Orientador: Gabriel Forato Anhê / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T14:26:05Z (GMT). No. of bitstreams: 1 Mesquita_CarolineCosta_D.pdf: 2692158 bytes, checksum: 5f07d43267a2e976f9b9818ec63ba452 (MD5) Previous issue date: 2015 / Resumo: Os ritmos circadianos de ingestão alimentar se estabelecem com objetivo de manter a homeostasia de nutrientes no meio celular, frente a variações intrínsecas ao ciclo claro/escuro. Neste sentido, a gliconeogênese de roedores é suprimida no período noturno, no qual há ocorrência de um surto alimentar bifásico que compreende aproximadamente 90% de todo o aporte calórico diário. Estes eventos apresentam, entre si, uma relação causal, onde o próprio aumento dos nutrientes circulantes, principalmente a glicose, controla a gliconeogênese. O padrão inverso é observado na fase clara do ciclo claro/escuro. Recentes estudos têm demonstrado que, a ativação farmacológica de vias da Unfolded Protein Response (UPR), no sistema nervoso central, resulta em resistência à ação anorexigênica da insulina e, consequentemente, aumento da ingestão alimentar através de um mecanismo não completamente esclarecido. A UPR é uma resposta celular adaptativa que atenua a taxa de tradução de mRNAs, aumenta a proteólise e, deste modo, recupera o fenótipo celular. Esta reposta, quando ativada cronicamente, pode resultar em morte celular programada e resistência à insulina. No entanto, ainda não estava claro se a via do ATF6 da UPR tem, de fato, uma relação com o ritmo alimentar. Para responder a esse questionamento, realizamos análises da variação circadiana das proteínas envolvidas na via da UPR, imunoprecipitações com animais adrenalectomizados, e aplicação de dexametasona subcutânea. Os resultados demonstram que o ATF6 teve um aumento noturno atingindo o máximo de transição da fase clara para fase escura. A partir desse fato, foram realizados ensaios de imunuprecipitações em ratos adrenalectomizados para evidenciar as associações dos complexos CRTC2/ATF6 e CRTC2/CREB1. Contudo, nossos dados suportam a hipótese que os níveis fisiológicos de glicocorticóides podem reprimir a expressão CRH e estimular a ingestão de alimentos, através de uma via dependente de ATF6. Estes eventos, por consequência, poderão reduzir a atividade transcricional do CREB1, sobre o CRH, corroborando com os dados da literatura que apontam que os glicocorticóides endógenos dos roedores (principalmente a corticosterona) exercem um conhecido papel no controle da ingestão alimentar, decorrente principalmente da modulação da expressão do neurotransmissor anorexigênico CRH / Abstract: The circadian cycles of food intake have an important role in the homeostasis of cellular environment, acting over intrinsic changes to the sleep/wake cycle. Therefore, mice gluconeogenesis is suppressed at night where a food outbreak, responsible by 90% of all daily caloric ingestion, occurs. These events are connected by a causal relation, where the current nutrient increasing, mostly glucose, controls gluconeogenesis. The oppose pattern is verified during the light stage of the sleep/wake cycle. Recent studies have indicated that pharmacological activation of Unfolded Protein Response (UPR) pathways, at central nervous system, implies in resistance to the anorectic insulin effect, and, consequently, increase of the food intake activity trough a not fully explained engine. UPR is an adaptive cellular response that reduces mRNA¿s transcriptional rate, increases proteolysis, and therefore, restores cellular phenotype. This response, when constantly enabled, may induce cellular death and insulin resistance. However, it was not clear yet if the UPR¿s ATF6 pathway has, indeed, a connection with the feed rhythm. To answer to this question, we did several analysis of the circadian variation of the proteins connected to the UPR¿s pathway, adrenalectomized animals immunopreciptations and subcutaneous dexamethasone applications. The results demonstrated that ATF6 has a nightly increase and has reached the maximum of transcription rate from the light to the dark cycle. From this point, it was made immunopreciptations tests in adrenalectomized mice to evidence the association between CRTC2/ATF6 and CRTC2/CREB1 complexes. However, our data support the hypothesis that the physiological levels of glucocorticoids may suppress CHR expression and stimulate food intake trough an ATF6 dependent pathway. Those events, therefore, may reduce CREB1¿s transcriptional activity, confirming other studies data that indicates that endogenous mice glucocorticoids (mainly corticosterone) play an well-known role in food intake control, mainly due from modulation of the expression of the anorectic neurotransmitter CRH / Doutorado / Farmacologia / Doutora em Farmacologia

The endoplasmic reticulum (ER) facilitates the synthesis, assembly and quality control of all secretory, transmembrane, and resident proteins of the endomembrane system. An accumulation of unfolded proteins or a disruption in the specialized folding environment within the organelle causes ER stress, thus impairing the folding capacity of the ER. In response to this stress, the ER initiates a signaling cascade called the unfolded protein response (UPR) in an attempt to restore ER homeostasis. The vertebrate UPR is propagated by three ER-resident transmembrane proteins (i.e., PERK, IRE1α, and ATF6α), each initiating a signaling cascade that ultimately culminates in production of a transcriptional activator. The UPR was originally characterized as a pathway for the upregulation of ER chaperones, and a comprehensive body of subsequent work has shown that protein synthesis, folding, oxidation, trafficking, and degradation are all transcriptionally enhanced by the UPR. However, UPR activation is also accompanied by extensive mRNA suppression. The mechanisms responsible for this suppression and its consequences for physiological processes beyond the realm of ER protein folding and processing are only now beginning to be described. The overall goal of my thesis work was to explore this process of UPR-mediated gene suppression by identifying the mechanisms involved and the cellular processes affected. As a result, I characterized a novel mechanism of UPR-mediated transcriptional repression involving the translational regulation of the transcription factor C/EBPβ resulting in the suppression of the gene Il4ra, encoding an essential subunit of the IL-4/IL-13 receptor. As a consequence of this suppression, a novel effect of ER stress was identified in the impairment of IL-4/IL-13 signaling, a finding of potential significance in the study of inflammatory disease. In addition to this mechanism, I validated a novel approach to the identification of UPR-regulated transcription factors using publically available bioinformatic software. Through this analysis, I identified the transcription factor HNF4α as a novel post-translational UPR-regulated transcription factor, the regulation of which, resulted in the suppression of a number of lipid metabolic genes. This analysis not only identified a novel UPR-regulated transcription factor, but also presented a new tool for the characterization of UPR-mediated gene suppression. My work represents an independent and original investigation into the process of UPR-mediated gene suppression; and reveals that the UPR facilitates transcriptional suppression through the transcriptional, translational, and post-translational regulation of multiple transcription factors, resulting in the coordinated attenuation of physiological pathways. This function of the UPR is likely to contribute to metabolic, inflammatory, and other chronic disease states.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20059号 / 薬科博第66号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Combining the nematode Caenorhabditis elegans with novel microfluidic technology has produced a phenotypic movement assay that is at once rapid, sensitive, and low-cost. The method is based on the neurophysiologic phenomenon of worms exhibiting robust, continuous, directed locomotion in response to mild electric fields inside a microchannel. As we demonstrate with the studies reported herein, our microfluidic electrotaxis platform is a unique tool for studying the effects of environmental and genetic manipulations on C. elegans’ movement behaviour, which in turn indicates the state of the organism’s neuronal and muscular systems. In one initiative to develop an inexpensive biosensor, we use the setup to measure the response of worms to common environmental pollutants. Results indicate that worms’ electrotactic swimming behaviour is particularly susceptible to metal salts. A comparison with traditional assays measuring fecundity, growth, and lifespan reveals that electrotactic speed shows a comparable level of sensitivity as a toxicity endpoint. Another study demonstrates that worms expressing a mutant form of α-synuclein, a familial Parkinson’s disease-related protein, show deficits in electrotactic swimming speed that coincide with dopaminergic neuron damage. We further show that both the electrotaxis and neuronal phenotypes can be ameliorated by treatment with curcumin, a putative neuroprotective agent. We have also used the platform to investigate the effects of other environmental and genetic stresses on electrotactic behaviour. Our findings indicate that the response can withstand many different insults but is affected by stresses that induce the mitochondrial and ER unfolded protein responses, which themselves play roles in preserving electrotactic swimming behaviour alongside the heat shock response. These data expand our knowledge of how the motor output component of C. elegans’ electrotactic response is perturbed by environmental and genetic manipulations, and also support the utility of microfluidic electrotaxis as a functional output of nematode locomotory circuits in a multitude of contexts. / Thesis / Doctor of Science (PhD)

Cholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host quality control mechanism called ER-associated degradation (ERAD) that facilitates the translocation of misfolded proteins into the cytosol for degradation. Cytosolic CTA1, however, escapes this fate and is then free to activate its target, heterotrimeric G-protein subunit alpha (Gsα), leading to adenlyate cyclase (AC) hyperactivation and increased cAMP concentrations. This causes the secretion of chloride ions and water into the intestinal lumen. The result is severe diarrhea and dehydration which are the major symptoms of cholera. CTA1’s ability to exploit vesicle-mediated transport and ERAD for cytosolic entry demonstrates a potential link between cholera intoxication and a separate quality control mechanism called the unfolded protein response (UPR), which up-regulates vesicle-mediated transport and ERAD during ER stress. Other toxins in the same family such as ricin and Shiga toxin were shown to regulate the UPR, resulting in enhanced intoxication. Here, we show UPR activation by CT, which coincides with a marked increase in cytosolic CTA1 after 4 hours of toxin exposure. Drug induced-UPR activation also increases CTA1 delivery to the cytosol and increases cAMP concentrations during intoxication. We investigated whether CT stimulated UPR activation through Gsα or AC. Chemical activation of Gsα induced the UPR and increased CTA1 delivery to the cytosol. However, AC activation did iv not increase cytosolic CTA1 nor did it activate the UPR. These data provide further insight into the molecular mechanisms that cause cholera intoxication and suggest a novel role for Gsα during intoxication, which is UPR activation via an AC-independent mechanism

<p> Cellular cholesterol homeostasis is a fundamental and highly regulated process. Transcription factors known as sterol regulatory element binding proteins (SREBP) are responsible for the expression of many genes involved in the uptake and biosynthesis of cholesterol. SREBP activation and lipid dysregulation has been associated with cellular endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). Our lab has previously reported a relationship between ER stress and SREBP activation causing lipid dysregulation and hepatic steatosis. This project was designed to elucidate the mechanism of ER stress-induced SREBP activation and determine its relationship with cellular pathologies associated with ER stress and lipid accumulation. My research has examined the mechanism by which ER stress activates SREBP-2 in various cell lines, including epithelial and macrophage cells. This research revealed that (1) ER stress-induced SREBP-2 activation is not dependent on caspases and occurs through the conventional sterol-mediated proteolytic pathway; (2) the mechanism of ER stress-induced SREBP-2 activation is sensitive to changes in ER calcium; (3) ER stress is associated with SREBP-2 activation and lipid dysregulation in a model of renal injury; and ( 4) ER stress-induced SREBP activation in vitro is not associated with lipid accumulation in macrophage foam cells. </P> <p> This project has also offered me the opportunity to further enhance our understanding of the mechanism by which ER stress causes SREBP activation in a sterolindependent manner. </P> / Thesis / Doctor of Philosophy (PhD)

<p>Atherosclerosis is a chronic inflammatory disease of the arterial wall and is the primary cause of coronary artery disease, the most common cause of death in western societies. Risk factors for cardiovascular disease include dyslipidemia, diabetes, smoking, and obesity. These risk factors have also been shown to promote vascular endoplasmic reticulum (ER) stress; a cellular response characterized by the accumulation of misfolded proteins in the ER. Thickening and decreased stability of arterial plaque can lead to thrombosis and subsequent clinical complications of myocardial infarction and stroke. However, the exact mechanisms that lead to the development of atherosclerosis remain unclear. Here we show that inhibition, as well as a deficiency of glycogen synthase kinase (GSK)-3α, can protect against accelerated atherosclerosis in a low-density lipoprotein receptor (LDLR) knockout mouse model. Compared to LDLR^-/- controls, mice deficient in GSK-3α showed a decrease in lesion volume in the aortic root as well as protection against diet-induced hepatic steatosis. In addition, necrotic core volume was significantly reduced in LDLR^-/-GSK-3α^-/- mice compared to controls, a characteristic indicative of advanced plaque formation. Furthermore, hepatic and vascular ER stress levels were unaffected by the deletion of GSK-3α, a result that is consistent with the hypothesis that GSK-3α functions downstream of ER stress. Macrophages isolated from GSK-3α deficient mice had a reduction in unesterified cholesterol accumulation as well as a significant increase in the expression of the anti-inflammatory cytokine IL-10. Finally, BMT experiments showed a significant decrease in plaque size in the aortic sinus of LDLR^-/-GSK-3α^+/+ mice transplanted with GSK-3α deficient bone marrow. These results demonstrate a possible link between ER stress-induced activation of GSK-3α and the downstream effects leading to atherogenic initiation and progression.</p> / Master of Science (MSc)

<p>Introduction: Mitochondrial dysfunction and oxidative stress increase with aging and may contribute to age-associated muscle atrophy (sarcopenia). Resistance exercise (RE) can promote the accretion of muscle mass, increase strength, and ultimately improve function in the elderly. Such beneficial effects are thought to be mitigated solely by increased muscle mass and strength; however, the contribution of the mitochondria to the beneficial effects of RE in aging have not been thoroughly characterized. While mitochondrial benefits have been established separately in both young and aged adults following chronic RE, the acute effects have not been well characterized. Methods: Sedentary young and aged adult males completed either an acute bout of fatiguing RE or endurance exercise (EE), and muscle biopsies were obtained at 3, 24 and 48 h post- exercise depending on the study. Results: Despite equivalent lean-body mass, increased age was associated with elevated mtDNA deletions, indicating potential for mitochondrial dysfunction. RE was associated with reduced mitochondrial content (transcripts, protein, and mtDNA copy number) at 48 h post-exercise, a response that did not differ with increasing age. Paradoxically, reduced mitochondrial content occurred alongside elevated total peroxisome proliferator-activated receptor γ coactivator one α (PGC-1α) mRNA; however, RE altered only the PGC-1α4 isoform post-exercise, a transcript that regulates myostatin and insulin-like growth factor one (IGF1) signalling and ultimately muscle hypertrophy and not mitochondrial adaptations. In addition, PGC-1α modulates the unfolded protein response (UPR), and RE was subsequently shown to elevate endoplasmic reticulum stress and elicit the UPR. Conclusion: PGC-1α mRNA increases regardless of exercise mode; however, differential expression or regulation of alternate PGC-1α isoforms or transcriptional binding partners co-activated by PGC-1α may dictate the specific phenotypic adaptations that occur following divergent modes of exercise. Furthermore, RE acutely decreases mitochondrial content despite elevated PGC-1α mRNA, and this response is not influenced by age.</p> / Doctor of Philosophy (Medical Science)

Tissue fibrosis occurs in the advanced stages of various chronic diseases and can account for 45% of all deaths related to chronic diseases worldwide. The extracellular matrix (ECM) components comprising the fibrotic scar are primarily derived from myofibroblasts, which are contractile fibroblasts arising from the trans-differentiation of several cellular progenitors. Disturbances in immune cell infiltration and function could lead to the uncontrolled production of pro/anti-inflammatory mediators, which may alter the phenotype, state, and function of myofibroblasts progenitors, leading to aberrant wound repair and pathological fibrosis. A great deal of knowledge has implicated macrophages in the pathogenesis and exacerbation of the fibrotic process. Nonetheless, much remains to be elucidated on the potential mechanisms regulating macrophage accumulation and pro-fibrotic polarization, and whether these mechanisms can be further investigated to modulate tissue repair. The Endoplasmic reticulum (ER) stress has recently been implicated as a key mechanism that propagates the pathogenesis of the fibrotic process. How ER stress precisely impacts the fibrotic process is still unclear. This thesis partly explored how modulating the outcome of ER stress – the unfolded protein response (UPR), would affect the severity of lung fibrosis and addressed the role of IL-6 signalling in macrophages during fibrosis. The data demonstrated that UPR activation in pro-fibrotic macrophages and partial deficiency of Grp78, the master regulator of the UPR, abrogated pulmonary fibrotic changes and reduced the accumulation of pro-fibrotic (M2-like) macrophages. These findings were later associated with high TUNEL levels, 7AAD positive cells, Chop and cleaved caspase 3 levels, which are suggestive of GRP78 mediated apoptosis in this population. On the contrary, mice lacking a terminal UPR mediator of apoptosis, called Chop, had increased ECM deposition and greater persistence of non-apoptotic macrophages. These findings suggest that UPR-mediated macrophage polarization and apoptosis may alter lung wound repair processes. As IL-6 synergized the effect of IL-4 to promote a hyper M2 macrophage state, it provided a unique and compelling model to study the dynamics of macrophage alternative programming, which has set the stage to investigate whether the UPR was implicated in the generation of a hyper pro-fibrotic macrophage phenotype. This hyper M2 macrophage model led to the identification of ER expansion program and the IRE1-XBP1 arm of the UPR in pro-fibrotic macrophage polarization, and suggested an unprecedented in vivo role of IL-6 in priming macrophages in the injured lungs to possibly potentiate pathological wound repair. Looking forward, many questions remain to be answered in order to precisely identify the vital UPR axis regulating ER expansion in macrophages during pathological wound repair and to get closer to the understanding of whether the UPR modulates the pro-fibrotic/pro-resolving capacity of macrophages. Insights on these mechanisms may facilitate the development of therapeutics that better manage chronic fibrotic diseases which pose fatal consequences and increase public concern. / Thesis / Doctor of Philosophy (PhD)

The unfolded protein response (UPR) is composed of three highly conserved pathways (ATF6, IRE1, PERK). Cellular stressors induce protein misfolding and aggregation in the endoplasmic reticulum (ER). This signaling pathway maintains protein homeostasis when there is stress in the ER. When the UPR is activated, the eukaryotic initiation factor 2 alpha (eIF2α) becomes phosphorylated, which inhibits global mRNA translation. If ER stress remains chronically unmitigated, the UPR induces apoptosis. GADD34 and CReP shift in expression when the UPR is activated and work as phosphatases and dephosphorylate eIF2α in a feedback loop, allowing protein synthesis to resume. Several human diseases, including fatty liver disease (FLD) are affected by cell stress from improper protein folding and accumulation, making the UPR a therapeutic target. Previous studies have indicated the UPR to both cause or become activated by FLD, depending on the duration of cellular stress. At least 25% of humans worldwide have steatosis, and zebrafish are a powerful model organism for FLD studies. Their embryos are easily obtained, and the liver develops quickly in their transparent larvae, which allows us to visualize the development of fat in the liver. It is unknown how exactly the UPR is involved in inducing lipogenesis in hepatocytes. We sought to better understand the link between UPR activation and steatosis. Pharmacological treatments with various drugs, some of which induce ER stress, were administered over different durations in zebrafish embryos and subsequently the expression of UPR network and lipogenesis genes were quantified through RT-qPCR. To visualize whether these drugs induced steatosis, zebrafish livers were stained with Oil Red O and imaged. Our results indicate that all chronic durations of pharmacological treatments resulted in fatty liver, and the expression of atf6 decreased in response to treatment that prevents the dephosphorylation of eIF2α. This data provides insight pertaining to the activity of the UPR network during FLD in zebrafish models.