Orientador: Helena Coutinho Franco de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:22:09Z (GMT). No. of bitstreams: 1 Paiva_AdrieneAlexandra_D.pdf: 2285275 bytes, checksum: e9d86e12d4ac3d041953799c130a314a (MD5) Previous issue date: 2015 / Resumo: A doença hepática gordurosa não alcoólica (Nonalcoholic fatty liver disease - NAFLD) é a principal manifestação hepática na obesidade e síndrome metabólica. A história natural da doença envolve a esteatose, estresse oxidativo, inflamação, fibrose e morte celular. Níveis plasmáticos elevados de lipoproteínas ricas em triglicérides são fatores de risco independentes para doenças cardiovasculares. Estudos clínicos e experimentais mostram forte correlação entre triglicérides plasmáticos (TG) e os níveis de apolipoproteína CIII. A apolipoproteína CIII também é aumentada no plasma de pacientes diabéticos. Ao comparar camundongos apoCIII transgênicos com controles não transgênicos irmãos (NTg), mostramos aqui que a super-expressão de apoCIII, independentemente da dieta rica em gordura (High fat diet - HFD), resulta em características NAFLD, ou seja, aumento do conteúdo de lípidos no fígado, diminuição poder antioxidante, aumento da expressão de TNF'alfa', receptor de TNF'alfa', caspase-1 clivada e interleucina-1'beta', diminuição do receptor de adiponectina-2 e aumento da morte celular. Além disso, os indicadores de inflamação sistêmica de TNF'alfa' e a proteína C-reactiva estão também elevados em camundongos apoCIII. Esse quadro é agravado e características adicionais da NAFLD são diferencialmente induzidas por HFD em camundongos apoCIII. A HFD induziu intolerância à glicose, juntamente com aumento da gliconeogênese, evidenciando a resistência hepática à insulina, induziu um aumento significativo da TNF'alfa' (8 vezes) e IL-6 (60%) no plasma em camundongos apoCIII comparados aos NTg. Os outros indicadores de inflamação (proteína C reativa plasmática e TNF?, TNFr, caspase-1 e IL-1'beta' hepática) mantiveram-se superiores em camundongos apoCIII em HFD. A ativação da via intrínseca (Bax / Bcl-2) e a proteina efetora (caspase-3) de apoptose foram aumentados em ambos os camundongos apoCIII com low fat diet (LFD) e HFD. Como esperado, o tratamento com fenofibrato reverteu vários dos efeitos da dieta e da apoCIII. No entanto, o fibrato não normalizou o padrão inflamatório nos animais apoCIII, tais como o aumento do TNF'alfa', TNFr, IL1'beta' e redução do adiponectina-R2, mesmo com total correção do teor de lipídios no fígado. Estes resultados indicam que a superexpressão apoCIII desempenha um papel importante na inflamação hepática e na morte celular e aumenta a suscetibilidade e a gravidade da esteatose hepática induzida pela dieta / Abstract: Nonalcoholic fatty liver disease (NAFLD) is the principal liver manifestation in obesity and metabolic syndrome. The natural history of the disease involves steatosis, oxidative stress, inflammation, fibrosis and cell death. Elevated plasma levels of triglyceride-rich remnant lipoproteins are independent cardiovascular disease risk factors. Clinical and experimental studies show strong correlation and causal links between plasma triglycerides (TG) and apolipoprotein CIII levels. Apolipoprotein CIII is also increased in the plasma of diabetic patients. By comparing apolipoprotein (apo) CIII transgenic mice with control non-transgenic (NTg) littermates, we show here that the overexpression of apoCIII, independently of high fat diet (HFD), results in NAFLD features, namely, increased liver lipid content, decreased antioxidant power, increased expression of TNF'alfa', TNF'alfa' receptor, cleaved caspase-1 and interleukin-1'beta', decreased adiponectin receptor-2 and increased cell death. In addition, systemic indicators of inflammation TNF? and C-reactive protein are also elevated in apoCIII mice. This picture is aggravated and additional NAFLD features are differentially induced by HFD in apoCIII mice. HFD induced glucose intolerance together with increased gluconeogenesis, evidencing hepatic insulin resistance. Marked increases in plasma TNF'alfa' (8-fold) and IL-6 (60%) were induced by HFD in apoCIII mice compared to NTg mice. The other inflammatory indicators (plasma C reactive protein, liver TNF'alfa', TNFr, caspase-1 and IL1 'beta') remained higher in HFD apoCIII mice. Cell death signaling (Bax/Bcl2), effector (caspase-3) and apoptosis were augmented in both low and HFD apoCIII mice. As expected, fenofibrate treatment reversed several of the diet and apoCIII effects. However, fibrate did not normalize apoCIII inflammatory traits, such as increased TNF'alfa1, TNFr, IL1'beta' and reduced adiponectin-R2, even under fully corrected liver lipid content. These results indicate that apoCIII overexpression play a major role in liver inflammation and cell death increasing susceptibility to and the severity of diet induced NAFLD / Doutorado / Fisiologia / Doutora em Biologia Funcional e Molecular

Autophagy is the major cellular, conservative, lysosomal catabolic process to eliminate and recycle intracellular waste and organelles through autophagosomes. Enhancing autophagy to promote the clearance of toxic proteins is developing as a promising approach to treat proteinopathy disorders like Alzheimer's disease (AD). AD is the most common aging-associated neurodegenerative disease. It is characterized by the aggregation of aberrantly hyperphosphorylated tau (p-Tau) and excessively produced Amyloid-beta (Aβ) into neurofibrillary tangles (NFTs), and amyloid plaques (AP) respectively. Reprogramming autophagy lysosomal pathway (ALP) through autophagy master controller, transcription factor EB (TFEB), is developing as an attractive strategy to treat AD. It is already proven that TFEB overexpression can promote Aβ and p-Tau lysosomal clearance, attenuate NFT and AP deposition and restore the behavioural deficits in AD mice models. Previously Song et al., 2016 have identified a small molecule curcumin derivative C1. They reported that C1 could bind to recombinant TFEB and enhance ALP both in vitro and in vivo conditions independent of mTOR inhibition. In the current study, C1 is systematically evaluated for its bioavailability, anti-AD efficacy in vitro, and in vivo AD experimental models. To validate TFEB mediated anti-AD efficacy of C1 in vitro, we tested the C1 effect on amyloid precursor protein (APP) and p-Tau degradation in vitro neuronal AD cell culture models. In N2a cells overexpressed with APP (695) and EGFP-P301L tau plasmids, C1 induced APP, CTFβ, and Tau lysosomal degradation. To demonstrate the TFEB dependent autophagic clearance effects of C1, TFEB is silenced in N2a cells with lentiviral shRNA particles. Under TFEB silenced condition, C1 induced reduction of FL-APP, CTFβ, and Tau was compromised. Overall In vitro experiments show that C1 induced lysosomal digestion of FL-APP, CTFβ, and p-Tau in a TFEB dependent manner. To further demonstrate C1 brain bioavailability, C1 and curcumin comparative pharmacokinetic studies (Pk study) in both mice (time course Pk study) and rat (single time point analysis) are conducted. In Pk studies, both C1 and curcumin are dosed at 10 mg/kg to determine their concentration in the whole brain (mice), separate brain regions (rat), CSF (rat), and plasma. The WinNonlin analysis of C1 and curcumin mice Pk study data revealed that C1 is significantly more bioavailable than curcumin in both brain and plasma, which is also corroborated by the single time point analysis in rats. To illustrate C1 anti-AD activity in vivo, C1 is screened in homozygous P301S (Tau), heterozygous 5xFAD (Aβ), and homozygous 3xTg (both Aβ and Tau) AD transgenic mice models. These mice were started to treat with C1 before the onset of AD pathology until the AD pathological phenotype is expressed to cause impairment in mice behaviour. In mice behavioural examination, C1 treatment has significantly improved mice motor function (Rotarod-P301S), restored cognitive impairment related to the cortex (contextual fear conditioning-5xFAD), hippocampus (Morris water maze-3xTg) and improved cholinergic activation (open field-3xTg). In the brain biochemical examination, C1 activated the TFEB mediated ALP pathway to degrade FL-APP, CTFα/β, Aβ, and p-Tau and reduced the amyloid plaque load, extra neuronal-NFT positive cells. Notably, C1 treatment in 5xFAD mice has significantly restored hippocampal synaptic function. In summary, the current study validates C1 as an orally bio-available potent small molecule TFEB activator which restores mice cognitive impairment, altered behaviour, and synaptic plasticity by reducing Aβ and tau levels in AD experimental models. Overall, the TFEB activator C1 can be a promising drug to treat AD.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13281号 / 論生博第19号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22742号 / 医博第4660号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 武藤 学, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ferroptosis is a form of regulated cell death that results in an accumulation of toxic lipid hydroperoxides. It has been implicated in several human disease models, including cancers, organ failure, and neurodegeneration. Identifying novel regulators and biomarkers for ferroptosis can result in identification of disease states where treatment with ferroptotic inducers or inhibitors can result in a positive outcome for a patient. In this thesis, I will describe several methods that were used to identify novel genetic regulators of ferroptosis. A CRISPR-Cas9-mediated, whole-genome loss of function screen identified several gene knockouts that resulted in a protective phenotype against ferroptosis. These genes were found to play a role in antioxidant response, glutathione bioavailability, and/or labile iron homeostasis consistent with the current model of ferroptotic cell death. Alternatively, a CRISPR-dCas9-mediated whole genome activation screen found Gch1 overexpression to have a potent anti-ferroptotic effect. Gch1 overexpression resulted in an increase to the basal levels of endogenous antioxidants including BH₄ and CoQ₁₀, as well as protection of a specific class of phospholipids containing polyunsaturated fatty acyl (PUFA) chains. This multipronged defense resulted in strong suppression of ferroptosis. Additionally, we were interested in the protection of this unusual class of phospholipids with two PUFA tails, and if their enrichment would result in a change in sensitivity to ferroptosis. We found that the degrees of unsaturation of the acyl chains, as well as differences in head groups of the phospholipids resulted in significant differences in the ability of the phospholipids to induce a strong ferroptotic response. The study of their uptake, subcellular localization, and remodeling pathways can further elucidate biological pathways of ferroptosis.

Dans le cadre d'un projet visant à développer des agents ciblant spécifiquement le facteur HIF1alpha inductible par l'hypoxie, BAYER Pharmaceuticals a identifié de façon inattendue de nouveaux inhibiteurs du complexe I, impliqué dans la respiration mitochondriale, qui étaient capables de tuer les cellules cancéreuses hypoxiques in vitro et in vivo. Lors de la modification d'un composé principal plusieurs dérivés ayant des propriétés pharmacodynamiques et pharmacocinétiques favorables ont été identifiés. Ces composés de deuxième génération comprennent BAY87-2243 (B87), que j'ai étudié au cours de mon travail de thèse. Au cours de ces dix dernières années, les mitochondries ont attiré une attention sans précédent en tant que cibles pour le développement de nouveaux traitements anticancéreux avec des applications cliniques. Ainsi, il est apparu important de caractériser les effets antinéoplasiques potentiels de B87 en détail. Mon premier objectif était de tester B87 in vitro, afin de déterminer les conditions dans lesquelles B87 pourrait inhiber la prolifération ou réduire la viabilité des cellules transformées. Dans des conditions de culture standard (disponibilité en nutriments et en oxygène normale), B87 limite la prolifération de plusieurs lignées cellulaires de cancer humain (y compris du carcinome du poumon non à petites cellules H460 et des cellules de carcinome colorectal HCT116), mais ne favorise pas la mort cellulaire. Ensuite, j'ai testé les effets anti-prolifératifs et cytotoxiques de B87 en combinaison avec des médicaments anticancéreux actuellement employés ou molécules naturelles avec des effets antinéoplasiques putatifs. Dans ce contexte, j'ai identifié un fort effet de synergie entre B87 et l'alpha-ketoglutarate (alphaKG), centrale intermédiaire du cycle de Krebs. Pour caractériser les mécanismes moléculaires sous-jacents d'une telle synergie, j'ai étudié la capacité de B87 en association avec l’alphaKG à moduler les grandes voies cellulaires bioénergétiques par l’analyse de flux extracellulaires et par spectrométrie de masse. Dans les cellules HCT116 et H460, l'administration combinée de B87 et alphaKG inhibe non seulement la respiration mitochondriale (comme observé avec l’action de B87 seule), mais aussi la glycolyse, en favorisant par conséquent une baisse létale des taux d'ATP et de glucose-6-phosphate. La combinaison de faibles doses de B87 avec l’alphaKG a également exercée des effets antinéoplasiques robustes in vivo. En conclusion, l’alphaKG s’avère augmenter les effets anticancéreux de B87 in vitro et in vivo, lors de la mise en place d'une crise bioénergétique létale dans les cellules malignes. / In the context of a project aimed at the development of agents that specifically target hypoxia-inducible factor 1 alpha, BAYER Pharmaceuticals unexpectedly identified novel inhibitors of respiratory complex I, which were able to kill hypoxic cancer cells in vitro and in vivo. Upon modification of a lead compound, several derivatives with favourable pharmacodynamic and pharmacokinetic properties have been identified. These second-generation compounds include BAY87-2243 (B87), which I studied during my PhD thesis work.Throughout the past decade, mitochondria attracted unprecedented attention as targets for the development of novel anticancer regimens with clinical applications. Thus, it seemed important to characterize the potential antineoplastic effects of B87 in detail. My first aim was to test B87 in vitro, in order to identify conditions in which B87 might inhibit the proliferation or reduce the viability of transformed cells. In standard culture conditions (normal nutrient availability and oxygen tension), B87 limited the proliferation of several cancer cell lines (including human non-small cell lung carcinoma H460 cells and human colorectal carcinoma HCT116 cells), but did not promote cell death.Next, I tested the antiproliferative and cytotoxic effects of B87 in combination with currently employed anticancer drugs or natural molecules with putative antineoplastic effects. In this context, I identified a strong synergistic effect between B87 and alpha-ketoglutarate (alphaKG), a central intermediate of the Krebs’ cycle. To characterize the molecular mechanisms underlying such a synergy, I investigated the ability of B87 plus alphaKG to modulate the major bioenergetic cellular circuitries by extracellular flux analysis and mass spectrometry. In both H460 and HCT116 cells, the combinatorial administration of B87 and alphaKG inhibited not only mitochondrial respiration (as expected from B87 alone) but also glycolysis, hence promoting a lethal drop in ATP and glucose-6-phosphate levels.Combining low doses of B87 with alphaKG also exerted robust antineoplastic effects in vivo, in both H460 and HCT116 xenografts growing on immunodeficient mice. In conclusion, alphaKG turned out to boost the anticancer effects of B87 in vitro and in vivo, upon the establishment of a lethal bioenergetic crisis in malignant cells.

As an organism develops, multiple cellular processes need to occur in order to specify and organize tissue. One essential process is the establishment of cell polarity, which drives cell fate specification and stem cell differentiation. Another key process is programmed cell death, which is important for tissue remodeling and clearing damaged or diseased cells from the body. A loss in cell polarity can lead to defects in tissue organization and carcinogenesis. Defects in programmed cell death can lead to autoimmune diseases and cancer. However, hyperactive programmed cell death can lead to neurodegeneration. The Drosophila ovary, which is composed of germline and somatic cells, is an excellent model to study both cell polarity and cell death. In the germ cells, oocyte fate is specified and maintained through the asymmetric localization of cell cycle and cell polarity RNAs, proteins, and organelles, such as mitochondria, to and within the oocyte. Additionally the somatic follicle cells, which surround the germ cells, require a specific apical-basal polarity to function. During oogenesis, programmed cell death can be induced within the ovary to prevent oogenesis from maturing under low nutrient, high stress or crowded conditions. When this occurs, the germline is cleared from the ovary by a process known as engulfment. Somatic follicle cells surrounding the germline synchronously enlarge and engulf the corpses of the dying germline cells. It is unknown what triggers the enlargement of the follicle cells. Previous research has shown that the apical side of a follicle cell is heavily marked by cell polarity proteins, to specify the apical side away from the lateral and basal sides. Since many important genes regulating both cell polarity and engulfment are conserved between Drosophila and other eukaryotes, we can study the establishment and maintenance of cell polarity and its role during engulfment to obtain a better understanding of these processes in mammals and their relevance to diseases. This dissertation investigates the role of cell polarity in both the specification of oocyte cell fate, and the organization and enlargement of the follicle cells during engulfment in the ovary. / 2016-11-03T00:00:00Z

The magnitude and effectiveness of CD8 response against intracellular pathogens is directed by survival and apoptotic signals that govern the fate of T cells. XIAP is a bona fide endogenous inhibitor of apoptotic signals. In this thesis, I have investigated the role of XIAP at various stages of CD8 T cell response. I used both in vivo and in vitro models to show that XIAP acts in a CD8 T cell extrinsic and intrinsic manner to regulate the expansion and contraction phases of the CD8 T cell response, respectively. During the expansion phase, XIAP prevents the cell death of APCs to promote APC-T cell interaction and cytokine release, which facilitates the proliferation and survival of activated T cells. During the contraction phase, XIAP functions in a cell-intrinsic fashion to inhibit the proapoptotic signals in the activated CD8 T cells to prolong the immune response. Finally, I also demonstrate that the expression of XIAP in T cells is critical for their differentiation in to memory subsets. Overall, I present that XIAP plays a critical role in generating an effective CD8 T cell immune response.

Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.

Cell clearance is critical for the resolution of inflammation. Defects in cell clearance can result in pathologies associated with chronic inflammation. Cells are cleared by phagocytes. These phagocytes can be either professional phagocytes whose main function is to engulf, or non-professional phagocytes, which have other tissue-resident functions but can engulf when needed. While the molecular biology of cell clearance has been heavily studied, the differences between mechanisms of engulfment by non-professional and professional phagocytes are not yet known. The study of cell clearance by non-professional phagocytes is important due to the presence of these cells in all tissues of the human body. The Drosophila ovary is a genetically tractable, in vivo model system to study engulfment by non-professional phagocytes. In response to protein deprivation, apoptosis is induced in the germline and the surrounding follicle cells engulf the dying corpses. The back and forth signaling that occurs between the dying germline and engulfing epithelial follicle cells is critical for efficient death and clearance. The germline must display eat-me signals for phagocyte recognition. The interaction of the eat-me signal and the engulfment receptor allows follicle cells to activate downstream signaling for internalization and corpse degradation. Interestingly, engulfment receptors have an additional role in driving germline death progression. The research in this dissertation focuses on the signals that drive the reciprocal signaling between non-professional phagocytes and dying cells. The results of this study demonstrate that Draper, an engulfment receptor, induces germline cell death by activating a Shark/JNK/NADPH oxidase signaling axis that is dependent on a YxxL motif. The results from this work indicate that the apoptotic machinery is activated and nurse cell nuclei are degraded in response to Draper overexpression, but Draper-induced cell death occurs independent of effector caspases, indicating that Draper induces cell death via other death pathways. An unbiased, high throughput approach to identify novel death-inducing receptors and ligands is also described. This study describes the reciprocal signaling mechanisms between engulfing and dying cells and opens up new avenues for targeting engulfment-mediated cell death. / 2021-11-08T00:00:00Z