Orientadores: Eduardo Rochete Ropelle, José Rodrigo Pauli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T03:45:50Z (GMT). No. of bitstreams: 1 Lenhare_Luciene_M.pdf: 1233501 bytes, checksum: 008c68d0add97d05e569e31f1aa030f4 (MD5) Previous issue date: 2014 / Resumo: SIRT1 são proteínas NAD+ dependentes que formam uma classe de proteínas com atividade desacetilase, que exercem um papel crítico na biogênese mitocondrial, homeostase da glicose e sensibilidade à insulina. No entanto o mecanismo pós-translacional de controle da atividade da SIRT1 permanece obscuro. No presente estudo investigou-se o papel da enzima Óxido Nítrico Sintase (iNOS) na S-nitrosação da SIRT1 no músculo de camundongos idosos. Primeiro identificou-se alto níveis da proteína iNOS no músculo esquelético de animais envelhecidos, tal fato foi acompanhado pelo aumento da S-nitrosação da SIRT1 e aumento da acetilação da PGC1? e FOXO1 levando ao comprometimento da biogênese mitocondrial. Foi demonstrado que o tratamento com óxido nítrico (NO) induziu a S-nitrosação da SIRT1 e reduziu a sua atividade desacetilase. Curiosamente, os camundongos knockout iNOS exibiram baixos níveis de S-nitrosação da SIRT1 e uma maior biogênese mitocondrial durante o envelhecimento, quando comparado com os camundongos do tipo selvagem. Finalmente, foi demonstrado que a S-nitrosação da SIRT1 é um mecanismo reversível, uma vez que a administração do inibidor farmacológico da iNOS (L-NIL) foi capaz de reduzir a S-nitrosação da SIRT1 e aumentar a atividade da SIRT1 incluindo a atividade dos seus substratos PGC1?, FOXO1 e AMPK no músculo de camundongos idosos. Coletivamente, o nosso estudo fornece evidências substanciais que a iNOS induz a S-nitrosação da SIRT1 e está associada com a diminuição da função mitocondrial no músculo esquelético de camundongos idosos / Abstract: SIRT1, an NAD + deacetylase -dependent, exerts a critical role on mitochondrial biogenesis, glucose homeostasis and insulin sensitivity. However, the post-translational mechanism that SIRT1 controls the activity remains unclear. Here we investigate the role of Nitric Oxide Sythase (iNOS) on SIRT1 S-nitrosation in muscle of old mice. First we identify high levels of iNOS protein levels the skeletal muscle of aged mice and it was accompanied by the augment of SIRT1 S-nitrosation, PGC1? and FOXO1 acetylation and impairment of mitochondrial biogenesis. We demonstrated that nitric oxide (NO) donor treatment induced SIRT1 S-nitrosation and it reduced deacetylase activity. Interestingly, iNOS knockout mice exhibited low levels of SIRT1 S-nitrosation and higher mitochondrial biogenesis during aging, when compared to wild type mice. Finally, we that demonstrated SIRT1 S-nitrosation is a reversible mechanism, once the pharmacological iNOS inhibitor (L-NIL) administration, reduced SIRT1 S-nitrosation and increase the activity of the SIRT1 substrates, including, PGC1?, FOXO1 and AMPK in muscle of old mice. Collectively, our study provides substantial evidences the SIRT1 induces iNOS S-nitrosation and it is associated with the impairment of mitochondrial function in the skeletal muscle of aged mice / Mestrado / Clinica Medica / Mestra em Clínica Médica

Abstract Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of energy metabolism and mitochondrial biology in high-energy cell types and tissues. The regulation of PGC-1α is versatile, and both transcriptional and post-transcriptional mechanisms play major roles. External stimuli affect PGC-1α-regulation which in turn adapts cellular signals to meet them. For example, conditions like fasting and diabetes mellitus (DM) are known to activate PGC-1α expression in the liver, resulting in enhanced de novo glucose production, gluconeogenesis. In the present study, the mechanisms of hepatic PGC-1α regulation and PGC-1α-regulated functions were elucidated. We found that PGC-1α was induced by oral type 2 diabetes therapeutic metformin, via AMPK and SIRT1, regulating the mitochondrial gene response, against previous assumptions. Simultaneously, gluconeogenesis was repressed by other means. Furthermore, PGC-1α upregulated the anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). PGC-1α also diminished interleukin 1β-mediated inflammatory response in hepatocytes. Novel, xenobiotic and endobiotic metabolizing Cytochrome P450 enzymes regulated by PGC-1α were also identified in this thesis. CYP2A5 was induced by PGC-1α through hepatocyte nuclear factor 4α (HNF-4α) coactivation. Also, vitamin D metabolizing CYP2R1 and CYP24A1 were identified as novel genes regulated by PGC-1α, suggesting a role for PGC-1α in the regulation of active vitamin D levels. The findings presented in this thesis provide insight into the pathology of glucose perturbations such as type 2 diabetes, and stimulate discovery of therapeutic agents to treat this disease. Furthermore, the findings suggest that vitamin D metabolism and energy metabolism are tightly linked, with PGC-1α emerging as a novel mediator. / Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori γ:n koaktivaattori 1α (PGC-1α) on merkittävä glukoosiaineenvaihdunnan ja mitokondrioiden toiminnan säätelijä korkeaenergisissä soluissa ja kudoksissa. PGC-1α:a säädellään monin tavoin: sekä transkriptionaalisella säätelyllä että transkription jälkeisellä muokkauksella on merkittävä rooli. Monet ulkoiset tekijät säätelevät PGC-1α:n aktiivisuutta, joka puolestaan säätelee solunsisäisiä signaalireittejä vastaamaan tähän signaaliin. Esimerkiksi paasto ja diabetes mellitus (DM) ovat fysiologisia tiloja, jotka lisäävät voimakkaasti PGC-1α:n ilmentymistä maksassa, jolloin glukoosin uudistuotanto eli glukoneogeneesi kiihtyy. Tässä väitöskirjassa tutkittiin PGC-1α:n säätelyä sekä PGC-1α -säädeltyjä signaalireittejä maksassa. Osoitimme, että tyypin 2 diabeteslääke metformiini indusoi PGC-1α:n ilmentymistä maksassa, vastoin aikaisempia käsityksiä. PGC-1α indusoitui AMPK:n ja SIRT1:n välityksellä, säädelleen edelleen mitokondriaalisten geenien aktiivisuutta. Samalla glukoneogeneesi kuitenkin repressoitui muilla mekanismeilla. Lisäksi osoitimme, että PGC-1α indusoi tulehdusreaktiota vaimentavaa interleukiini 1 reseptorin antagonistia (IL1Rn). PGC-1α esti interleukiini 1β:n aiheuttamaa tulehdusvastetta hepatosyyteissä. Lisäksi väitöskirjassa tunnistettiin uusia, PGC-1α -säädeltyjä lääkeaineita ja elimistön sisäisiä yhdisteitä metaboloivia sytokromi P450 -entsyymejä (CYP). Hiiren CYP2A5:n ilmentymisen osoitettiin olevan PGC-1α- ja HNF4α-välitteistä. Lisäksi osoitettiin, että D-vitamiinia metaboloivat CYP2R1 ja CYP24A1 ovat uusia PGC-1α -säädeltyjä geenejä. Tämä löydös viittaa siihen, että PGC-1α:lla on rooli aktiivisen D-vitamiinin säätelyssä. Tämän väitöskirjan löydökset lisäävät tietoa glukoosiaineenvaihdunnan häiriöiden kuten tyypin 2 diabeteksen molekulaarisista mekanismeista, joita voidaan hyödyntää mahdollisten uusien lääkeaineiden kehittämisessä. Lisäksi väitöskirjassa osoitettiin, että D-vitamiinimetabolia on kytköksissä energia-aineenvaihduntaan ja että PGC-1α:lla on tässä rooli, jota ei aiemmin ole tunnettu.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Faustová Supervisor: Prof. Doutor Jorge Miguel de Ascenção Oliveira PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: The impact of SIRT1 inhibition on zebrafish morphology and behavior After discovery of connection between yeast Silent Information Regulator 2 (Sir2) and its ability to alter lifespan, Sir2 and its seven mammalian orthologs became very attractive therapeutic target. These so called sirtuins are members of a histone deacetylase family. They possess unique catalytic activity having nicotinamide adenine dinucleotide as a cofactor and their function can be influenced by environmental factors. The aim of this diploma thesis was to extend knowledge of Sirtuin 1 (SIRT1), which is from all mammalian sirtuins considered to have the closest relation to yeast Sir2. At first we tested the impact of SIRT1 inhibition on early developmental stages of zebrafish (Danio rerio) embryos and larvae, finding out that SIRT1 is important for normal development and SIRT1 inhibition or malfunction result in cardiovascular defects, delayed development, and death. Additionally, we tried to learn more about SIRT1 and its connection with Parkinson's disease by combining nontoxic doses...

Background: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by increased pulmonary artery pressure, leading to right ventricle hypertrophy and ultimately heart failure and death. Sirtuin 1 (SIRT1) is an NAD+ dependent protein deacetylase that has been strongly implicated as a crucial link between longevity, stress response and maintenance of vascular health. In this thesis, we investigated the role of SIRT1 in the pulmonary vascular hypoxic response and the pathogenesis of pulmonary hypertension (PH) working under the hypothesis that SIRT1 plays a protective role in the pulmonary vasculature and that lack of SIRT1 would lead to worsening of PH in a model of chronic hypoxia (CH). Results: We determined that global SIRT1 knockout or SIRT1 catalytic inactivation resulted in a marked increase in right ventricle pressure and remodeling compared to wildtype mice in CH. Furthermore, hypoxia-induced erythrocytosis and pulmonary vascular remodeling were profoundly increased in both SIRT1 mouse lines. Subsequent molecular assessment revealed that SIRT1 knockout, but not inactivation, led to a significant increase in mRNA levels of hypoxia inducible factor (HIF)-1α and significantly higher activity in hypoxia, leading to elevated lactate dehydrogenase A (LDHA) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) in the lungs. Interestingly, both knockout and inactivation of SIRT1 enhanced the activity of HIF2α in the hypoxic lungs and kidneys, leading to increased erythropoietin (EPO) and plasminogen activator inhibitor-1 (PAI-1). Moreover, SIRT1 knockout or inactivation was associated with a trend towards hypoxic-independent increases in HIF3α mRNA in the lungs. Prevention of glycolytic shift using dichloroacetate (DCA) did not result in improvement in this model, yet resveratrol (RSV), a SIRT1 activator/mimic, partially prevented PH only in absence of SIRT1 activity. Finally, selective endothelial cell SIRT1 deletion was sufficient to cause worse PH in the CH model. Conclusions: SIRT1 plays a protective role in the hypoxic response through transcriptional and non-transcriptional control of the hypoxia inducible factors, thus protecting against worse hypoxia-induced PH. SIRT1 could be a novel target for future therapies in PAH.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18882号 / 医博第3993号 / 新制||医||1009(附属図書館) / 31833 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮本 享, 教授 小泉 昭夫, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR‐181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV‐infected individuals compared with age‐ and sex‐matched healthy subjects. Mechanistic studies revealed that up‐regulation of transcription factor ΔNp63 led to the decline of miR‐181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV‐infected individuals. Either reconstituting miR‐181a or silencing ΔNp63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence‐associated β‐galactosidase (SA‐β‐gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV‐induced T cell senescence is counterregulated by the ΔNp63–miR‐181a–Sirt1 pathway. An increase of IL‐2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3− effector T (Teff) cells upon manipulating the ΔNp63–miR‐181a–Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

Yes / Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3′ end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3′-DNA/ds siRNA and includes a 3′-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3′-DNA/ds siRNA, (II) passenger-3′-DNA/ds siRNA, (III) guide-3′-DNA/ss siRNA, and (IV) passenger-3′-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3′-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3′-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3′-DNA/siRNA constructs are discussed.

Sorafenib is a multi-kinase inhibitor and one of the few systemic treatment options for patients with advanced hepatocellular carcinomas (HCCs). Resistance to sorafenib develops frequently and could be mediated by the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin (SIRT)1. We aimed to test whether sorafenib efficacy is influenced by cellular NAD levels and NAD-dependent SIRT1 function. We analyzed sorafenib effects on apoptosis induction, NAD salvage, mitochondrial function, and related signaling pathways in HCC cell lines (HepG2, Hep3B, und HUH7) overexpressing SIRT1 or supplemented with the NAD metabolite nicotinamide mononucleotide (NMN) compared to controls. Treatment of HCC cell lines with sorafenib dose-dependently induced apoptosis and a significant decrease in cellular NAD concentrations. The SIRT1 protein was downregulated in HUH7 cells but not in Hep3B cells. After sorafenib treatment, mitochondrial respiration in permeabilized cells was lower, citrate synthase activity was attenuated, and cellular adenosine triphosphate (ATP) levels were decreased. Concomitant to increased phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), sorafenib treatment led to decreased activity of the mechanistic target of rapamycin (mTOR), indicative of energy deprivation. Transient overexpression of SIRT1, as well as NAD repletion by NMN, decreased sorafenib-induced apoptosis. We can, therefore, conclude that sorafenib influences the NAD/SIRT1/AMPK axis. Overexpression of SIRT1 could be an underlying mechanism of resistance to sorafenib treatment in HCC

No description available.

Obesity is a rapidly growing epidemic. It is associated with preventable chronic disease and vast healthcare cost in the United States (about 200 billion per year). Therefore, dissecting pathogenic mechanisms of obesity would provide effective strategies to prevent its development and reduce related cost. Obesity is characterized by excessive expansion of white adipose tissue (WAT). Autophagy, a cellular self-digestive process, is associated with WAT expansion and may be a promising target for combating obesity. Both hormone signaling (e.g., ERα) and energy sensing factors (e.g., Sirt1) control metabolism and prevent adiposity, and in which they have been shown to play collaborate roles. However, how autophagy is involved in ERα and Sirt1's inhibitory roles on adiposity is unknown. These questions have been addressed in my dissertation studies. To address this fundamental questions, I have established a method to monitor autophagy flux during adipocyte differentiation, which better reflected the dynamic process of autophagy. Compared with preadipocytes, autophagy flux activity was increased in mature adipocytes after differentiation. And then, my thesis project has addressed three main questions. Firstly, the gender difference in visceral fat distribution (Males have higher deposit of visceral fat than females) is controlled by an estradiol (E2)-autophagy axis. In C57BL/6J and wild type control mice, a higher visceral fat mass was detected in the males than in the females, which was associated with lower expression of estrogen receptor  (ER) and more active autophagy in males vs. females. ER knockout normalized this difference. Mechanistically, E2-ER- mTOR-ULK1-autophagy signaling contributed to the gender difference in visceral fat distribution. Secondly, in vitro and in vivo studies demonstrated that Sirt1 suppressed autophagy and reduced adipogenesis and adiposity via inducing mTOR-ULK1 signaling. Specific activation and overexpression of Sirt1 induced mTOR-ULK1 signaling to suppress autophagy and adipogenesis. And knockdown of Sirt1 exhibited opposite effects. The first and second studies revealed that ER and Sirt1 acted on mTOR-ULK1 signaling pathway, underlying the importance of their interaction in inhibiting autophagy and adipogenesis. As such, the third study was conducted and it unraveled that ER acted as upstream of Sirt1, possibly through its direct binding to Sirt1 promoter. Specifically, E2 signaling suppressed autophagy and adipogenesis. But when Sirt1 was knockdown, the effects of E2 on autophagy and adipogenesis were abolished. Taken together, my dissertation project underscores the importance for future research to consider gender difference and how E2-ER-autophagy axis contributes to this difference in other metabolic diseases. Also, the unraveled interaction between ERα and Sirt1 might lead to new therapeutic approach to adiposity and metabolic dysfunction in post-menopausal women or individuals with abnormal estrogen secretion. For example, dietary intervention or exercise challenge to activate Sirt1 may partially compensate estrogen deficiency. / Doctor of Philosophy / Obesity is a rapidly growing epidemic, which is associated with chronic disease and vast healthcare cost in the United States. Understanding the pathogenic mechanism of obesity is of critical importance. Recent studies have implicated autophagy, a cellular self-digestive process, in WAT development and expansion. It was also shown that hormone (e.g., via estrogen receptor ERα) and energy (e.g., via Sirt1) signaling control metabolism and adiposity. However, it is unclear whether and how autophagy interacts with ERα and Sirt1 in the regulation of adiposity. My dissertation project unraveled the mechanism of how hormone signaling (e.g., ERα) and energy sensing factors (e.g., Sirt1) interacted with autophagy to control adipogenesis and adiposity. My thesis project has addressed three main questions. Firstly, the gender difference in visceral fat distribution (Males have higher deposit of visceral fat than females) is controlled by an estradiol (E2)-autophagy axis, ER knockout normalized this difference. Mechanistically, E2- ER- mTOR-ULK1-autophagy signaling contributed to the gender difference in visceral fat distribution. Secondly, in vitro and in vivo studies demonstrated that Sirt1 induced mTOR-ULK1 signaling, suppressed autophagy and reduced adipogenesis and adiposity (ER similar effects). As such, the third study was conducted and it unraveled that ER acted as upstream of Sirt1, possibly through its direct binding to Sirt1 promoter. Taken together, my dissertation study has explored how hormone signaling (ER) and energy signaling (Sirt1) interact with autophagy to control adipogenesis and adiposity individually and collaboratively, which may provide new therapeutical approach to control obesity.