG-1 illuminates how negotiations of whiteness are constructed within United States dominant cultural discourses as a means to exclude the “other”.

Interferon Regulatory Factor -1 (IRF-1) is a transcription factor that acts as a tumour suppressor in cancer cells. The inactivation or deletion of IRF-1 either in one or both allele has been frequently reported in leukaemia and myelodysplasia (MDS). On the other hand nucleophosmin (NPM), a nucleo-cytoplasmic shuttling phosphoprotein is also known to be aberrant in some form of leukaemia. NPM was first proposed as a binding partner of IRF-1 in 1997 and suggested to inactivate IRF-1 by inhibiting its DNA binding ability. No further researches on the interaction between IRF-1 and NPM1 was reported prior to the start of my PhD. In the research presented here the interaction and mechanism by which IRF-1 might be inactivated by NPM was studied. Under the context of both NPM and IRF-1 being frequently associated with leukaemia and MDS, the study was done to determine the role of NPM under its naïve condition and a most frequent mutated condition (NPMc+), where the C-terminal of NPM was frequently mutated to give rise to a cytoplasmic NPM in certain leukaemia. In this current research, the direct interaction between IRF1 and NPM was further confirmed both in vitro as well as within the cells. Following this, the effect of this interaction in respect to the leukaemic condition having NPMc+ mutation was done, by comparing the end results on AML2 (leukaemic cells with intact wild type NPM) and AML3 (leukaemic cells having a single NPM allele mutated to form NPMc+) cells. In this research, overexpression of wild type NPM (NPMwt) was found to increase IRF-1 transcriptional activity. On further analysis, the DNA binding activity of IRF-1 due to the presence of NPMwt or NPMc+ was not always inhibited, instead it shows a change in binding specificity, where NPMwt bound IRF-1, lacks DNA binding ability and DNA bound IRF-1 has a reduced binding towards NPM. This is being studied further in terms of NPM overexpression and increased IRF-1 transcriptional activity, as the order of addition (order of interaction in vivo) plays a major role in activating or deactivating IRF-1. This along with the increased transcriptional activity of IRF-1 suggests a novel function of NPM, where it could act in favour of IRF- 1 activity. Additionally, the NPM induced change in IRF-1 localisation was confirmed by the cytoplasmic localised IRF-1 in NPMc+ expressing cells and nucleolar sequestration in NPMwt overexpressing cells. This gives a novel mechanism by which NPM regulates IRF-1. Further, the NPMc+ specific colocalisation of IRF-1 urges to study the other proteins that may have been re-localised in AML cells due to the NPMc+ specific interaction. A mass spectrometric analysis on the cellular distribution of total proteins were analysed between AML2 (cells with NPMwt) and AML3 (cells containing NPMc+). Specific proteins related to cancer have been identified to be differentially distributed rather than being a random translocation. With this being said, a peptide phage display technology coupled with next generation sequencing was done to identify NPMwt binding peptides that can be used in drug discovery process or as small molecule inhibitors or activators. Three different peptides were selected at the end of the study that bind very effectively to NPMwt. These peptide can either aid or restrict NPM activity and need to be validated and studied in the future.

Breast cancer is the most common malignancy among women in the U.S. While many successful treatments exist for primary breast cancer, very few are available for patients with metastatic disease. The purpose of this study was to understand the role of Insulin Receptor Subtrate-2 (IRS-2) in breast cancer metastasis. IRS-2 belongs to the IRS family of cytoplasmic adaptor proteins that mediate signaling from cell surface receptors, many of which have been implicated in cancer. Although the IRS proteins are highly homologous in structure and have some complementary functions, growing evidence supports that the IRS proteins have unique roles in cancer. IRS-1 has been shown to promote tumor cell proliferation, while IRS-2 has been positively associated with cancer cell invasion, glycolysis and tumor metastasis. In the current work, we identified IRS-2 as a novel hypoxia-responsive gene in breast carcinoma cells. In contrast, IRS-1 expression does not increase in response to hypoxia, supporting the notion of their non-overlapping functions. Hypoxia promotes the adaptation and resistance of cancer cells to chemo- and radiation therapy, and also promotes tumor cell survival, invasion and metastasis by selecting for aggressive tumor cells that can survive under stressful low oxygen conditions. We have shown that IRS-2 upregulation in response to hypoxia promotes Akt signaling and tumor cell viability and invasion. We identified a cell context-dependent role for Hypoxia Inducible Factor (HIF) in the regulation of IRS-2 expression in hypoxia, with HIF-2 playing a more dominant role than HIF-1. We also demonstrate that binding of Snail, a regulator of the EMT, to the IRS-2 promoter keeps the chromatin in an open conformation that is permissive for HIF-dependent transcription of IRS-2 in hypoxia. IRS-2 is not upregulated by hypoxia in well-differentiated epithelial-like carcinoma cells that do not express Snail, implicating IRS-2 gene expression as part of the EMT programming. In summary, we have identified an endogenous mechanism by which cancer cells can shift the balance of IRS-1 and IRS-2 to favor IRS-2 expression and function, which promotes survival, invasion, and ultimately metastasis. Understanding the mechanism of IRS-2 regulation by hypoxia may reveal new therapeutic targets for metastatic breast cancer.

The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic factors are all under the control of IRF-1, which effects both transcriptional activation and repression. In addition to these cell autonomous tumour suppressor activities, IRF-1 is also a key regulator of the immune system and, as such, mediates immune surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of IRF-1 is a key factor in several different types of cancer. Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions that this protein warrants further investigation as a drug target, very little is known about its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the development of autoimmune diseases, it is particularly important that drugs be able to decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects. This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1 domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies were developed as tools to study the C-terminal phosphorylation. Using these, it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the C-terminal Mf1 domain. Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory activity of IRF-1 at various promoters, but did not affect repressor activity. Gel shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding and suggested this was through increased interaction with the cofactor/histone acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding. Acetylation by p300 appears to be important although it is not yet clear whether this directly or indirectly affects IRF-1 activity. Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently suppressed colony formation in both anchorage dependent and independent assays, and may improve inhibition of invasion in Transwell assays. Thus, cell treatment with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective tumour suppression and inhibition of metastasis.

Une des caractéristiques des cellules cancéreuses réside dans la modification de leur capacité à répondre à divers stress par rapport à des cellules saines. La modulation afférente de l'expression de gènes peut se produire à deux niveaux : transcriptionnel et post-transcriptionnel. La plupart des efforts de compréhension ont été axés sur l'étude la régulation transcriptionnelle. Néanmoins, une façon efficace et rapide de modifier l'expression des gènes est la capacité de réguler le " pool d'ARNm " pré-existant en intervenant au niveau post-transcriptionnel. Aussi, les modifications de la stabilité de l'ARNm et/ou de l'efficacité de traduction dans un contexte particulier tel que le stress tumoral et faisant intervenir des interactions ARN/protéines sont de plus en plus étudiés dans le cas des cancers. Une compréhension plus approfondie de ces mécanismes permettra de mieux comprendre 1/ l'implication des protéines liant l'ARN (RBP) dans le développement tumoral et 2/ considérer le développement de thérapies ciblées contre le cancer. Nous nous sommes concentrés sur l'étude d'une RBP pertinente vis à vis du cancer, mais très peu étudiée sur cette problématique : la protéine Staufen1 (Stau1). Stau1 est un membre de la famille des protéines qui lient l'ARN double-brin. Cette RBP contribue à la régulation post-transcriptionnelle de nombreux gènes par son implication dans des mécanismes qui vont promouvoir le transport, la dé-répression de la traduction et l'induction de la dégradation d'ARN messagers (ARNm) spécifiques. Stau1 apparait également comme un régulateur spécifique de l'expression génique intervenant dans un contexte particulier de stress cellulaire, contexte familier aux tumeurs en cours de développement. Les transcrits régulés par Stau1 se classent dans un large éventail de catégories fonctionnelles et il est intéressant de noter qu'une grande proportion d'entre eux code pour des protéines impliquées dans la régulation de processus biologiques cellulaires critique dans le développement de cancers. De part son rôle de régulateur de l'expression génique et son implication dans la réponse au stress cellulaire, nous avons émis l'hypothèse que la modification de l'expression de Stau1 puisse avoir un impact à différent niveaux sur le développement et la progression tumorale. Ce projet de thèse s'est orienté sur deux axes 1) l'étude de l'expression de Stau1 en condition de stress cellulaire et 2) l'effet de la répression de Stau1 sur le développement tumoral
One of the characteristics of cancer cells lies in the modification of their ability to adapt to various stresses compared to healthy cells. The afferent modulation of gene expression can occur at two levels: transcriptional and post-transcriptional. Most of the efforts for understanding the gene expression process are focused on transcriptional regulation study. However, a quick and effective way to modify gene expression is the ability to regulate the "mRNA pool" by intervening in post-transcriptional level. Besides, changes in mRNA stability and/or translation efficiency in a context such as cellular stress in tumors and interactions involving RNA / protein are increasingly studied in the case of cancers. A deeper knowledge of these mechanisms will allow a better understanding of 1 / the involvement of RNA binding proteins (RBP) in tumor development and the 2 / the consideration of the development of targeted cancer therapies. We focused on the study of a relevant RBP for cancer development process, but very few studies have been undertaken on this issue: the Staufen1 protein (Stau1). Stau1 is a family member of double-stranded RNA-binding proteins. This RBP contributes to the post-transcriptional regulation of many genes through its involvement in mechanisms that will promote the transport, the derepression of translation and the induction of degradation of specific RNA transcripts (mRNA). Stau1 also appears as a regulator of specific genes expressed in respond to cellular stress induced by an unfavorable tumor microenvironment. The transcripts regulated by Stau1 can be divided into a wide range of functional categories. Interestingly, a large proportion of Stau1 targets encode proteins which regulate many critical biological cell processes in cancer development. Regarding Stau1 regulatory role and its involvement in the response to cellular stress, we made assumptions that the modification of Stau1 expression could have an impact on various levels of development and tumor progression. This project will be considered from two aspects 1/ the study of Stau1 expression under cellular stress 2/ the impact of Stau1 repression on tumor development

H-REV107-1 wird in normalen Geweben ubiquitär exprimiert, während die Expression in humanen Mamma-, Ovarial-, und Lungentumoren unterdrückt ist. H-REV107-1 hemmt das Tumorwachstum in vitro und in vivo. Die Expression und Regulation des H-REV107-1 Gens wurde in verschiedenen, humanen Zelllinien untersucht. In Tumor Zelllinien wird die H-REV107-1 Expression durch IFNgamma induziert Eine Korrelation der H-REV107-1 und der IRF1 Expression nach Induktion mit IFNgamma wurde gezeigt. H-rev107-1 konnte nach konditionaler IRF1 Expression, Proteinsynthese-unabhängig, nachgewiesen werden und ist ein direktes Zielgen von IRF1. Die H-rev107-1 Expression ließ sich durch Unterdrückung des MEK/ERK Signalwegs mit dem MEK1 Inhibitor PD98059 aktivieren. Dies bedeutet, dass H-REV107-1 durch mindestens zwei verschiedene Signalwege, IFNgamma und MEK/ERK, reguliert wird. Der in vitro amplifizierte H-REV107-1 Promoter enthält keine TATA-Box, sondern ein Initiator Element sowie, in dem für eine TATA-Box definierten Abstand, eine ATF2 Bindungsstelle. Die Inkubation von transient transfizierten Zellen mit TNF alpha, cAMP und IFN gamma steigerte die Luciferase Aktivität. Mit Hilfe von Deletions- und Mutationskonstrukten wurden die regulatorischen Bereiche des Promoters bestimmt. Eine Mutation der cRel-Bindungsstelle, potentiell über NFkappaB reguliert, resultierte in einer Luciferase Aktivität von nur 9% des Wildtyp Promoters. Die Mutation der CREB/ATF2 Bindungsstelle reduzierte die Luciferase Aktivität auf 37%. Die Ko-Transfektion eines NFkappaB Suppressor reduzierte die Luciferase Aktivität um 53%. Diese Ergebnisse legen nahe, dass NFkappaB und ATF2 die H-REV107-1 Expression positiv regulieren. In einem EMSA wurde die Bindung von ATF2 an die CREB/ATF-2 Bindungsstelle gezeigt. Die Ergebnisse lassen vermuten, dass H-REV107-1 durch eine IFNgamma induzierte, möglicherweise PKR vermittelte, Signalkette von den Faktoren IRF1 und ATF2 direkt, sowie von NFkappaB indirekt, reguliert wird.
The H-REV107-1 class II tumor suppressor gene is ubiquitously expressed in normal tissues and down regulated in human breast, ovarian and lung tumors. H-REV107-1 has the capacity to suppress growth of tumor cells in vitro and in vivo. H-REV107-1 is up regulated after treatment with IFN gamma. A NIH3T3 cell line harboring an estrogen inducible IRF.1/hER fusion protein showed a protein synthesis independent up regulation of H-rev107-1 expression after induction of IRF-1. H-rev107-1 is a direct target of IRF-1. Inhibition of the MEK/ERK pathway, using the MEK1 inhibitor PD 98059, leads to a restored expression of H-rev107-1. Therefore, H-REV107-1 can be a target of the MEK/ERK-pathway. Thus, H-REV107-1 is regulated by at least two different pathways. To understand the regulatory mechanisms of the expression of the H-REV107-1 gene, the putative promoter region was analyzed in silico. The sequence was amplified and cloned. Induction of the promoter constructs with TNF alpha, cAMP and IFN gamma increased the luciferase activity. Several deletions constructs and constructs with putative transcription factor binding sites mutated were used to narrow down the important regulatory elements of the promoter. The mutations of a cRel binding site and a CREB/ATF-2 binding site decreased the luciferase activity by 91% and 63%, respectively. Co transfection of the full length promoter construct with a repressor of NFkappaB activation, reduced the luciferase activity to 47%. As a result of the investigation H-REV107-1 is directly regulated by IRF-1 and probably indirectly regulated by NFkappaB and the MEK/ERK signaling pathway. In an Electro Mobility Shift Assay (EMSA), the binding of ATF-2 to the CREB oligonucleotid was demonstrated by the use of a specific antibody. The ATF.2 binding site in the posititon –30 bp - 23 bp of the human, TATA-less H-REV107-1 promoter replaces the TATA-like element, which can be found in the H-rev107-1 promoter of rat and mouse.